key: cord-0005596-n0kg6jmy authors: nan title: Physicians Poster Sessions date: 2008-03-26 journal: Bone Marrow Transplant DOI: 10.1038/bmt.2008.31 sha: eb4cd01d77c95ab377fc8d2a5d9a97e348309895 doc_id: 5596 cord_uid: n0kg6jmy nan Fanconi anemia (FA) is an autosomal recessive chromosomal instability disorder with a very high risk of head and neck squamous cell carcinoma (HNSCC), notably after hematopoietic stem cell transplantation (HSCT). Risk factors of HNSCC are well known: HSCT conditioning with chemotherapy and radiotherapy, prolonged immunosuppressive treatment and chronic graft versus host disease (GvHD). However, HNSCC characteristics in FA patients after HSCT are not well described. We thus analyzed 13 cases of HNSCC in FA patients who underwent HSCT in Saint-Louis Hospital, between 1976 and 2007. Patients' age at time of transplantation ranged from 4.5 to 19.2 years (median: 9.7 years). All patients received bone marrow as source of stem cells and 10 patients were transplanted from related donors. Concerning the conditioning all patients received irradiation. All patients developed extensive chronic GvHD with lichen of oral mucosa. HNSCC was diagnosed at a median time of 10 years after HSCT. There were diverse tumors' sites: tongue (n=8), palate (n=5), gingival (n=2), jugal (n=2), hypopharynx (n=1), oropharynx (n=1). Three patients had 2 tumors' sites. The TNM classification was: T1 (n=6), T2 (n=2), T3 (n=2), T4 (n= 3). Four patients were lymph nodes positives, 6 were N0. Concerning the treatment, 10 patients had surgery, with clear margins for seven. Neck dissection was carried out for 6 patients. Three patients had irradiation; 1 patient had chemotherapy, and one patient had cryotherapy. Chemotherapy and radiotherapy were associated with toxicity. Five patients presented progressive disease during or immediately after the treatment and five patients relapsed within a median of 5.9 months from diagnosis. For those 10 patients, death occurred with a median time of 6.4 months after diagnosis. At the end of the study, 3 patients were alive without any evidence of recurrence or metastasis. The time of follow up after diagnosis for these patients was: 2.5 months, 3.5 months and 16.5 months, respectively. HNSCC in FA patients after HSCT is aggressive and related with very poor prognosis. A systematic oral follow-up is essential to allow early surgery which remains the only curative treatment. It is very important to attempt to prevent this cancer by reducing chronic GvHD and using conditioning without irradiation. Idarubicin containing regimen for HSCT prolongs mucosal toxicity A.H.E. Herbers*, J.P. Donnelly, N.M.A. Blijlevens Radboud University Nijmegen Medical Cent (Nijmegen, NL) Introduction: We explored the kinetics of citrulline among recipients of an allogeneic or autologous haematopoeitic stem cell transplant (HSCT) who had been given different myeloablative regimens to determine whether the conditioning regimen can be seen as an independent risk factor for gastrointestinal mucosal barrier injury. Citrulline is a quantitative marker for the loss of functional small bowel epithelial cells following cytotoxic therapy. Low citrulline reflects severe mucosal barrier injury. Methods: From March 2004 to June 2007 weekly citrulline was measured among 106 recipients of allogeneic (VUD) or autologous HSCT recipients who had been given different myeloablative regimens. Results: See table: demographic data and citrulline concentrations at baseline and week 1-5 (mean in microM ± 95%CI) for each conditioning regimen. All patients had a lower baseline citrulline concentration (mean ± 95%CI: 23.7 microM ± 1.8) in respect to normal volunteers, probably due to prior chemotherapy and/or active malignant disease. Citrulline levels significantly decreased by the first week after starting myeloablative regimens (13.5 microM ± 1.0; p<0.01), reaching a nadir averaging after 2 weeks of therapy (6.3 microM ± 0.6). Citrulline concentrations, then only increased gradually. Citrulline was the lowest 2 weeks after starting myeloablative therapy with regimens that did not contain idarubicin (6.8 microM ± 0.7). In the conditioning regimen containing idarubicin, citrulline stayed low between week 2 and 3, respectively citrulline 4.9 microM ± 0.7 after 2 weeks and 4.9 microM ± 0.9 after 3 weeks. The citrulline concentration after 3 weeks in the idarubicin containing regimens was significantly lower than in the regimens without idarubicin (citrulline in not idarubicin containing regimens after 3 weeks was 10.2 microM ± 1.1; p<0.01). There were no data for HDM, BEAM and CyTbi at week 4 and 5 because patients had already been discharged. Conclusion: Citrulline concentrations rapidly declined after the start of myeloablative therapy and reached a nadir after 2 weeks. This pattern was independent from the used conditioning regimen. Thereafter two patterns of mucosal barrier injury emerged depending on whether or not idarubicin was used. Patients treated with idarubicin containing regimens experienced prolonged and worsened mucosal barrier injury, whereas patients treated with regimens without idarubicin showed a quicker although gradually increase of citrulline. Reduced-intensity conditioning (RIC) has been increasingly used over the last decade in patients (pts) not eligible for myeloablative conditioning (MC). RIC allows consistent engraftment with reduced toxicity, yet the long-term effects, duration of immunosuppressive therapy (IST) and quality of life in long-term survivors are less defined. We compared outcomes of 50 pts given RIC to 61 pts given MC from 1/2000 to 1/2003. The median age was 49 (range, 16-65) and 36 (18-65) years in the RIC and MC groups, respectively (p< 0.001). The MC group included more pts with acute leukemia/MDS (56% vs 30%, p=0.005) while pts with myeloma were given only RIC (28% of the RIC group, p< 0.001). 48% of pts in the RIC and none in the MC group had a prior autologous SCT (p< 0.001). After a median follow-up of 6.2 years (range, 5.0-7.9) 45 pts are alive, 25 after RIC and 23 after MC with estimated survival of 44% (95CI, 31-57) and 45% (95CI, 30-59), respectively (p=NS). Long-term survival with RIC was achieved across all diagnoses including 6 of 18 pts with acute leukemia/MDS, 8 of 9 pts with CML, 5 of 16 pts with lymphoid malignancies and 6 of 17 pts with myeloma. The corresponding rates in the MC group were 16 of 33, 3 of 4, 4 of 14, and 0 of 0, respectively. Chronic GVHD occurred in 30 pts after RIC [cumulative incidence 51%(39-66)] and in 32 pts after MC [66%(53-81), (p=0.08)]. 16 pts with chronic GVHD after RIC were eventually able to stop IST, 12 died on IST (relapse-8, non-relapse-4) and only 2 of 25 long-term survivors were still on IST at last follow-up. The median duration of IST was 18 months and the probability of stopping IST after 5 years was 72%. In the MC group 11 pts with chronic GVHD were able to stop IST, 10 died on IST (relapse-8, non-relapse-2) and 11 of 23 long-term survivors were still on IST at last follow-up. The median duration of IST was 45 months (p=0.01) and the probability of stopping IST after 5 years was 40% (p=0.007). Two women gave birth in the RIC group while 2 men in the MC group fathered children spontaneously. There was one secondary malignancy in the MC group and 2 pts sustained myocardial infarction (one fatal). One pt in the RIC group had reversible nephrotic syndrome. In summary long term survival is similar after RIC and MC SCT, however IST is significantly shorter after RIC. All 25 pts surviving more than 5 years after RIC sustained excellent quality of life and only two still required IST. These observations require further confirmation in larger registry studies. Short tandem repeats (STR) marker systems are used in the monitoring of hematopoietic chimerism in patients after allogeneic haematopoietic cell transplantation (AHCT). The biological effect of STR disparity on AHCT outcome has rarely been studied. Therefore, we aimed to evaluate the impact of STR disparity on allo-HCT outcome in our single center. Between Sept 2001 and Feb 2007, 150 patients (81M/69F, median age: 34 ys) underwent AHCT (Stem cell source: 119 PB/31 BM) were retrospectively analyzed. Their diagnoses were 90 acute leukemia, 30 CML, 6 MDS and 24 other diseases. Multiplex PCR method was performed to amplify 16 STR loci (D3S1358, HUMvWA, D16S539, D2S1338, Amelogenin, D8S1179, D21S11, D18S51, D19S433, THO1, FGA, D7S820, CSF1PO, D13S317, TPOX, D5S818) (ABI Prism 3130). The loci examined were classified as complete matched (CM), partially matched (PM), and fully mismatched (FMM) between donors and recipients. Results: The loci of D13S317, D18S51 and D2S1338 were the most informative, while the loci of TPOX and CSF1PO were the least. The incidence of acute GvHD was 46.7 % (n=69), which acute severe GvHD (grII-IV) was observed in only 31 patients. Chronic GvHD was developed in 63.4 % patients. The incidence of grII-IV GvHD was higher in patient with CM in TPOX loci (p=0.02). Chronic GvHD was more frequent in the patients with PM in D7S820 loci than those with CM or FMM (p=0.016). While PM in D21S11 locus increased TRM, FMM or PM in D5S818 and FGA loci decreased the TRM. In our cohort analysis, 2-year probability of disease-free survival(DFS) and overall survival(OS) were 58.1±5.5% and 67.5±4.4%, respectively. The CM in D21S11 locus (p=0.07) and PM in D5S818 locus (p=0,009) prolonged the probability of DFS. In multivariate analysis, these loci had an impact on DFS (p=0.055 ve p=0.005). D19S433 and D5S818 loci had an effect on the OS. We repeated similar analyses into two groups, mismatched (MM) group, which FMM and PM was accepted as a whole group or CM group, while the incidence of grII-IV GvHD was higher in patients with CM of D18S51 and TPOX loci, the chronic GvHD was more frequent in those with CM D7S820 loci. Similar as the first analyses MM in D21S11 increased TRM, while MM in D5S818 and FGA decreased the incidence of TRM. The impact of D5S818 on the OS also continued, additionally D19S433 had minimal effect on the OS. In conclusion, some disparities of STR loci might affect the transplantation outcome; however, these results should be analyzed together with other co-variates and on multicenter basis. Therapeutic drug monitoring of oral vs intravenous busulfan in recipients of reduced-intensity conditioning allogeneic stem cell transplantation M. Arnan*, C. Muñoz, J.M. Sancho, T. Peralta, B. Patiño, R. Parody, J.M. Ribera, A. Clopés, R.F. Duarte Institut Catalá d'Oncologia (Barcelona, ES) Pharmacokinetically targeted therapeutic drug monitoring (TDM) may improve the outcome of patients conditioned for SCT with oral (PO) busulfan, primarily through a reduction of early drug-related toxicity. Intravenous (IV) administration of the drug prevents erratic gut absortion and, although it does not fully eliminate interpatient drug exposure variability, it has led to an apparent advantage in toxicity and survival in some studies. Direct comparison of TDM patterns for PO and IV busulfan has not been analyzed to date. We have prospectively performed TDM in 18 consecutive patients, median age 57 years (53-67), who received busulfan-based reduced intensity conditioning allogeneic SCT for myeloid malignancies (11 AML, 5 MDS, 2 CML) between February 2006 and September 2007. Patients received RIC with 150mg/m² IV fludarabine and either 10mg/kg PO (n=7) or 8mg/kg IV (n=11) busulfan divided in 10 doses 6-hourly. All patients received peripheral blood progenitors from matched related (16) or unrelated (2; 1 PO and 1 IV) donors, and GVHD prophylaxis with cyclosporine and either methotrexate (related) or Alemtuzumab (unrelated). There were no differences in patient characteristics between the busulfan PO and IV groups. Analysis of the busulfan area under the curve (AUC) and the concentration at steady state (Css) for both groups is shown in the table. Only 3 cases in the IV group (27%) required dose reduction, and all 3 had at least two concomitant factors that interfere with busulfan elimination (e.g. obesity, voriconazole treatment and other drug interactions). However, 5 of the cases of PO busulfan (71%), with and without such factors, had Css levels above the target (>900ng/mL) and required a median 22% dose-reduction. Neutrophil engraftment and early conditioning-related toxicities were similar in both groups, with no cases of venoocclusive disease. Early mortality at day +100 was very low overall, with only 1 death from gram negative sepsis. In conclusion, busulfan TDM is required in over 2/3 of the cases treated with oral drug in order to avoid potentially toxic levels above the therapeutic target. IV busulfan, however, makes the majority of patients fall within the therapeutic range, and so, TDM monitoring would remain necessary only for those with important concomitant factors that affect busulfan elimination. Morbidity and mortality were overall very low in this series, although sample size prevents statistical comparison of clinical outcomes. Prospective evaluation of quality of life and psychosocial issues associated with chronic graft-versus-host disease -results of an interim analysis F.H.A. Mumm* (1) , D. Wolff (2) , P.Y. Herzberg (3) , J. Tischer (1) , G. Ledderose (1) , H. Greinix (4), S. von Harsdorf (5), K. Rieger (6) , J. Hahn (7) , E. Holler (7), H. J. Kolb (1) , W. Hiddemann (1) , P. Heussner (1) (1)LMU Munich (Munich, DE) ; (2) University of Rostock (Rostock, DE) ; (3) University of Leipzig (Leipzig, DE) ; (4)University of Vienna (Vienna, AT); (5)University of Ulm (Ulm, DE) ; (6) University of Berlin (Berlin, DE); (7)University of Regensburg (Regensburg, DE) Purpose: The intention of this prospective multicentre project is to analyse the National Institute of Health (NIH) consensuscriteria for chronic GvHD in correlation with physical functioning, quality of life (QOL) and a variety of psychosocial determinants as distress, anxiety, depression and personality factors in a long-term perspective. As a specific QOL assessment tool for the population of BMT patients the German version of the Functional Assessment of Cancer Therapy -Bone Marrow Transplantation (FACT-BMT) will be validated. Methods: Eligible are patients after allogeneic hematopoetic stem cell transplantation (HSCT) without GvHD from day 100 to 365 or with GvHD symptoms from day 100 without any time limit. Self-assessment instruments include cGvHD Activity Assessment based on NIH-criteria, Lee cGvHD Symptom-Scale, FACT-BMT, Human Activity Profile (HAP), SF36, Hospital Anxiety & Depression Scale (HADS), NCCN-Distress-Thermometer, Berliner Social Support Scale (BSSS) and 24-item Adjective Measure (24-AM). Follow-up surveys are conducted 1, 2, 5, 8, 12 and 18 months after baseline. At all points of measurement disease status, medication and comorbidities are documented. For clinician rating of GvHD the NIH-Symptom-Scale is applied additionally. Results: Actually 102 patients (mean age 45 years, range 18-67) were evaluated. 65 had cGvHD (mild n=18, moderate n=26, severe n=21). cGvHD NIH consensus grading correlates with FACT physical (r=.41, p<0.0001) and functional well-being (r=.31, p<0.01), p= p<0.01), mental health (r=.38, p<0 .001) and inverse with HAP maximum score (r=.35, p<0.01). The HADS Depression score correlates with pain (r=.36, p<0.0001), impaired physical (r=.23, p=0.023) and functional well-being (r=. 32, p≤0 .001) as well as social/family well-being (r=.25, p=0.019), while the HADS Anxiety score did not. Emotional well-being however correlates with HADS Anxiety score (r=.36, p<0.0001). Conclusion: Preliminary analysis demonstrates that severity of cGvHD correlates with impairment of physical functioning as well as daily activities. Moreover that leads to impairment of various aspects of QOL. cGvHD patients with depression experience reduced QOL concerning social/ family well-being as well as physical and functional well-being, where as anxiety is only linked to impaired emotional well-being. Our results suggest that cGvHD is an important determinant of QOL of survivors after HSCT. Updated results will be presented. Objectives: The hemophagocytic syndrome (HPS) is a clinicopathological entity characterized by macrophage or histiocyte activation and marked hemophagocytosis in bone marrow and other parts of the reticuloendothelial system, resulting in cytopenias. After allogeneic stem cell transplantation (ASCT), patients are at risk of HPS because of viral infections due to immunodeficiency, administration of steroids and immunosuppressants for the treatment of graft versus host disease. However, HPS is a rare complication after ASCT. The aim of this prospective observational study was to evaluate the incidence of HPS after ASCT. Methods: Between August 2006 and November 2007, all patients who received an ASCT, were included in this prospective study. All the following criteria were needed for the diagnossis of HPS: -sustained fever over 7 days -cytopenia (neutropenia and/or thrombocytopenia) -presence of more than 3% mature macrophages in bone marrow -hyperferritinaemia (> 1000 ng/ml) Results: During this study, 68 patients (38 male, 30 female) received an ASCT. The median age was 23 years (3-49 years) . ASCT was performed for the following indications: aplastic anemia (n=28), acute leukemia (n=26), multiple myeloma (n=6), Hodgkin lymphoma (n=3), Gaucher disease (n=3), myelodysplastic syndrome (n=1), sickle cell disease (n=1). We observed 6 cases of HPS (6/68; 8.8%): 1 case of EBV-related HPS, 1 case of CMV-related HPS, and 4 cases with no evidence of bacterial, fungal or viral infections. Three patients died despite aggressive supportive care. Conclusion: To our knowledge, this is the first prospective observational study conducted with the aim to evaluate the incidence of HPS after ASCT. This study provides a relatively high incidence of HPS after ASCT. When sustained fever with progressive cytopenia and hyperferritinaemia are observed, HPS should be suspected, and bone marrow aspirate considered. The rapid diagnosis of HPS and the early initiation of an appropriate treatment is essential for patient management. Refractory haemorrhagic cystitis after allogenic haematopoeitic stem cell transplantation: clinical benefit of early factor XIII infusion N. Chaumard, F. Legrand, F. Larosa, E. Deconinck CHU Jean Minjoz (Besançon, FR) Hemorrhagic cystitis (HC) is a severe complication after haematopoietic stem cell transplantation (HSCT) . The aim of this study was to evaluate the clinical benefit of Factor XIII (FXIII) infusion in affected patients remaining symptomatic after conventional conservative treatments, independently of the use of aetiological treatment (antiviral drugs). Between 1998 and 2007, 22 patients with HC were identified among 290 HSCT recipients. In 12 patients HC resolved in a few days spontaneously or after cidofovir infusion. Nine patients with persistent symptoms received infusions of 50 UI/kg of FXIII ; the results for the 4 first patients were published in 2002 (Demesmay et al) and completed with 5 additional cases. Hematuria was evaluated according to National Cancer Institute toxicity criteria. Clinical improvement was defined as complete resolution of macroscopic hematuria, changes in the irritative urinary symptoms, and decrease of platelet transfusions. Patients' characteristics are summarized in the table. All patients presented with platelets counts <50 G/L and daily platelet transfusions at the onset of HC. Only 1 patient showed a low FXIII seric level. Since 2002 BK virus was evaluated in HSCT recipients and cidofovir treatment initiated in all patients with urinary symptoms. FXIII concentrate was administered between 8 and 15 days after the onset of HC except for two patients (Day 30 ; Day 46). In 4 cases a second infusion was necessary. For 7 patients, urinary clots quickly disappeared (< 48h) and hematuria resolved in a maximum of 10 days. Platelet transfusions requirements completely disappeared for 5 patients. Only 2 patients presenting with grade IV HC and uncontrolled grade III GVHD failed to respond. FXIII associated with standard treatment of HC allow the complete resolution of urinary symptoms in < 4 days in 4/9 cases and <10 days in 7/9 cases. No patient died as a direct result of HC, and no adverse event was observed following FXIII administration. Our observation suggests that patients may benefit more from this treatment if it was initiated rapidly after the onset of urinary symptoms. Only one previous study reported the potential benefit of factor XIII infusion in the setting of HC after HSCT (Sakuma et al). In France treatment of HC with FXIII is an off-label use requiring the authorization of regulation authorities. Demesmay et al. Factor. Transplantation 2002 74:1190 Sakuma et al. . Jap J Clin Hematol 1994; 35: 279-85 P424 Haemorrhagic cystitis after allogeneic haematopoietic stem cell transplantation F. Al Sabty*, E. Demeckova, E. Bojtarova, B. Czako, M. Hrubisko, S. Kubalova, M. Mistrik University Hospital (Bratislava, SK) Objectives: To determine the incidence of hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (HSCT) and the related risk factors. Design:Retrospective chart review. Methods: From 1989 to 2007, a total of 240 consecutive patients underwent allogeneic HSCT for a variety of malignant and non malignant hematological diseases. 123 recieved bone marrow transplant (BMT), 117 recieved peripheral blood stem cells transplant (PBSCT). 21 matched unrelated donor (MUD), 219 matched related donor (MRD). These were 132 males, 108 females. Median age 33y (15-60y). All patients received high-dose cyclophosphamide (CY)-containing regimens as preparative therapies. 50 patients recieved total body irradiation (TBI) with CY. They all received 2mercaptoethane sulphonate sodium (mesna) combined with hyperhydration 3 liter/day as prophylaxis for HC. Results: 33 patients developed HC with a cumulative incidence of 13.8%. The median onset of HC was posttransplant day 35 (range 1-146). Significant risk factors for HC by univariate analysis include graft versus host disease GvHD (n=22/33, 66.7%, p=0.033), MUD (n=6/21, 28.6%, p=0.039), BMT (n=23/123, 18.7%, p=0 .02), diagnosis of acute myeloblastic leukemia (p=0.05), myelodysplastic syndrome (p=0,02), and positive cytomegalovirus antigenemia (n=7, p=0.001). Late-onset HC ( >30 days after HSCT) was more frequently associated with GvHD than early-onset HC (p<0.001). Age of the patient, donor age and sex, platelets count at onset, addition of TBI to the conditoning regimen and GvHD prophylaxis were analysed with no significant results. Multivariate analysis showed that GvHD was the most important risk factor for the development of HC after allogeneic HSCT (p=0.025). Conclusions: 1) The prophylactic measure with mesna and hyperhydration to be effective enough to prevent cyclophosphamide-induced HC of early onset. 2) Patients with GvHD whether acute or chronic have a high risk for developing HC. 3) HC is less after PBSCT than after BMT. 4) HC is more in MUD than in MRD. 5) There is no evidence that age of the patient, donor age, donor sex, or GvHD prophylaxis can influence the development of HC. 6) There is no evidence that the addition of TBI to the conditioning regimen can increase risk of HC. 7) Thrombocytopenia alone can not cause HC, but it contributes to the severity of HC. Decreased and increased basal metabolism may relate to early deaths after allogeneic stem cell transplantation S. Nishiwaki* (1), A. Seto (1) , K. Watanabe (1) , N. Imahashi (1) , M. Yanagisawa (1) , M. Shinba (1) , T. Yasuda (1) , T. Oba (1) , S. Terakura (2) , K. Miyamura (1) , Y. Kodera (1) (1)Japanese Red Cross Nagoya First Hospital (Nagoya, JP); (2) Nagoya University Graduate School of Medicine (Nagoya, JP) Early deaths after allogeneic stem cell transplantation (allo-SCT) are major concern and to be avoided. Recently hematopoietic cell transplantation-specific comorbidity (HCT-CI) is used for risk assessment before allo-SCT but it wouldn ft note for early deaths. We have an impression that a patient who looks older than his actual age tends to involve in early death. It is known that aging relates to decreased basal metabolism (B.Met) and loss of organ reserve. Agerelated decline in B.Met is partially explained by a reduction in the metabolic activity of tissue components. Organ age of those who looks older than his actual age may also be older than his actual age. This may relate to decreased B.Met, decreased organ reserve and eventually leads to early deaths. It is also said that B.Met was increased in some cancer patients and this leads to weight loss and cachexia. According to these, we assumed that both decreased and increased B.Met may relate to early deaths after allo-SCT. We examined 360 adult patients receiving allo-SCT at our institute between 1997 and 2006 and analyzed risk factors (RF) for overall survival (OS) after 30 days from allo-SCT (OS30) and OS after 60 days (OS60). We use the Harris-Benedict equation to calculate B.Met. Comparing patient fs B.Met (PBM) with standard BM (SBM) calculated from ideal body weight, we took note of B.Met ratio (BMR) as a parameter (BMR=PBM/SBM). Twenty-eight patients (7.8%) died within 30 days from allo-SCT and 56 patients (15.6%) died within 60 days. Multivariate analysis (MA) for OS30 showed BMR≤0.95 (low BMR; LBR) (hazard ratio (HR):3.60; p=0.01), BMR >1.05 (high BMR; HBR) (HR:3.49; p=0.02) and non completeremission (nonCR) (HR:8.55; p=0.0006) as RF. MA for OS60 showed LBR (HR:3.17; p=0.0005), nonCR (HR:3.79; p<0.0001), HLA class1 mismatch (class1MM) (HR:2.15; p=0.007), and unrelated donor (HR:2.06; p=0.02) as RF. OS60 was 90.3% in 0.950,5G/l and platelet count >50G/l) occurred in median time of 21 days after transplant. The graft failure occurred in 9 patients (27%) within the follow-up (median time 333, range 44-899 days). Prior to graft failure, progressive mixed chimerism in T-cells was detected in 56%, in monocytes in 67% and in granulocytes in 57% of patients. Despite a small number of patients, a correlation between graft rejection and a mean degree of donor chimerism on day 30 after SCT in T-cells (57% vs. 93%, p=0,013) and in monocytes (59% vs. 98%, p=0,003) was found. The significant correlation between graft rejection and the degree of donor chimerism in monocytes < 85% (median value for CD3+ and CD14+ cells) on day 30 and 60 after stem cell transplantation was confirmed. The same was not confirmed for CD3+ cells. Moreover, in all cases with graft rejection split chimerism over 5% between lymphoid (CD3+) and myeloid cell lines (CD14+ or CD15+) was detected. Conclusion: The degree of donor chimerism in monocytes early after stem cell transplantation correlates with graft rejection. The effects of high-dose therapy on salivary profile and oral integrity in myeloma patients undergoing autologous stem cell transplantation I. Avivi* (1), S. Avraham (1) , M. , T. Zuckerman (1) , A. Nagler (2) , J.M. Rowe (1) , R. Nagler (3) (1)Rambam Medical Center (Haifa, IL); IL) ; (3) Bruce Rappaport Faculty of Medicine, Technion (Haifa, IL) Background: Oral mucositis is the most common complication of high-dose therapy (HDT) followed by autologous stem cell transplantation (ASCT). Its underlying mechanism and severity may be partly mediated via alterations in the normal salivary composition. Objective: Analysis of changes, both clinical and biochemical, occurring in the saliva of myeloma patients suffering from HDT-related mucositis. Patients and methods: 25 consecutive myeloma patients treated with HDT/ASCT at Rambam Medical Center, Haifa and Sheba Medical Center, Tel-Aviv (Israel) between 12/2005-10/2006 were included in the study. Patients underwent a daily assessment for oral mucositis (questioner focusing on oral sensorial complaints and physical examination) and were requested to collect salivary samples on days -3 and +7 of transplantation. Saliva samples were analyzed for antibody content (particularly IgA), anti-oxidant capacity and the amount of various proteins -reflecting the degree of mucosal oxidative damage. Results: HDT resulted in oral mucositis in 100% of patients, which appeared to be most severe on day 8 after transplantation (range:+2-+14). Clinical mucositis was associated with significant changes in saliva characteristics (day -3 vs+ 7). They included a reduction in the salivary pivotal antibody IgA by 54% (p=0.05) and a decrease in salivary antioxidant activity measured by total antioxidant status ( 40%; p=0.0004), anti-oxidant capacity (ImAnOx) (23%; p=0.002) and uric acid level (51%, p=0.006).These changes were accompanied by prominent oxidative damage, reflected in a significant increase in various salivary proteins, i.e., carbonyls by 28% (p=0.047) and albumin by 119% (p=0.024). Conclusions: HDT-related mucositis observed in myeloma patients undergoing ASCT, is associated with an oxidative mucosal damage. Interestingly, there is a significant reduction in salivary antioxidant and immunological activity. These alternations might have a dramatic impact on mucositis development and severity. Therapeutic interventions, enhancing antioxidative and immunological activities are warranted in myeloma patients suffering from mucositis after ASCT, and probably patients undergoing ASCT for other disorders. Purpose: Transfusion associated iron overload is known to increase the complications of hematopoietic stem cell transplantation (HSCT) . In this study, we investigated the association between iron overload and HSCT complications, and also evaluated the efficacy of iron chelating therapy before HSCT. Materials and methods: We retrospectively analyzed patients who underwent HSCT at Seoul National University Children's Hospital from September 2003 to December 2006. The patients were devided into 3 groups. The group A was consisted of patients whose ferritin levels were maintained below 1,000 ng/mL from diagnosis to HSCT. Patients with ferritin level above 1,000 ng/mL at the time of HSCT were classified into group B. The group C was consisted of patients whose ferritin levels decreased less than 1,000 ng/mL after iron cheating therapy with deferoxamine before HSCT. Results: Total 111 patients were evaluated. The number of patients were 47 (42%) in group A, 33 (29.7%) in group B, and 31 (30%) in group C, respectively. In the comparison between group A and B, hepatotoxicity was more common in group B (P=0.017). Also, group B was associated with increased adult respiratory distress syndrome (ARDS) (P=0.029) and treatment-related mortality (TRM) (P=0.023). In the survival analysis, group B showed decreased event free survival (P=0.007). In the comparison between group A and C, there was no significant difference of HSCT complications and TRM. Group C also showed decreased incidence of hepatotoxicity, ARDS and TRM compared to group B. Conclusions: Elevated serum ferritin level was associated with increased HSCT complications and decreased event free survival, and iron chelating therapy could improve the outcome of HSCT. Further prospective study is necessary to confirm the benefit of iron chelating agents. P432 CMV and cGvHD: major players in the development of obstructive pulmonary function changes after allogeneic stem cell transplantation R. Rabanus*, J. Hahn, R. Andreesen, E. Holler, G.C. Hildebrandt University of Regensburg Medical School (Regensburg, DE) Pulmonary complications after allogeneic stem cell transplantation (allo-SCT) significantly contribute to morbidity and mortality. We analyzed 207 patients (pts) receiving allo-SCT for the development of restrictive (PRC) and obstructive (POC) pulmonary function changes. Pulmonary function tests (PFT) were performed before and routinely every 3 to 6 months after allo-SCT. After follow-up >2 years, PFTs were obtained yearly. 142 pts were included according to the availability of PFTs; 65 pts were excluded due to early mortality and/or the lack of PFT data. PRC were defined as a FVC less than 90% and POC as a FEV1 less than 80% of pretransplant values. Median time of follow-up was 1071 days (107-2924). Of PRC,median time of first onset of was at 180 days and of POC at 270 days (60-1800) after allo-SCT. The cumulative incidence (CI) for PRC was 70.7% and for POC 34.2%. Univariate risk factor analysis included patient/donor age and gender, type of transplant, stem cell source, conditioning regimen intensity, TBI, T cell depletion, CMV serology of donor and recipient, acute (aGVHD) and chronic GVHD (cGVHD) and previous thoracic irradiation. Positive patient CMV serology was associated with the development of both PRC (CI 81.5%; p<0.01) and POC (CI 46.1%; p<0.01), whereas CMV+ serostatus of the donor affected only the risk of POC (CI 47.5%; p<0.01). In comparison, PRC in CMV seronegative (CMV-) pts with a CMV-graft occurred in 61.2% and POC in 23.0% of cases. When both donor and recipient were CMV+, CI of PRC was 81.4% (p<0.05), and CI of POC rose to 57.3%, (p<0.01). CMV reactivation was seen at least once in 63.0% of CMV+ pts receiving a CMV-graft, in 41.9% of CMV+ pts with a CMV+ graft and in 14.8% of CMV-pts receiving a CMV+ graft, resulting in a CI for POC of 51.4% in pts with proven CMV reactivation (p=0.06). cGVHD increased the risk of POC (42.9% vs. 15.4%; p<0.01), but was not associated with PRC or the CMV serostatus of donor or recipient. After controlling for potential confounders in multivariate analysis, only cGVHD (p=0.037; HR 2.3), CMV+ donors only (p=0.026; HR 2.3) and both CMV+ donors plus CMV+ recipients (p=0.004; HR 3.7) were independent risk factors for POC. Therefore, our data confirm the previously described association between cGVHD and the development of POC. In addition, this analysis strengthens the concept, that anti-CMV immunity and CMV reactivation contribute to pulmonary function impairment after allo-SCT. Patients and Methods:We estimate QL in 34 patients after allo-HCST with follow-up 1-15 years. HSCT from related donor was performed in 10 (29,4%), from unrelated donor -in 24 (70,6%) patients. Myeloablative conditioning regimen was used in 29 (85,3%), reduced intensity conditioning regimenin 5 (14,7%) patients. Quality of life and intensity of symptoms was estimated by questionnaire: 1.Pediatric Quality of Life Inventory (PedsQL) for children and adolescents ages 2-18 years old (y.o). 2.Medical Outcomes Survey Short Form 36 (MOS SF-36)-for patients ages older than 18y.o. 3.NJ Children Cancer Symptom Inventory (NJC-CSI) -for recipients ages 8-18 y.o. 4.MD Anderson Symptom Inventory (MDASI) -for patients ages more than 18 y.o. Results: 1. Patients ages 8-18 y.o. demonstrated worse meanings in QL in all domains than healthy peers but the worst results were revealed in school domain in patients ages 8-18 y.o. 2. We revealed unfavorable influence teen's age on QL, patients 13-18 y.o. have more statistical significant difference in the meanings in QL in comparing with healthy peers, than recipients younger age. Also patients 13-18 y.o. have more moderate and severe symptoms than children 8-12 y.o. 3. Patients older than 18 y.o. have reduced meanings in physical domain, but psychological domain is accorded with results in healthy people, which shows good adaptation of this patients. 4. Recipients with chronic graft versus host disease (GvHD) have statistically significant worsening in all domains of QL in comparing with patients without this complication. Also recipient. with chronic GvHD have worse meaning of different symptoms such as weakness, pain, sleep disturbance, shortness of breath, loss of appetite, psychological problem, than recipients without this complication. Conclusion: regular estimation QL in long-term survivors especially in adolescents after allo-HSCT helps to early revealing of changes in physical and psychological functioning. Chronic GvHD and a female donor are predictive factors for cellular senescence of haematopoietic cells in longterm survivors after HSCT G.M. Baerlocher* (1), A. Rovo (2) , A. Mueller (1) , S. Matthey (1) , M. Stern (2) , J. Halter (2) , , C. Arber (2) , A. Gratwohl (2) , A. Tichelli (2) (1)University Hospital Berne (Berne, CH); (2)University Hospital Basel (Basel, CH) Human hematopoietic stem cell (HSC) transplantation (HSCT) is an ideal setting to study the same HSCs in different backgrounds. The establishment of the donor-derived hematopoiesis involves extensive proliferation of HSCs which could lead to cellular senescence. Telomere shortening serves as marker of the cellular mitotic history and has been described after HSCT. Telomere attrition has mainly been observed during the first year after allogeneic HSCT; thereafter, telomere length dynamics of recipients appear not to differ from their donors. The aim of our cross-sectional study was to evaluate the telomere attrition in leukocyte subsets of very long survivors after HSCT in relation to donor/recipient age, sex, cell counts of peripheral blood, number of transplanted nucleated cells, and occurrence of acute and chronic graft versus host disease (GVHD). Automated multicolor flow-FISH was used to measure the telomere length in diverse leukocyte subsets of 44 long-term survivors (20 females/24 males) and their respective HLAidentical sibling donors (24 females/20 males). At transplantation the median age of donors and recipients was 25.8 (2-46) and 26.8 years (5-50) respectively. The median follow-up after HSCT was 17.5 years (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) . Four patients received HSCT for aplastic anemia, 40 for hematological malignancies. All patients received bone marrow as source of HSC and total body irradiation was part of the conditioning in 39 (89%). Acute GVHD was observed in 31 (70%), and chronic GVHD in 22 (50%) patients (15 females/7 males). The telomere length (mean ± std) was significantly shorter in recipients as compared to their donors (p>0.01) for granulocytes (6.5±0.9 vs 7.1±0.9), T-cells (5.7±1.2 vs 6.6±1.2), B-cells (7.1±1.1 vs 7.8±1.1) and NK/NKT-cells (4.8±1.0 vs 5.6±1.3) and it was shorter for males compared to females even though this difference was small (range 0. 1-0.8 kb) . Age, sex of the recipient, number of transplanted nucleated cells, and acute GVHD had no impact on telomere attrition. For all cell types we found a significant telomere shortening in recipients transplanted with a female donor (p<0.04) and in those with chronic GVHD (p<0.04). The telomere length difference between donor/recipient was most pronounced for recipients with the combination of a female donor and chronic GVHD ( Figure 1 ). In summary, our data reveal a female donor and chronic GVHD to be predictive factors for a higher telomere attrition in long-term survivors after HSCT. Five-year experience of early enteral feeding in patients undergoing allogeneic stem cell transplantation after myeloablative conditioning I. Yakoub-Agha* (1) , M. Ben Rejeb (2) , H. Baudelle (2) , E. Tresch (1) , C. Berthon (1) , V. Coiteux (1) , C. Dendonker (2) , JB. Micol (1) , JP. Jouet (1) , D. Seguy (2) (1)Maladies du Sang (Lille, FR); (2) Nutrition (Lille, FR) Since the early 2003 with the encouraging preliminary results of our pivotal study (Seguy, transplantation 2006) , all patients refereed to our unit for myeloablative Allogenic Stem Cell Transplantation (allo-CST) were offered right away early enteral feeding via a naso-gastric tube (NGT). The aim of this work was to investigate the evolution of our practices regarding nutritional support and its impact on early patients' outcome over the last five years. After insertion of the NGT at day 1 post-transplant, patients received cyclic nocturne enteral feeding up to patient's discharge. Patients who refused, were intolerant or those developing gastro-intestinal GVHD, received parenteral nutrition in case of unsatisfactory Early outcome of patients and factors influencing 100-day overall survival were prospectively assessed. The practices evolved comparing the periods 2001-02 and 2003-05: decrease of patients with parenteral nutrition (73% vs. 31%), increase of number and median duration of enteral nutrition realized (49% vs. 93%) and (10 d vs. 15 d; P = .001). The group of patients with enteral nutrition compared with the group without it had more often normal (> 35 g/L) serum albumin level (76% vs. 45%, P = .005), shorter neutropenia (20 d vs. 25 d; P = .0001) and thrombopenia (27 d vs. 56 d; P = .014) durations, less often acute grade III/IV graft-versushost disease (37% vs. 9%; P = .001). In addition, patients with enteral feeding had better survival during the 100-day period following transplantation (92% vs. 67%, P = .001) with less death secondary to infection (1% vs. 15%; P = .009). Multivariate analysis confirmed that enteral nutrition was the foremost factor influencing positively 100-day survival (CI 95%: .067 to 0.646; P < .007). Conclusions: Enteral Nutrition has been well tolerated and dramatically reduced the proportion of patients requiring parenteral nutrition. This study confirms the positive impact of enteral nutrition on early outcome of patients undergoing myeloablative allo-CST. When possible enteral nutrition should be preferred to parenteral nutrition. Myeloablative conditioning regimen with intravenous versus oral busulfan plus cyclophosphamide: role of cyclophosphamide dose D. Parent (1) , G. Damaj (2) , A. , E. Tresch (3) , V. Coiteux (3) , L. Terriou (3) , , M. Yilmaz (1) , I. Yakoub-Agha* (3) (1)CHR Pharmaceutique (Lille, FR); (2) Maladies du Sang (Amiens, FR); (3) Maladies du Sang (Lille, FR) The main objective of this retrospective study was to investigate the impact of Cy dose on pts' outcome. This study compared the standard association of oral Bu (16 mg/kg) plus Cy (200 mg/kg) (oral-group; n=24) to the association of intravenous Bu (12/mg/kg) plus Cy (120 mg/kg) (IV-group; n=16). The two groups were comparable in terms of transplantation modalities, and pts' characteristics except for the age of recipients (median at transplantation was 25.6 years and 42.9 years for oral and IV groups, respectively; p=.015) and the period of transplantation (between Sep 01 and Oct 04 and between Feb and Nov 05 for oral and IV groups, respectively). Underlying diseases were, AML (n=22), CML in chronic phase (n=9) and myelodysplastic syndrome (n=9). All pts received an HLA-matched related graft and the same prophylaxis for GVHD (cyclosporine and short-course methotrexate), infections and veno-occlusive disease (VOD) (intravenous heparin 100 IU/kg from admission to day +28 post-transplant). T-cell chimerism was performed serially over one year for all pts using a real time PCR (sensitivity <1%). At the date of analysis, median follow-up was of 729 days (379-729). All pts engrafted but 2 of the IV-group who experienced secondary graft rejection (p=ns). Furthermore, pts of IV-group had less often mixed chimerism at days 30 and 60 posttransplant while those of oral-group had more often full-donor chimerism (p=.002 and p=.05 for d30 and d60, respectively). No pts developed VOD. There were no significant differences between the 2 groups in terms of other post-graft events including mucositis, acute and chronic GVHD, hemorrhage, infections, relapse and intensive care unit transfer. Of note, oral Bu was given using weight-adjusted capsules that could explain in part the good tolerance in this group. Estimated 2year overall survival (OS) was 79% and 87% for oral and IV groups respectively, (ns). In multivariate analysis, group of conditioning had no influence on pts' outcome. The only factor observed to influence adversely OS and DFS was the age of patient > 34.5 years (HR =10.33, p=.03 and 8.7, p=.007, respectively) . Conclusion: This study showed comparable results in both groups. Surprisingly, patients of IV-group experienced less often full-donor T-cell chimerism than did those of oral-group Background: Chronic inflammation is increasingly recognized as a potent carcinogenetic mechanism. Recently models in rodents have suggested that cancer from hematopoietic stem cell origin can occur in the setting of chronic inflammation. However, no such proof exists in humans. Methods: We have analyzed a unique set of squamous cell carcinomas arising in long terms survivors of allogeneic stem cell transplantation. A combination of fluorescent in situ hybridization (FISH) analysis and of micro-satellite analysis of micro-dissected tumor cells, were used. Results: We obtain conclusive evidence that four epithelial tumors among eight studied arose from the engrafted donor cells. Conclusion: Donor-derived hematopoietic stem cells recruited to sites of chronic inflammation yielded epithelial tumors in some recipients after allogeneic hematopoietic stem cell transplantation. Hyperferritinaemia and the metabolic syndrome in longterm survivors of stem cell transplantation J. Shankari*, E. Olavarria, E. Kanfer, D. Marin, A. Rahemtulla, J.F. Apperley, N. Salooja Imperial College (London, UK) An elevated ferritin is commonly noted in patients following stem cell transplantation for leukemia.It is commonly assumed that the elevation is related to iron overload secondary to transfusions received before or in the peri-transplant period. Transferrin saturations are frequently in the normal range, however, raising the possibility that raised ferritin levels represent a manifestation of the metabolic syndrome. This is typically characterised by obesity, dyslipidaemia, and elevated fasting glucose. Other associations are hypertension, elevated transaminases and raised ferritin. We have noted a high incidence of features of the metabolic syndrome in our longterm survivors of transplant. In this study we have documented the incidence of raised ferritin and looked for coexisting dyslipidaemia, hypertension, raised fasting glucose or elevated transaminases ± fatty liver on ultrasound. We have investigated 56 long term survivors of transplantation, with the median age of 48 years (range 21-71yrs) and the median transplant follow-up time of 15 years (range 5-26). 43 patients had CML, 7 ALL, 4 AML and 2 SAA. 21 patients had a ferritin greater than 300ug/l (11/43 CML, 5/7 ALL, 3/4 AML and 2/2 SAA), the median ferritin was 972ug/l (range 315 -2000) and 19/21 had transferrin saturations that were in the normal range with a median of 30% (range 9-51). Among the 11 patients with CML who had a raised ferritin, 10 had at least one additional feature of a metabolic syndrome, 9 had an elevated cholesterol > 5.5/l or triglycerides and were on statins, 5 were hypertensive, 3 had diabetes and fatty liver on ultrasound, 7 had elevated transaminases and 7 patients had 2 or more features. Among 10 patients transplanted for SAA, AML or ALL, five patients had one feature only of a metabolic syndrome and a further five had none. 3 patients had an elevated cholesterol >5.5 mmol/l, or triglycerides and were on statins, one patient was hypertensive and a further patient had diabetes. In this series, ferritin was more commonly raised in patients transplanted for acute leukaemia than those transplanted for chronic myeloid leukaemia. This may reflect the comparatively higher transfusion requirements of patients with acute leukaemia prior to SCT. In patients transplanted for CML only 25% had raised ferritin levels and among this group features of the metabolic syndrome were common. This association merits further study. Evidence of endothelial dysfunction in haematopoietic stem cell transplantation: differential timing depending on the type of transplantation M. Palomo*, M. Díaz-Ricart, C. Carbó, M. Rovira, F. Fernéndez-Avilés, G. Ghita, G. Escolar, E. Carreras Hospital Clínic de Barcelona (Barcelona, ES) Introduction: Intensive conditioning regimen and allo-reactivity seem to be the main causes of the endothelial dysfunction observed after allogeneic hematopoietic stem cell transplantation (HSCT). This dysfunction may be the origin of some complications observed early after HSCT. To evaluate if a less intensive conditioning without allo-reactivity produces endothelial dysfunction, we analyzed comparatively the effect of incubating endothelial cells in vitro with sera samples from patients receiving autologous vs. allogeneic HSCT. Methods: Sera samples were collected before (pre) , and at days 0, 7, 14 and 21 after HSCT from patients receiving BEAM (auto group, n=11) or CyTBI (allo group, n=12) conditioning. Human umbilical vein endothelial cells (HUVEC) in culture were grown with media containing pooled sera from the different collection points. Expression of the adhesion receptors VCAM-1, ICAM-1, and E-selectin, on endothelial cells, and von Willebrand factor (VWF) and tissue factor (TF), on the extracellular matrix (ECM), after 7 days of culture, was analyzed by immunocytochemistry. Leukocyte and platelet adhesion on EC monolayers and on ECM, respectively, was explored under flow conditions. Cell growth was controlled morphologically. Results: Exposure of HUVEC monolayers to sera from both groups of patients caused an increased in the expression of all adhesion receptors, more significantly for VCAM-1 and ICAM-1, on the cell surface with different timing. In the auto group, the increase was progressive from sample Pre and maximal at day 14, whereas in the allo group there were two peaks of maximal expression at day 0 and at day 21. A similar tendency was observed for VWF on the ECM, and no changes were detected for TF. In experiments performed with flowing blood, results of leukocyte adhesion on EC paralleled those observed with the expression of the adhesion receptors. Platelet adhesion on the ECM augmented with the expression of VWF. Cell growth was altered in both groups specially from day 14. Conclusions: The increase in both the expression of adhesion receptors on HUVEC in culture and the concentration of VWF in the ECM indicate that there is endothelial damage after HSCT. This damage seems to have a differential timing depending on the type of HSCT. This differential timing could be attributed to the use of G-CSF in auto-HSCT, and to the allo-reactivity after engraftment in the allo-HSCT. Incidence of hepatic veno-occlusive disease following stem cell transplantation: systematic review of literature from 1979- 2007 J.A. Coppell (1) , P.G. Richardson (2) , P.L. Martin (3) , M. Iacobelli (4), E. Carreras (5), T. Ruutu (6) , T. Barbui (7), R. Soiffer (8), D. Niederwieser (9) (1)Imperial College Healthcare NHS Trust (London, UK); (2) Introduction: The incidence of hepatic veno-occlusive disease (VOD) after stem cell transplantation (SCT) is highly variable in published reports, and is dependent on a number of factors including type of transplant, conditioning regimen, and the clinical criteria used to make the diagnosis. Historically, the incidence has been reported to be as high as 60%. Severe VOD is defined by high mortality and is frequently associated with progression to multi-organ failure (MOF), including renal and pulmonary dysfunction. Whilst several other features of VOD have been explored to assess severity, such as the rate of rise of bilirubin and weight gain, outcome using these parameters can also be variable. The purpose of this review was to calculate the mean overall incidence of VOD and the mortality of severe VOD or VOD with MOF from published reports, and to examine how the incidence may have been affected by changes in SCT practice over time. Methods: Incidence data from 135 reports of VOD among SCT recipients published between 1979 and October 2007 were analyzed. A sub-analysis was undertaken to determine the mean incidence of VOD for studies reporting on patients (pts) transplanted before and after 1994, in order to compare the effects of high resolution HLA typing and use of less hepatotoxic conditioning regimes on the incidence of VOD since that time. Finally, mortality rates from severe VOD or VOD with MOF were assessed from publications where this was specifically addressed. Results: The overall mean incidence of VOD was 13.7% (95% CI: 13.3 -14.1%), with most studies (129/135) reporting an incidence of between 0 and 40%. The mean incidence of VOD after 1994 was significantly higher (14.6%; 95% CI: 14.0-15.2%) [70 studies with 12,234 SCT pts] than that before 1994 (11.5%; 95% CI: 10.9-12.1%; p≤0.05) [51 studies with 10,943 SCT pts]. The overall mortality rate among pts with severe VOD or VOD with MOF was 84.3% (95% CI: 79. 6-88.9%) [19 studies with 235 'severe' VOD pts]. Conclusion: The overall mean incidence of VOD was 13.7% (range 0-40%), suggesting that it is less common than early reports indicated. The incidence was significantly higher among pts transplanted after 1994 than before this time, despite recent advances in SCT and the advent of reduced intensity conditioning (p<0.05). The evolution of MOF in the setting of VOD after SCT can be considered as a reliable indication of severity and predictor of poor outcome. Ability of palifermin to decrease incidence and severity of oral mucositis in patients with haematologic malignancies receiving high-dose chemotherapy conditioning for autologous stem cell transplantation C. Depau* (1), D. Baronciani (1) , M. Pettinau (1) , A. Fanni (1) , M. Micheletti (2) , M. Drera (2) , C. Cattaneo (2) , G. Rossi (2) , E. Angelucci (1) (1)Ematologia Cagliari (Cagliari, IT); (2) Ematologia Brescia (Brescia, IT) Oral mucositis is a devastating side effect of intensive chemotherapy. Aim of this study was to compare Palifermin to standard treatment as prophylactic treatment for oral mucositis in patients receiving high dose chemotherapy before autologous HSCT. Materials and methods: since October 2005 to September 2006, in the course of Palifermin italian multicentric EAP trial 29 patients (19 male,10 female; median age 60, range19-73) with haematologic cancers (12 with Multiple Myeloma, 4 with Acute Myeloid Leukemia, 2 with Hodgking disease and 11 with non Hodgking Lymphoma) received Palifermin (60 micrograms per kilogram of body weight per day iv) for 3 consecutive days immediately before the initiation of conditioning therapy and after autologous stem cell transplantation. We compared palifermin group with a matched control group (ratio 1:2) of 57 patients (30 male, 27 female; median age 60, range 21-74) with haematologic cancers (27 with Multiple Myeloma, 6 with Acute Myeloid Leukemia, 4 with Hodgking disease and 20 with non Hodgkin Lymphoma) with the same characteristics in terms of age, type of disease, conditioning therapy, source and number of haematopoietic stem cells, who undergone autologous stem cell transplantation from March 2003 to February 2007 in the same two Italian Centres but not received Palifermin. Oral mucositis was evaluated using the WHO oral toxicity scale. Outcomes assessed were post-transplantation incidence and severity of oral mucositis, necessity of oppioid, time to engraftment, length of hospital stay. Main results: Main adverse events in Palifermin group were cutaneous rash, pruritus, taste alteration, transient increase of serum amylase and skin iperpigmentation. All these events were transient and mild in severity. Overall incidence of oral mucositis was 10/29 vs 37/57 in Palifermin and control group, respectively (P=0.02). Incidence of severe mucositis (WHO grade 3-4) was 1/29 vs 18/57 in study and control group, respectively (p= 0.004). Two over 29 patients received oppioid in the Palifermin group and 15/57 in the control group (p=0.04). There was no significant difference in time to engraftment, in length of stay between groups. Conclusions: Palifermin reduced the incidence and severity of oral mucositis in patients receiving high dose chemotherapy and autologous stem cell transplantation confirming Spielberg data in an multicentric Italian population with a lower risk of severe mucositis. Second myeloablative allogeneic stem cell transplantation using cord blood for leukaemia relapsed after first allogeneic SCT T. Konuma*, J. Ooi, S. Takahashi, A. Tomonari, N. Tsukada, S. Kato, S. Kasahara, K. Uchimaru, A. Tojo, S. Asano Institute of Medical Science, University of Tokyo (Tokyo, JP) Background: Relapsed leukemia is a serious complication after allogeneic stem cell transplantation (SCT). Treatment options for leukemia relapsed after SCT include chemotherapy, donor lymphocyte infusion and second SCT. In such treatments, second SCT is the only potentially curative therapy. The rapid availability of the cord blood (CB) graft may be one of the most advantages for patients who require urgent SCT, such as second SCT. Here, we report the results of myeloablative unrelated CB transplantation as second SCT in 6 adult patients with leukemia relapsed after first SCT. Patients and methods: First SCT following conditioning regimen using 12 Gy total body irradiation were performed with unrelated CB in 3 patients, matched related bone marrow (BM) in 1, partially matched related BM in 1 and matched unrelated BM in 1. The median time between first and second SCT was 41 (range, 12-57) months. At second SCT, the median age was 24 (range, 19-41) years, the median weight was 53.5 (range, 37-76) kg, and the median cryopreserved nucleated cell dose was 2.75 (range, 2.09-2.96) x 10 7 /kg. Three of the patients were transplanted for the diagnosis of acute myeloid leukemia, 2 for acute lymphoblastic leukemia and 1 for chronic myeloid leukemia. Three patients were in complete remission, 3 in relapse at the time of second SCT. In all cases, myeloabrative conditioning consisted of busulfan 4 mg/kg/day p.o. for 4days, fludarabine 25 mg/m²/day i.v. for 4days and cyclophosphamide 60 mg/kg/day i.v. for 2days. Graft-vesus-host disease (GVHD) prophylaxis was consisted of cyclosporine with or without methotrexate. Results: Five of 6 patients had myeloid reconstitution and the median time to more than 0.5 x 10 9 /L absolute neutrophil count was 17 days. A self-sustained platelet count more than 50 x 10 9 /L was achieved in four of 6 patients at a median time of 54 days. Grade II-IV acute GVHD occurred in 2 of 5 and extensive type chronic GVHD occurred in 1 of 5 evaluable patients. Although 2 patients died of relapse on 2 and 13 months and 1 patient died of encephalitis on 18 months, 3 patients are alive and free of disease on 14, 27 and 33 months after second SCT, respectively. Conclusion: This study suggests that second myeloablative SCT using CB may be feasible and useful in selected patients with leukemia relapsed after first SCT. The quick availability of the CB graft compared to other grafts and myeloablative conditioning regimen might contribute to better prognosis. Induction of psoriasis-like lesions after unrelated cord blood transplantation following non-myeloablative conditioning B. Rio (1) , C. Bachmeyer (2) , P. Moguelet (2) , K. Khosrotehrani (2) , S. Lapusan (1) , J.P. Marie (1) , S. Aractingi (2) (1)Hotel-Dieu (Paris, FR); (2) Hopital Tenon (Paris, FR) Psoriasis is characterized by T-lymphocyte reaction against keratinocytes, after stimulation of macrophages and dendritic cells in the interfollicular region of the skin, a cutaneous site specific of humans. Any animal models do not exist and pathophysiological researches are limited in absence of animal models. Occurrence of inflammatory skin, psoriasislike plaques after unrelated cord blood transplantation (UCBT) led us to assess the frequency and characteristics of the lesions. Material and methods: The patients without related or unrelated donors were proposed for unrelated cord blood transplantation from October 2003 . Until January 2007, 33 patients received a single (n=31) or double cord blood units (n=2) after non myeloablative conditioning regimen consisting in cyclophosphamide 50 mg/kg, fludarabine 200 mg/kg and 2 Gy total body irradiation (n=30) or 140 mg/m² melphalan in 2 patients with previous high dose irradiation. Graft vs host disease was prevented by CsA and MMF. Systematical observation of skin lesions and biopsies were made and materials were analysed in universitary pathological service. Results: Skin inflammatory lesions occured beyond 100 days after UCBT in 12 patients in absence of other criteria for acute or chronic graft versus host disease (cGVHD). Two of them were characterized as eczema and the 10 other ones were consistent clinically and histologically with psoriasis with variable expression of elementary lesions. In any case, diagnosis of GVHD could be suggested on biopsies. The first patient received systemic corticosteroid because of a possible unusual presentation of cGVHD, with dramatic response and treatment was promptly stopped. The remainder patients received topical steroid with rapid response. New lesions were seen in 8 patients with quick reponse after topical steroids. After 1 year, no lesion could be seen in our patients. Discussion and conclusion: The high incidence of psoriasislike lesions after UCBT in our series was surprising. The role of T-lymphocytes was suggested by late occurence of psoriasis when ciclosporine were decreased or stopped. Different specificities could be discussed : 1) a specific stimulation of myeloid dendritic cell by G-CSF used from D+1 to 5 G/L WBC after UCBT could be questionned; 2) the rapid recovery of NK cells after UCBT could induce a host dendritic cells lysis; 3) the donor dendritic cells or NK could stimulate skin macrophages. Chimerism and cells characterization are ongoing. Objective: Patients in isolation for Hematopoietic Stem Cell Transplant (HSCT) face several weeks of heavy physical and emotional discomfort.It has been difficult to identify the most suitable supportive interventions.At Memorial Sloan Kettering Cancer Center (MSKCC), New York,art therapy and massage are regularly offered to these patients. Some of their results lead to the hypothesis that patients in isolation for HSCT may benefit from adding complementary therapy. In connections with the experience at MSKCC, the BMT Unit, in "San Martino" Hospital, Genoa, (Italy), has devised the "Magnolia Project", to assess the response of HSCT patients to a integrated program of art therapy and physiotherapy added to standard care. Method: From January 2005 until July 2007 art therapy and physiotherapy have been added to standard care for the patients in isolation. The hematologist refers to the team of complementary therapy the patients who are assessed as vulnerable or distressed. So far, 60 patients have been seen weekly by both the art therapist and the physiotherapist during their time in isolation. The art-therapy sessions are based on free drawing and painting, and facilitating art therapy techniques, which include patients' comments and verbal narrative. The physiotherapy sessions are based on methamorphic massage, relaxing and active muscular distension, sensorial stimulations, respiratory exercises. The evaluation is based on self-assessment questionnaires, which are filled by the patients twice: at the time of admission, and at the end of the hospitalization. Results: From the analysis of the questionnaires, the benefits received by the patients from the art therapy sessions seem to be the followings: 1) Increased relaxation; 2) Feeling creative; 3) Being able to express and share personal emotions. The benefits received from the physiotherapy sessions seem to be the followings: 1) Pain control and decrease; 2) Experiencing body-mind wellbeing; 3) Increased relaxation. Conclusion: These preliminary results from the Magnolia Project support the hypothesis that providing a combination of art therapy and physiotherapy to patients in isolation may fulfill most important physical and emotional needs.Art Therapy has offered a space for dealing with phantasies and with both positive and negative emotions; physiotherapy has encouraged relaxation and the dialogue with one's body at a psycho-physical level. S94 days to neutrophils >0.5; 11 days to neutrophils >1.0; 16 days to platelets >50) were not significantly different to those of the 67 non-Group O patients (11 days to neutrophils >0.5; 11 days to neutrophils >1.0; 16 days to platelets >50) by the Mann-Whitney-Wilcoxon rank sum method. Because Hoffmann et al. suggest that the effect of Group O on engraftment was most marked with lower CD34+ transplant doses, we investigated engraftment times by blood group for the subgroup of 23 patients transplanted with CD34+ doses below 3.5 x 10 6 /kg. Within this subgroup, there were 10 Group O and 13 non-O patients. Median engraftment times still showed no significant differences between Group O patients and non-O patients. Conclusions: We see no evidence of any effect of ABO blood group on engraftment times in our patients. Although our usual minimum acceptable dose for autologous transplant, 2.5 x 10 6 /kg, may be higher than that used at some other centres, we were still unable to show any effect of ABO group on engraftment even in patients transplanted with relatively lower cell doses. Clinical evaluation of patients with refractoriness to platelet transfusions due to alloimmunisation to HLA antigens after haematopoitic stem cell transplantation , C. , A. Pérez-Corral (1), J. Anguita (1) , A. , M. Kwon (1) , G. , D. Serrano (1) , R. Carrión (1) , I. Buño (1) , P. Balsalobre (1) , B. Ortega (2) , R. Moreno (2) , J.L. Díez-Martín (1) ( Background and Objectives: haematopoitic stem cell transplantation (HSCT) can be complicated by refractoriness to platelet transfusions due to alloimmunisation to HLA antigens (RTAHLA). We report the clinical evolution and platelet transfusion requirements in patients (pts) with RTAHLA and HSCT in our hospital. Methods: A retrospective analysis of pts who underwent HSCT from January 1997 to September 2007 in our HSCT unit was performed. HLA alloimmunization was assessed by testing the presence of antibodies against HLA antigens in patients with suspected refractoriness to random donor platelet transfusion. Bleeding complications, platelet transfusion requirements and HLA platelet compatible units transfused in the first 100 days post HSCT were registered. Results: From January 1997 to September 2007, 421 patients received a HSCT. 7/421 (1%) showed RTAHLA (table1). The median age of such patients was 45 (range 34-59) and all of them were women. 3/7 pts (43%) received AutoHSCT, 3/7 (43%) received AloHSCT, and 1/7 (14%) underwent a cord blood transplantation. The median number of platelet units transfused in patients with RTAHLA was 176 (range 21-615), the median of platelet transfusion previously reported in our hospital in HSCT were 26 units. 3/7 pts developed hemorrhagic complications, pt 2 developed mild transitory genitourinary bleeding on day +45, pt 3 developed mild transitory upper digestive hemorrhage on day +40, and pt 1 developed persistent moderate mucosal nasal bleeding post HSCT, despite immunoglobulin therapy. Only pt 1 received immunoglobulin treatment. 6/7 pts received HLA compatible platelet units. Conclusions: Pts with RTAHLA have higher platelet transfusion requirements during HSCT than non refractory pts and may have a tendency to suffer more bleeding episodes. However, our 7 pts didn't have severe or life-threatening bleeding complications. Larger pt series are warranted to draw more representative conclusions. Echodobutamine stress test in patients after bone marrow transplantation: more information about late cardiotoxicity G. Lucchini*, P. Corti, E. Cavatorta, S. Bonanomi, L. Di maio, G. Trocino, C. Uderzo University Milano Bicocca (Monza, IT) Introduction: Acute cardiac toxicity in bone marrow transplanted patients is well known but few data concerning late cardiac effects in children are reported. This study evaluates cardiac function by echocardiography before and after administration of adrenergic agonist in patients surviving bone marrow transplantation (BMT) with normal basal ultrasound scanning. Patients and Methods: 29 patients (24 males, median age at BMT 9,2 years), underwent BMT (6 autologous, 20 related, 1 haploidentical, 1 unrelated, 1 singeneic donor) for haematological diseases (27 malignant, 2 non malignant). 24 of them received pre-BMT anthracyclines as front line chemotherapy (median cumulative dose = 300 mg/mq). All received cyclophosphamide (median dose = 135 mg/Kg), 23 of them with Total Body Irradiation (TBI) at the dose of 1200 cGy (fractions?), as conditioning regimen. All patients performed a cardiac ultrasound scanning as pre-BMT screening and once a year during the follow-up. At a median time of 10 years post-BMT, patients were evaluated with echocardiography before and after administration of dobutamine (5-10 mcg/Kg/min). The test was considered suggestive for good cardiac contractile reserve if the ejection fraction (EF) had increased of 10% or more compared to the basal value 3 minutes after dobutamine infusion. Results: The median age of the patients at the test time was 20.9 years (range 14-32) with a median follow-up of 14.8 years (range 9.9-23.3) from BMT. All patients completed the test without discomfort. Systolic function was normal in all but one patients, with a median basal EF of 56.8%. A positive response to dobutamine stress test was observed in 22 patients (median EF increase = 18.9%), whereas 7 patients had a limited EF increase (median increase = 5.8%), associated to low left ventricular end diastolic volume and diameter increase, low left ventricular posterior wall and interventricular septum systolic thickening. No significant risk factors (anthracycline dose, TBI, age at BMT, acute or chronic graft versus host disease) were found (p = ns). Conclusions: With this safe and feasible stress test we showed a reduced inotropic cardiac reserve in 25% of BMT survivors with apparent normal cardiac function at basal ultrasound scanning. This study suggests to include an exercise test for routine assessment of BMT survivors who reach the adult age and should have all the information about the quality of life. Palonosetron is a serotonin antagonist with a 40 hours halflife, requiring only one dose for single-day chemotherapy and one administration every other day for multiple-day regimens. Limited data are available for the activity of palonosetron in a high-dose setting, particularly in multiple-day regimens. We prospectively evaluated patients undergoing high-dose chemotherapy (HDCT) ± autologous hematopoietic stem-cell transplant (ASCT) treated with palonosetron and dexamethasone for prevention of acute and delayed emesis. Between March and November 2007, 46 consecutive patients who underwent HDCT received a single dose of palonosetron 0.25 mg IV every-other-day until the end of chemotherapy plus dexamethasone 8 mg IV twice-a-day for the entire treatment duration. Diagnosis was non-Hodgkin's lymphoma (NHL; n=28), Hodgkin's lymphoma (HL, n=6), multiple myeloma (n=5), sarcoma (n=1) testicular (n=3) and breast cancer (n=4). HDCT regimens consisted of high-dose single day-chemotherapy: cyclophosphamide, metothrexate, etoposide, melphalan; or multiple-day schemes as cytarabine, BEAM (carmustine, cytarabine, etoposide and melphalan), busulphan + melphalan, mitoxantrone + melphalan, metothrexate + cytarabine + cyclophosphamide and a slightly modified BEP regimen (bleomycin, etoposide, cisplatin) . Nausea and vomiting were evaluated over the following 5 days by monitoring the number of vomiting episodes and daily nausea as referred by patients. Major endpoints included complete response (CR) (no emesis or nausea) and no emesis (NE) during both the acute phase and the delayed period. A total number of 71 HDCT were administered, 42 of them (57%) consisting of a 1-day treatment. The treatment was very well tolerated without significant adverse events. CR was observed in 56/71 courses (79%) while NE in 59/71 (83%). The control of nausea and emesis was not correlated with the duration of HDCT since 12 emetic episodes occurred independently of treatment schemes. Nine of them were observed over the first 24 hours (75%), the other 3 were associated with multiple-day HDCT. Three patients experienced severe nausea, which lasted for more than 2 days in 2 of them. In conclusion, the combination of palonosetron and dexamethasone demonstrated to be safe and effective in preventing acute and delayed emesis induced by highly emetogenic HDCT. In addition, no differences in terms of CR and NE were found between 1-day and multipleday HDCT using an alternating day administration. Central nervous complications after reduced-intensity conditioning stem cell transplantation M. Uzunov (1) , S. Lapusan (2) , T. Storme (2) , S. Wittnebel (3) , C. Boccaccio (3) , J.H. Bourhis (3) , J.P. Marie (2) , B. Rio (2) (1)Hopital Pitie Salpetriere (Paris, FR); (2) Hotel Dieu (Paris, FR) ; (3) Institut Gustave Roussy (Villejuif, FR) Transplantation following reduced intensity conditioning (RIC) results in lesser transplant related morbidity and mortality, due to shorter duration of neutropenia, earlier immune reconstitution and lesser regimen related toxicity. Previous clinical series in adults reported central nervous system (CNS) complications occuring in more 37% patients after standard HSCT. Reported incidence after RIC HSCT varies between 11% and 44%. To evaluate the nonrelapse CNS complications we have retrospectively reviewed the medical records of 131 consecutive pts receiving RIC HSCT for various hematologic malignancies in two transplant centers from Nov 1999 to Dec 2005. Underlying disease was AML-45pts, NHL-16 pts, myeloma-26 pts, CLL-13 pts, MDS-10 pts, HL-7 pts, ALL-6 pts, CML-5 pts, amylosis-1 pt, myelofibrosis -1 pt, Waldenstrom-1pt. 124 patients received a fludarabine based conditioning regimen associated to TBI2Gy,Busulfan,ATG,Endoxan,Ida,Ara-C or Melphalan. The total dose of fludarabine varied between 90 and 200mg/m 2 , according to each conditioning regimen. 91 pts (69.4%) received SCT from an IS, 20 pts (15.2%) from an MUD and 17 pts (12.9%) received un unrelated cord blood. Immunosuppression consisted in CSA, MMF and/or short course MTX. A total of 15 pts (11.4%) developed various CNS complications with a median onset of 21 days (range 8-160). Subtypes comprised infectious, cerebrovascular , metabolic and unknown complications. Symptoms included seizures, impaired consciousness, headache, nausea, vomiting hemiparesis and coma. In all, 3 pts had cerebral toxoplasmosis, 4 patients cerebral hemorrhage, 2 pts presented hemiparesis and amyotrophy after prolonged intensive care, 1 pt presented a pyamidal syndrome of unkown etiology, 1 pt impaired consciousness due to metabolic disturbances and 4 patients developed limbic encephalitis (the clinical picture associated lethargy, cognitive dysfunction with severe memory disturbance, seizure and psychiatric symptoms -personality change, irritability). Limbic encephalopathy occurred in 2 pts receiving RIC CBT (11.7%), Objectives: Long-term survivors after autologous stem cell transplantation (ASCT) are increasing over the years. Furthermore, outcome improvements and treatment options for acute myeloid leukemia have enhanced the interest for the late effects and quality of life (QoL) after ASCT. Beside the standard outcomes, new indexes have been identified: late effects, QoL, physical and psychological wellbeing. Different instruments have been developed and diffused to obtain a standard measure and interpretation of QoL. We have designed a study to measure QoL in long-term survivor acute myeloid leukaemia (AML) patient (pts) in continuous complete remission after ASCT. Methods: The measure of QoL was obtained using the Medical Outcome Survey -Short Form questionnaire. This is a generic instrument: it assesses health concepts that are not age, disease or treatment specific; it is thus adapted to all types of population. This questionnaire has been shown to be reliable and valid, and is currently being used in several studies and languages. The concepts for measure include 8 scales of self-evaluation: physical functioning (PF), role-physical (RP), body pain (BP), general health (GH), vitality (VT), social functioning (SF), roleemotional (RE), mental health (MH). Between 1982 and 2006, 430 pts with AML in first and second remission underwent an ASCT at our institution. Ninety-four pts have a survival time from ASCT > 10 years, with a median follow-up of 16 years (10-23); we contacted these 90 pts, 24 males and 16 females, and asked the consent to participate in this study; 40 returned the questionnaire. The median age at ASCT was 40 years (16-54) and 46 years at interview; Results: All 40 questionnaires were evaluable. The mean score of values in every scale obtained in our population is compared with the mean values reported in a representative Italian population pattern, respectively: PF 87.5 vs 84.46, RP 80.63 vs 78.21, BP 74.88 vs 73.67, GH 72.6 vs 65.22, VT 66.13 vs 61.89, SF 77.19 vs 77.43, RE 80.83 vs 76.16, MH 73.25 vs 66.59 . In all scales, similar values were recorded in both populations analyzed. Conclusions: In this study, we have shown the QoL after a median follow-up of 16 years from ASCT in AML pts. These data are relevant because although in the literature there are some reports describing the QoL normalization after different treatment strategies, the follow-up reported is in general not very long. We hope this study may help the physicians in the different treatment choices. Psycho-social actions to contain emotions in patients treated with bone-marrow transplantation S. Marsicano*, A. Romanelli, G. Sala, L. Pucci, A. Castiglioni, M. Vinci, D. Tabiadon, L. Tedeschi San Carlo Borromeo Hospital (Milan, IT) Introduction: Psychological stress in BM transplanted isolated pts has been studied for many years and literature shows interesting results. Many researches have however been carried out cognitive tests, easy to be self-submitted by pts and therefore cheaper to be developed. Cognitive self-submitted tests allow a limited emotional situation view, as the individual sensitivity to fear and anxiety changes according to single threshold on feelings variation. Materials and methods: 12 pts affected lymphoma or multiple myeloma and candidate to autologous bone marrow transplantation (ABMT) were submitted to Rorschach culture free projective test at three different times : 1) diagnosis 2) harvesting PBSC phase 3) one year after bone-marrow transplantation (BMT). The research is going on, but the evaluation of tested pts at first and second time show already an interesting perspective. Results and discussion: Emotional status, that might impact cure efficiency during isolation, is referred to psychical activity coming out from Rorschach tests at first and second subministration. Emotions are then faced with actions, planned by psychologists, to contain cure negative impact. Permanent psychic characters may be enlarged by isolation, increasing fear and anxiety and leading to reactive behaviours. Some actions to face these feelings have been therefore suggested. They are usefull to contain pts's anxious comments and complaints, for istance throw psycho-socialeducational personnell's actions as even-silent-proximity. Following table shows results. Some psychic activities expressed by impotence/anxiety feeling result to change at diagnosis and harvesting time; the psyco-social actions developed and reported in "Action Plan" section , allowed an increase in customer satisfaction for better cure efficiency and quality of life. Serious adverse events during haematopoietic stem cells infusion are determined by the number of infused granulocytes T. Bernal*, J.M. Garcia, D. Carrera, G.M. Albaiceta Hospital Univ. Central de Asturias (Oviedo, ES) Background: Toxicity related to autologous cryopreserved peripheral blood stem cells (PBSC) is generally attributed to DMSO and, in recent series, to the number of granulocytes in the infused product. Serious adverse events are uncommon as well as neurological adverse events. Methods: A sample including 9 cases of infusion-related severe adverse events and 18 matched controls was selected. The following data were collected: Demographics (age, sex, weight), main diagnosis and comorbidities, apheresis-related data (date of apheresis, white blood cell count, granulocyte and platelet count per bag at the end of the collection procedure, final DMSO concentration and number of bags), transplantation characteristics (number of stem cell transplant, premedication, conditioning, outcome) and characteristics of the adverse event. Univariate analyses were done using chi-square, T-tests or Mann-Withney tests, according to variable types. Variables with a p<0.1 in the univariate comparison were introduced in a logistic regression analysis. Using this model, odd ratios with 95% confidence intervals and ROC curves were computed. A final p<0.05 was considered significant. Results: Six of 9 cases presented neurological symptoms, including blindness, hemiparesis, aphasia and stupor. There were no differences in age (cases: 55±10, controls 50±14), diagnosis, comorbidities, conditioning regimens or premedication before infusion. Adverse events were more common in women (6 of 9 cases; 6 of 18 controls, p=0.1). White blood cell count per bag was higher in cases than controls (360±153 vs 234±68 x 10 6 /ml, p=0.006), so were granulocytes (230±30 vs 122±72 x 10 6 /ml, p=0.001). DMSO (48±44 vs 53±14 ml, p=0.348) and platelet number (2021±1941 vs 1278±675 x 10 3 /ml, p=0.29) were similar in cases and controls. In the multivariate analysis, the only variable significantly related to adverse events was the granulocyte count p=0.021] . Area under the ROC curve was 0.92. A cut-off point of 185 granulocytes/ per bag was related to a sensitivity of 100% and a specificity of 89%. These results did not change significantly when other apheresis-related variables (DMSO, platelets) were introduced in the model. Conclusions: The increase in granulocyte number in the apheresis product for PBSC is related to an increased risk of severe adverse events during infusion. A limit of 185 x 10 6 granulocytes/ml seems to be safe for serious adverse events. Impact of escalated dose antithymocyte globulin application scheme on acute and short-term toxicity compared to a fixed dose regimen in allogeneic stem cell transplantation D. Mougiakakos *, S. Feihl, M. Grube, B. Holler, K. Landfried, J. Hahn, R. Andreesen, E. Holler University Hospital Regensburg (Regensburg, DE) Antithymocyte globulin (ATG) is a common component of conditioning regimes in allogeneic haematopoietic stem cell transplantation (HSCT), especially if the donor is not a HLAidentical sibling. Clinical studies have shown its important role in the prevention of graft rejection and acute GVHD. Its application is often associated with side-effects resulting from cross-reactions with lymphocytes and macrophages and a subsequent cytokine release leading to symptoms such as fever, chills, tachycardy and thrombocytopenia. In this context it is very meaningful to investigate whether an escalated dose application of ATG is superior especially in terms of acute toxicity as compared to the standard regimen with an equal dosage every day based on the hypothesis that a stepwise desensitization results in a lower acute toxicity. To address this question we performed a retrospective analysis of 50 patients with hematologic disorders that underwent an allogeneic HSCT in our department in the period 1998-2007 and received ATG either escalated (n=34) (10, 20 and 30 mg/kg bodyweight on d1-3) or in a fixed dose regimen (n=16) (20 mg/kg bodyweight on d1-3). We evaluated the clinical (fever, chills, tachycardy, body weight) and laboratory (thrombocytes, transaminases, creatinine, quick, CRP) parameters from d1-3 of the ATG treatment and 24h following the last application (d4). In addition we looked for any unexpected impact on acute GVHD (grade > 1), day of engraftment (ANC > 500/µl), CMV reactivation and transplant related mortality (TRM). Our statistical analysis (t-test, chisquare-test) showed a significant (p < 0,05) lower occurrence of fever and chills in the group receiving fractioned ATG (26% and 23% vs. 70% and 62%). Other parameters were not affected and there was no impact on engraftment day or GVHD indicating that this regime might cause less acute P460 Severe affection of the hypothalamus-pituitary-peripheral gland axis as an early complication after allogeneic stem cell transplantation M. Ringhoffer *, M. Schmitt, S. von Harsdorf, A. Wochnik, T. Zenz, S. Stilgenbauer, D. Bunjes University Hospital (Ulm, DE) Background: Durable impairment of the endocrine function is a common complication after allogeneic stem cell transplantation and has largely been attributed to long-term survivors. Here we report 3 patients (pts) with pituitary gland and subsequent peripheral endocrine insufficiency diagnosed at a mean of 105 (56-164) days after transplantation. Patients: All pts (2 females and 1 male) underwent haematopoetic stem cell transplantation for acute myeloid leukaemia at a median age of 56 (50-67) years. 2/3 pts had refractory disease, the other patient was in CR1 at the timepoint of transplantation. Donor types were HLA-identical siblings in 2 cases and a haploidentical family donor in the third case. Two pts received total body irradiation during conditioning, the third patient with an age of 67 years received a reduced intensity conditioning regimen in combination with an Yttrium-90 based radioimmunotherapy. All patients received intrathecal prophylaxis before and supportive therapy with aciclovir, levofloxacin, itraconazole, and trimethoprim/sulfamethoxazol after transplantation according to the local SOPs. One pt had acute GvHD grade II at the timepoint of diagnosis. Results: The patients clinically presented all with fatigue or lethargy and depression; one patient tried to commit suicide. The primarily leading laboratory finding was a lowered TSH (mean: 0,036 mIU/l) in combination with a subnormal fT3 (mean: 2,75 pmol/l) and a normal fT4 (mean: 15,6 pmol/l). The extended analysis of the other endocrinological axes showed severe affection of the gonadal axis in 2/3 pts and of the adrenal axis in 3/3 pts. MRI scans of the sella and pituitary gland did not detect a morphologic abnormality in any of the patients at timepoint of diagnosis. All patients have been substituted with L-thyroxine and hydrocortisone, the male patient received additional testosterone replacement. The pts recovered from the clinical symptoms and achieved normal peripheral hormone levels, however, the affection of the pituitary gland was ongoing at different degrees in all patients. Discussion and conclusion: Although there is some rationale, the pathogenesis of the pituitary affection in our patients remains unclear. However, severe affection of the hypothalamus-pituitary-peripheral gland axis should be taken into consideration as a differential diagnosis also shortly after transplantation. Palonosetron in prevention of nausea and vomiting following highly emetogenic chemotherapy before haematopoietic stem cell transplantation -single-centre experience P. Rzepecki*, J. Barzal, T. Sarosiek, C. Szczylik Institute of Health Services (Warsaw, PL) Objective: A clinical study of palonosetron was carried out to evaluate its efficacy in preventing both acute and delayed emesis after high-dose chemotherapy [HDC] before hematopoietic stem cell transplantation [HSCT] by using a historical control group of patients treated with ondansetron as the comparative drug. Methods: 23 patients were treated with palonosetron. Ten of them suffered from lymphoma and received BEAM as conditioning regimen. Eight patients were treated with CARBOPEC because of relapse of their disseminated germ cell tumors. Five patients with acute myeloid leukaemia received BuCY regimen before HSCT. Patients received palonosetron (0,25 mg i.v. bolus 30 min before chemotherapy) on the first day of conditioning regimen before HSCT. Historical control group of patients was treated ondansetron 32 mg i.v. daily until the end of HDC. Patients from both groups also received dexamethasone. The patients' groups were comparable for statistical analysis. The observation period started with the initiation of HDC and continued for 24 h after completion of the chemotherapy for acute emesis, and during five days after finishing treatment for delayed nausea and vomiting. The severity of nausea was evaluated according to the following 4-grade scale every 6 h after administration of the study drug: none (no nausea); mild (slight nausea but no disruption to daily activities); moderate (nausea and some disruption to daily activities);and severe (extreme nausea and severe disruption to daily activities).The emetic response rate was tabulated for patients using the following criteria: complete (no emetic episode); major (1 to 2 episodes); minor (3 to 5 episodes); and failure (>5 episodes). The response rate of the study drugs was evaluated by the following 4-grade scale based on the condition of nausea and vomiting (highly effective, moderately effective, slightly effective and not effective). [table] . Results: Patients treated with palonosetron 0.25mg plus dexamethasone had significantly higher response rates than those receiving ondansetron plus dexamethasone during the the both: acute and delayed phases [highly+ moderately effective: acute phase 15% vs 5% for CARBOPEC; 70% vs 35% for BEAM and for 32% vs 20% BuCY; delayed phase: 60% vs 30%for BuCY; 100% vs 50% for BEAM and 25% vs 10% for CARBOPEC. Conclusions: Single-dose palonosetron was more effective than ondansetron treatment in preventing acute and delayed nausea and vomiting following HDC before HSCT. (2) , P. McCarthy (2) , I. Demidova (1) , A. Misiurin (1) , E. Parovitchnikova (1) , V. Savchenko (1) (1)Russian Research Center for Hematology (Moscow, RU); (2) Roswell Park Cancer Institute (Buffalo, US) Sometimes the striking difference in regimen related toxicity (RRT) and efficacy of drug therapy can be observed in pts from different ethnic groups who underwent chemotherapy and hematopoietic stem cell transplantation (HSCT) for hematological malignancies. In our study we proposed that genetic variability of polymorphisms in some drug detoxifying enzymes (glutathione-S-transferase M1 (GSTM-1) and glutathione-S-transferase T1 (GSTT-1) )possibly can determine these differences in RRT between pts. 54 pts were treated by first allogeneic or autologous HSCT from 1/1998 to 6/2007 in Russian Center for Hematology. All of them were transplanted for high risk hematological malignancies. The median age was 43 yrs (18-65), there were 20 women/34 men. 41 pts belonged to heterogenic Slavish ethnic population (Russian, Ukrainian and Belarus) and 13 pts were from other ethnic groups, which included Turkish, Armenian and North Caucasian ethnicities, who are less genetically diverse. All of them received HLA-identical HSCT from sibling donors; the source of transplanted cells was BM in 47 pts and PBSC in 7 pts. Median follow-up was 13 months . DNA was extracted from fresh BM or PB samples of recipients before HSCT and genotypes were investigated using multiplex PCR for GSTM-1 and GSTT-1 and amplification products were visualized by electrophoresis in 1,5% agarose gel. We identified homozygous null genotypes for each of GSTM-1 and GSTT-1 leading to absence of protein translation and for both of them, trying to reveal some ethnic differences and their influence on RRT in pts after HSCT. Analyzing the RRT in two ethnic groups of pts we revealed the statistically significant difference in toxicity events of grade 2-4 (Bearman RRT criteria) for non-Slavish group of pts (12/13 pts (92%) versus 25/41 (60%), p= 0.04). The same trend was seen for stomatitis grade 2-4 (10/13 pts (76%) versus 24/41 (58%)) while this difference was not significant (p= 0.3) . In this study we did not identify any difference in frequency of GSTM-1 or GSTT-1 null genotype or their combination between two ethnic populations. However, the GSTM-1 deletion was associated with increased risk in grade 2-4 RRT for the whole population with p= 0.02, while GSTT-1 deletion had no statistical significance. Genotype analyses of polymorphisms in additional drug-metabolizing enzymes are ongoing now as well as multivariate statistical analyses which may help to identify the reasons for different frequency of RRT events. The salivary neutrophil level as an early indicator of engraftment after autologous transplantation for myeloma and lymphoma. The role of patient education and preventive dentistry for oral mucositis rescue J. Vondrakova, R. Pink, E. Faber, I. Skoumalova, I. Maresova, M. Koupilova, J. Vitkova, J. Pazdera, K. Indrák University Hospital (Olomouc, CZ) Objectives: Analysis of neutrophil levels in the saliva as the early marker of stem cells engraftment after autologous transplantation (ASCT) myelomas and lymphomas patients. Evaluation of risk factors for oral mucositis (OM) to improve stomatological care before transplantation. Methods: Results of prospective study (started January 2006) after obtaining ethics commitee approval are reported. Patients treated for multiple myeloma (MM), Hodgkins (MH) and non-hodgkins lymphoma (NHL) were stomatologically examined before, during and after planned high-dose chemotherapy with ASCT to evaluate OM according to NCI-CTC and WHO classfication. Before the beginning of hematooncological treatment all potential sources of odontogenic infection and/or local irritation of oral mucosa were removed. The conditioning regimen included high-dose melphalan for MM and BEAM regimen for NHL, MH. Supportive care was administered according to standard institutional practise. Neutrophil levels in saliva were monitored daily and compared with the neutrophil level in peripheral blood and degree of OM. Salivary sampling and salivary neutrophil (SN) determination were standartised (rinse of oral cavity, laboratory process, examination using light microscope and Fuchs-Rosenthal chamber, engraftment defined as SN > 25x10 6 /l). Results: Data of 27 patients (15 male, 12 female, 16x NHL, 9x MM, 2x MH, median age 53 years, range 26-63) were analyzed. The median SN level before chemoterapy was 123x10 6 /l, on day 0, +5, +8, +11, +13 was: 70, 45, 5, 30 and 100x10 6 /l. All patients engrafted with WBC>1,0x10 9 /l on day 12 (10-18), PLT>20x10 9 /l on day 11 (7-14), SN on day 11 (9-17), median days with OM was 4 (0-10), median severity OM grade I (0-II). Myelomas patients engrafted with WBC>1,0x10 9 /l on day 12 (11-13), SN on day 10 (9-14), median days with OM was 2 (0-2), median severity OM grade I (0-II). Lymphomas patients engrafted with WBC>1,0x10 Background: Avascular necrosis of the femoral head (ANFH) in patients (pts) with chronic Graft Versus Host Disease (cGVHD) has been described as particularly related to highdose or long term steroid treatment or hormonal disorders, producing destructive lesions which require hip replacement. Here we described 4 pts who presented ANFH without a long term steroid treatment nor hormonal disorders, observed in our centre in the last 3 years. Reports: 4 male pts, median aged 46 years (range 24-59), were submitted to reduced intensity (3/4) or conventional (1/4) haematopoietic stem cells transplantation for different haematological diseases (2 myelodisplasia, 1 lymphoma, 1 chronic myeloid leukaemia). Similar features for every pts were: cyclosporine (CSA) based GVHD prophylaxis (4/4 pts, but 1 discontinued early CSA for neurological intolerance) a previous short term treatment with low dose steroids for acute or cGVHD (median term was 85 days, range 66-111; median prednisone dosage was 1mg/Kg/day, range 0. 3-1.5 ) and a long term treatment with micofenolate-mofetile (MMF). MMF was employed for steroid-CSA refractory cGVHD in 3/4 pts and for GVHD prophylaxis in pts with intolerance to CSA. MMF treatment started at full dosage 15 mg/Kg twice a day, median period of full dosage was 185 days (range 127-375) median term of tapering up to complete suspension was 573 days (range 330-1065). None pts had collateral effects due to MMF treatment or intolerance. No GVHD reactivation was observed during MMF tapering. After suspension of every immunosuppressive treatment (2 pts) or during MMF tapering (2 pts), signs and symptoms suggestive for ANFH, in term of persistent hip pain and difficulties in deambulation appeared. Diagnosis of ANFH was confirmed by Nuclear Magnetic Resonance. One patient was submitted to total hip arthroplastie. No alteration in hormonal assessment have been found in our pts. Discussion: ANFH is a long term complication observed in transplant setting, particularly in pts with previous long term steroid treatment or hormonal disorders. Our pts were not included in these categories, because they experienced only a short course of low-dose steroid therapy. Long term MMF treatment is the only common feature, also if a strictly relation with ANFH is not demonstrated, a careful surveillance on orthopaedic situation is recommended in this setting, because it is associated with significant morbidity and required replacement surgery in 25% of affected pts. Recommendations for palifermin use in the prevention of oral mucositis in patients undergoing haematological autologous stem cell transplantation A. Olivieri* (1) , E Gallo (2) Background: Oral mucositis (OM) is an acute, severe, and often dose-limiting toxicity in patients undergoing haematological stem cell transplantation (HSCT). Palifermin, a recombinant human keratinocyte growth factor, has been shown to decrease the incidence, duration, and severity of OM in patients with haematologic malignancies receiving myeloablative therapy followed by autologous HSCT. Up to now, no algorithm or guidelines regarding the recommended use of palifermin have been published. Methods: A panel of four Italian haematologists with experience in stem cell transplantation reviewed the published literature with the aim of defining recommendations for the use of palifermin. Computerised literature searches of PubMed were undertaken to identify relevant publications using the following keywords: autologous transplantation, transplantrelated mortality (TRM), mucositis, non-haematological toxicity, myeloma, lymphoma, risk factors. Eosinophilia is know to appear post HSCT. However, its significance is still obscure. This study evaluated the association of post-allo-sib-HSCT blood eosinophilia and the outcome of transplantation. Methods: a retrospective analysis of 89 patients (AML, ALL, CML, SAA) who received BMT or PBPCT from matched sibling donors in whom in addition to the routine blood work up also a mean and median eosinophilia were calculated based on blood smears done in 3 day intervals from day 0 to +50 and weekly until +100. Results: eosinophil count >100 and < 350 was seen in 20% of cases , eo >350 and <500 in 6% of cases with no significant difference among patients with different diseases or age groups (0-16 yoa , >16-30 , >30) or transplant material (BM vs PBPC) ; mean eo>500/ul was not observed and rather normal values of eo<100/ul were seen in 74% of cases. Higher eo counts (100-500 / ul) were recorded in patients after BuCy conditionong (39% of pts) and BuCyVp (21%) as compared to those receiving Cy/ATG (13%). Notably, severity of acuteGvHD was inversely correlated with the number of eosinophils: aGvHD of grade>I was seen only in patients with a low eosinophil number (with mean eo count below 350/ul; 26% of low eo cases) while aGvHD grade I was prevalent among high eo (eo>350/ul ; 75% of high eo cases). All patients with a high eo count had cGvHD at later time of observation, however also low eo count patients contributed to the cGvHD group but to lesser extent. Observation of changes in eosinophil numbers in periods directly related to the onset of aGvHD symptomatology revealed a dynamic rise in eo count in aGvHD grade I cases , not seen in aGvHD=0 or aGvHD>I patients. No difference in overall survival (OS) was seen between high (eo>100/ul) and low (eo<100/ul) eosinophil groups when all patients were analised , but when disease types were compared the OS in acute leucaemia low eosinophil group was 0,8 while in CML high eosinophil group was 0,2 (OS in CML low eo group was similar to that in acute leucaemia high eo group = 0,5). Evaluation of deoxycholate amphotericin-B toxicity after HSCT C. Annaloro*, C. Olivares, E. Tagliaferri, P. Usardi, A. Della Volpe, F. Onida, G. Lambertenghi Deliliers Fondazione IRCCS Ospedale Maggiore (Milan, IT) The use of deoxycholate amphotericin B (D-AMB) in the empiric treatment of febrile neutropenia was abandoned by our BMT Unit in 2006, but we here review the data concerning the 29 HSCT recipients (21 autologous, 9 allogeneic, 13 males and 16 females, median age 57, range 22-66) treated for this indication over the previous two years. D-AMB was administered at a dose of 0.7 mg/kg over 6-8 hours, starting with a 5 mg and a 25 mg administration on the first day; all of the patients received intravenous hydration exceeding 3000 mL/day, including at least 1000 mL of normal saline solution. Antihistamines were administered to lessen the degree of acute infusion reaction. Creatinine clearance was calculated from creatininemia using the Cockroft formula. Treatment was discontinued after the resolution of the febrile episode, or in the case of failure, or after the development of toxic events requiring a switch to other antifungal drugs. Median creatinine clearance was 95 mL/m (range at baseline, and 58 mL/m (range 30-121) at the end of D-AMB treatment. The median maximum intravenous KCl supplementation required was 240 mEq/day (range 140-400); if the daily requirement exceeded 200 mEq/day, intravenous kanrenoate was regularly added. The median duration of D-AMB treatment was nine days (range 5-15). D-AMB treatment was followed by lasting defervescence in 18 cases; it was discontinued because of failure or toxicity in respectively four and seven cases, being replaced by either liposomal amphotericin B or caspofungin. Although, strictly speaking, D-AMB is not licensed for the empiric treatment of febrile neutropenia, its low cost encouraged many efforts to minimise toxicity, including hyperhydration and continuous infusion; the extent of the renal damage was sometimes simply overlooked. Allogeneic HSCT is commonly considered a contraindication to D-AMB; our preliminary data show that a striking reduction in renal function and a need for high levels of intravenous potassium supplementation are also common findings in autologous HSCT patients, despite regular overhydration. The need for continuous infusions raises further doubts about its efficacy, and leads to a considerable reduction in renal function of about 30%. Although D-AMB has been a cornerstone in the history of oncohematology, it probably now deserves a place the memories of the past. S102 Case: The patient was diagnosed with Addison disease due to X-ALD at the age of 10 y. Neurological symptoms included unsteady gait and fatigue. Brain scan showed minimal signs of periventricular demyelination. He received a 10/10 matched cord blood HSCT at the age of 11.5y with conditioning according to the EBMT inborn errors protocol (busulphancyclophosphamide). Graft versus host disease prophylaxis consisted of methylprednisolone and cyclosporine. Neutrophils engrafted at D+20. Donorchimerism was mixed (66%) at D+30 and complete at D+66. At D+117 he was admitted with fever, lymphadenopathy and hepatosplenomagely. Blood EBV PCR showed 1281694 copies/ml, rising to >5000000/ml D+124. Scan and PET-scan of abdomen and thorax showed multiple lymphoproliferative lesions in lung, mediastinum, liver, spleen and retroperitoneum. Brain scan was unchanged. Lymph node biopsy showed evidence of EBV+ PTLD with plasma cell differentiation. Rituximab was initiated at D+124; immunosuppression was stopped. At D+146 EBV copies had declined to 173589/ml. PET scan showed a partial response. However the patient's mental status declined accompanied by spasticity, yet no signs of X-ALD New brain scan showed cerebral T2 hyper-en T1 hypo-intense foci suggestive of lymphoma with involvement of medulla oblongata and cerebellum. Cerebrospinal fluid (CSF) EBV copies were 20482/ml. Twoweekly intrathecal (IT) injections of methotrexate (MTX) and methylprednisolone were initiated together with weekly cidofovir. Blood EBV and CSF copies declined to <500/ml and undetectable at D+164 but brain imaging showed increase of PTLD lesions. 4 monthly CHOP courses (cyclophosphamide, doxorubicin, vincristine, prednisone) in association with monthly IT MTX were planned. PET scan at D194 (after 2 CHOP) showed complete remission of the peripheral PTLD and partial remission of central PTLD. Cidofovir was stopped, 2 more CHOP were given resulting in complete remission at 5m follow-up. At 9m follow-up, the patient is in complete remission, but with residual hemiplegia and frontal signs. Conclusion: Therapy for central PTLD is difficult and caseoriented. In this patient, combination of rituximab, cidofovir, IT MTX and CHOP led to complete remission, yet with important sequellae. Outcome after allogenic stem cell transplantation and intensive care in a historical comparison H. Lellek*, S. Kluge, G. de Heer, R. van de Loo, T. Zabelina, N. Kröger, A.R. Zander University Hospital Hamburg (Hamburg, DE) The transplantation of hematopoetic stem cells (SCT) can lead to severe complications with a transfer to an intensive care unit (ICU). The necessity for ICU-treatment is associated with aggravation of the outcome. In the last decade there were a lot of improvements in the treatment both with hematopoetic stem cells and of critical ill patients. We compared the outcome in the years from 1990 till 1996 and from 2006 till June 2007. The number of patients undergoing a stem cell transplantation and ICU-treatment was nearly the same. Between 1990 and 1996 315 patients were undergoing an autologous or allogenic SCT in our department, 43 ( 13,7 %) were transferred to ICU. Recently from 2006 till June 2007 patients were treated in the ICU. Compared to previous time the patients are older. The average age was 31 and is 49 years now. The underlying disease is not a risk factor for a critical aggravation. The time point for transfer to the ICU was previously 80 and now 66 days after SCT, mainly because of respiratory failure. In the nineteens nearly every patient was invasive ventilated, today only 50%. Otherwise nearly every patient is treated with non invasive ventilation both in a therapeutic manner and as bridging before intubation. The principal reason for ICU-treatment was severe sepsis (50%), followed by GvHD (17%), toxic organ failure (17%) and others (17%). From 1996 From till 1999 survived the ICU-treatment and 2 (4,7 %) were long term survivors (longer then 9 months). In our days 22 (52,4 %) patients could leave the ICU in a improved condition and 9 (21,4 %) are long term survivors. Despite higher age the outcome of patient treated on an ICU after a SCT has clearly improved. Using non invasive ventilation helps to avoid long weaning time connected with complications. Life-threatening complications occur after engraftment during the immunreconstitution. The patients are in risk of critical aggravation and should be monitored carefully. Treosulfan-compared to total body irradiation-based preparative regimens before allogeneic stem cell transplantation for acute myeloid leukaemia: a retrospective long-term follow-up analysis D.W. Beelen* (1), T. Gromke (1) , R. Trenschel (1) , D. Wolff (2) , L. Kordelas (1) , M. Freund (2) , J. Casper (2) (1)University Hospital of Essen (Essen, DE) ; (2) University Hospital Rostock (Rostock, DE) Background: Treosulfan (Treo), a bifunctional alkylating agent with profound stem cell toxicity, is emerging as a promising new element of preparative regimens before allogeneic stem cell transplantation (alloSCT). Currently, no data are available, which allow an assessment of the tolerability and efficacy of Treo-based compared to standard myeloablative preparative regimens in disease-specific transplant settings. Methods: In a bi-center retrospective analysis, a total of 203 patients (pts) with acute myeloid leukemia (AML), who underwent alloSCT between 1999 and 2005, were evaluated with regard to major transplant endpoints after a Treo-based preparative regimen (46 pts) compared to a myeloablative total body irradiation (TBI)-based regimen (157 pts). Both cohorts were comparable in terms of patient age (median 48 vs 43) years, donors (matched unrelated 52% vs 57%) and cytogenetic disease characteristics, as well as the post transplant follow-up period (median of 48.6 months). Results: For pts transplanted in complete remission (CR) (125 pts), non-relapse mortality (NRM) at 100 days, 1 and 3 years after transplant was 6%, 13% and 23% after Treo-compared to 10%, 16% and 22% after TBI-based conditioning (n.s.). Similar, no significant difference of NRM between the two regimens was detectable in the 78 pts with more advanced disease stages. The post transplant risk of relapse (RR) increased with the disease stage (CR vs advanced stages) and the cytogenetic risk category of AML (low-intermediate risk vs high-risk), but was not influenced by the conditioning regimen: The RR for CR pts at 1 and 3 years was 13% and 17% after Treo-compared to 24% and 33% after TBI-based conditioning (n. s.). The corresponding figures for advanced disease stages were 43% and 57%, and 29% and 33%, respectively (n.s.). Overall survival (OS) in CR pts at 1 and 3 years was 77% and 60% after Treo-compared to 69% and 50% after TBI-based conditioning (n. s.). In more advanced disease stages OS at 1 and 3 years declined to 29% and 21% after Treo-, and 31% and 18% after TBI-based conditioning, respectively (n.s.). Major determinants of OS were the disease stage (CR vs. advanced stages) and the cytogenetic risk category of AML (low-intermediate risk vs high-risk). Conclusion: This retrospective long-term follow-up analysis supports that Treo-based conditioning leads to equivalent results regarding the major endpoints of alloSCT compared to a myeloablative TBI-based preparative regimen. Second transplant in the management of AML relapse after first allogeneic stem cell transplantation: Results from a retrospective analysis by the German transplant cooperative group and the German registry for stem cell transplantation (DRST) M. Verbeek*, M. Bornhaeuser, J. Finke, D. Beelen, H. Kolb, R. Schwerdtfeger, J. Kienast, A. Zander, L. Kraut, S. Schoenland, R. Mayer, H. Sayer, J. Hahn, D. Bunjes, M. Christopeit, M. Eder, R. Repp, R. Arnold, H. Oettinger, H. Schrezenmeier, C To evaluate the present role of a second transplant (SCT2) for the treatment of AML relapse after first allogeneic stem cell transplantation (SCT1), a retrospective analysis was based on data from the German registry for stem cell transplantation (DRST) between 1998 and 2007. Regardless of the intensity of the conditioning, a second transplant was defined as transfusion of a stem cell containig graft, followed by prophylactic immunosupression. The study was approved by the DRST board, and written consent was obtained from all participating centers, before their respective patients were included. Data were obtained from the DRST database, missing details were collected using a specific questionnaire. 166 patients were identified, and 149 were evaluable for overall survival, which was the primary endpoint of the study. Median duration of remission after SCT1 was 133,5 days. Identical twin, matched sibling, mismatched family, matched unrelated and mismatched unrelated donor were used for SCT2 in 0,6%, 35%, 12,4%, 42%, and 12%, respectively. For SCT2, 1/3 of the patients had switched to a different donor. The conditioning for SCT2 was reduced in 9%, intermediate in 71%, and standard in 9%. 60% of patients achieved a remission after SCT2, however, more than half of them developed second relapse after a median of 101 days. At last contact, 31 patients (18,5%) were alive, whereas the majority had died from leukemia (38%) or transplant-related causes (43,5%). With a median follow up of 9 months among survivors, the two-year overall survival was 13,4%. In a Cox regression model, a matched (family or unrelated) vs. mismatched donor (p=0,023), a longer remission after SCT1 (p=0,004) and a stage of remission at time of SCT2 (p=0,001) were associated with superior survival. In contrast, no influence on outcome could be detected for sex, age, type of conditioning, year of SCT2, or using an alternative donor for SCT2. In conclusion, results after second allogeneic transplant for relapsed AML after SCT1 have not improved during the last decade. Neither modern conditioning regimen, nor switching to an alternative donor have shown so far to alter the unfavorable course of post transplant relapse. Induction of remission by cytoreductive chemotherapy prior to SCT2 might be a way to improve the results. However, in early and chemotherapy-refractory relapse post SCT1, alternative stretegies including palliative care should be considered outside of a clinical trial. Matched related allogeneic haematopoietic stem cell transplantation for acute leukaemia in Eastern Europe: better results in recent years S. Giebel*, B. Labar, J. Holowiecki, M. Labopin, M. Komarnicki, V. Koza, M. Mistrik, A. Lange, A. Hellmann, A. Vitek, J. Pretnar, T. Masszi, J. Mayer, J. Wojnar, M. Krawczyk-Kulis, F. Frassoni, V Allogeneic HSCT from HLA-matched related donor (MRD-HSCT) is widely used for the treatment of high risk acute myeloid leukaemia (AML) and acute lymphoblastic leukemia (ALL). The goal of this study was to analyze the results of MRD-HSCT for acute leukemia in Eastern Europe, as well as to determine prognostic significance of various donor-, recipient-and procedure-related variables. Six-hundred-forty patients (AML, 459; ALL, 181) treated with MRD-HSCT in first complete remission in 10 countries, between 1990-2006, were included in the analysis. The median recipient age was 25 (16-69) years. The conditioning regimen was myeloablative in 578 (90%) patients and bone marrow was used as a source of stem cells in 346 (54%) cases. Ex vivo T-cell depletion was performed in 7 (1%) patients. The probability of OS at 2 years equaled 65% (±-2). The LFS increased from 57% for MRD-HSCT performed between 1990 -2002 to 60% between 2003 -2006 , as a consequence of reduced non-relapse mortality (NMR) (22% vs. 15%, p=0.02), while the relapse incidence remained stable. The LFS rate was higher for bone marrow compared to peripheral blood (PB) used as a source of stem cells (63% vs. 51%, p=0.015) and for TBI-compared to chemotherapy-based conditioninig (63% vs. 55%, p=0.03). The differences were observed for both AML and ALL. In a multivariate analysis the following factors adversely affected LFS: year of HSCT before 2003 (HR=1.4; p=0.045), chemotherapy-based conditioning (HR 1.56; p=0.007), PB as a source of stem cells (HR=1.34; p=0.048), recipient age >40 years (HR=1.33; p=0.03), interval from diagnosis to HSCT <9 months (HR=1.45; p=0.035), and the diagnosis of ALL (HR=1.43; p=0.02). Factors associated with increased risk of relapse were: chemotherapy-based conditioning (HR 1.92; p=0 .007), PB as a source of stem cells (HR=1.63; p=0.01), interval from diagnosis to HSCT <9 months (HR=2.27; p=0.003), and donor/recipient gender combination other than female/male (HR=1.59; p=0.01). The risk of NRM was increased for patients aged >40 years (HR=1.48; p=0.045) and those treated with MRD-HSCT before year 2003 (HR=1.72, p=0.02). Conclusions: Results of MRD-HSCT for acute leukemia in Eastern Europe improved over time, as a consequence of decreased NRM. Leukemia relapse remains the major obstacle for successful transplantation. Use of TBI-based regimens appears advantageous, however the reason why results of bone marrow transplants compared to PB seem better has to be further investigated. Outcomes after allogeneic stem cell transplantation in adults patients with AML using Intravenous busulfanbased conditioning regimen. An ALWP-EBMT survey A. Nagler, M. Labopin, A. Shimoni, D. Bunjes, P. Pimentel, G. Socié, K. Boudjedir, A.R. Zander, A. Torres Gomez, M. Baccarani, H. Goker, A. Fassas, C. Kenzey, B. Samey, V. Rocha* on behalf of the ALWP EBMT Oral Busulfan (Bu) is the historical backbone of pre-allogeneic stem cell transplantation (alloSCT) conditioning regimen. However, oral Bu has an erratic and unpredictable absorption with wide inter and also intra-patient (pt) variability. In S104 contrast, I.V. Busulfan (IV Bu) has more predictable pharmacokinetics and favorable toxicity profile. The ALWP-EBMT performed a survey in order to assess the outcomes of AML pts who received IV-Bu as part of their pre-alloSCT conditioning regimen. 36 EBMT centers participated in this study. Overall, 269 alloSCT were analyzed. Median age was 44y . Disease status at alloSCT was CR1 in 54%, CR2 in 17%, and 29% in non-remission . 77% of the pts were with intermediate, 12% with poor and 10% with good risk cytogenetics, respectively. Overall, conditioning was myeloablative (MAC) in 78% and reduced-intensity (RIC) in 22%. MAC consisted of IV Bu and cyclophosphamide (IV BuCy) in 67%, IV Bu and fludarabine (IV BuFlu) in 22% and in 11% IV BuCy+VP16. RIC consisted in 98% of IV BuFlu. Donors were identical siblingsin 61%, matched unrelated in 28%, mismatched unrelated in 10%, mismatched family donors-1%, syngeneic 1%. 81% of the pts were transplanted with mobilized PBSC grafts while 17% received BM grafts and 2% cord blood. GVHD prophylaxis consisted of CSA+MTX in most of the transplants. With median follow up of 24 moths , two years-overall survival was 54%. Day-100 mortality was 3% in RIC transplants and 6% in MAC. Twenty patients presented sinusoidal obstructive syndrome (SOS) of the liver (5 mild, 7 moderate and 8 severe) with a median onset time of 25 days. The cumulative incidence of SOS at 1-year was 8.4% and it was observed in 6/29 (21%) HLA mismatched transplants, 8/75 (11%) in MUD and in 6/153 (4%) pts transplanted from sibling donors. It was also more common after MAC than RIC (9%vs.4%) and in patients transplanted in non-remission of the disease than those in-remission (12%vs6%). Three patients died of SOS. Two years LFS for pts transplanted in CR1 was 55%, 58% for pts transplanted in CR2 and 20% for those transplanted in advanced disease. In summary, use of IV BU in the conditioning regimen reduces early mortality, incidence and severity of SOS after allogeneic HSCT in patients with AML. The 2-year relapse incidence is acceptable in this non selected AML population. In spite of small number of patients developing SOS after IV Bu, type of donor, type of conditioning and status of the disease at transplant were associated with risk of SOS also in this setting. Salvage therapy for refractory and relapsed acute myeloid leukaemia: comparison between a sequential strategy (IDA-FLAG immediately followed by reduced-intensity conditioning allotransplant with high-dose melphalan) and a standard approach M. Pratcorona* (1) , M. Torrebadell (1) , S. Brunet (2) , M. Tormo (3) , J.M. Sancho (4) , R. F. Duarte (5), M. Rovira (1) , C. Martinez (1) , F. Fernández-Avilés (1) , P. Marín (1) , L. Rosiñol (1) , E. Giné (1) , J. Sierra (2) , E. Carreras (1) , E. Montserrat (1) , J. Esteve (1) ( Prognosis of patients with refractory (ref) and relapsed (rel) acute myeloid leukemia (AML) is poor, with allogeneic stemcell transplantation (alloSCT) being the option with the highest antileukemic potential. Nonetheless, only a minority of such patients ultimately undergoes alloSCT after salvage therapy. Therefore, a sequential strategy integrating a cytoreductive treatment immediately followed by reduced intensity conditioning, might increase the number of patients who benefit from alloSCT. In this regard, we compared the outcome of a series of patients with ref or rel AML treated with standard salvage therapy or with a sequential protocol. The control arm ("standard") was constituted by 47 patients with ref (n=27) or rel (n=20) AML from a single institution consecutively treated with the IDA-FLAG regimen. On the other hand, 24 additional patients received a sequential protocol ("sequential") for AML not in complete remission (ref=8; rel=12; up-front high-risk AML/MDS=4), consisting of an initial cytoreductive phase with IDA-FLAG (days -12 to -8) followed by high-dose melphalan (70 mg/m2 on days -3 & -2). Donor was an HLA-identical sibling in 14 patients, a matched unrelated donor in 8, a mismatched sibling in one patient, and cord blood in the remaining case. Graft-versus-host disease prophylaxis was based on CsA and mycophenolate mofetil. Distribution of main variables among subgroups ("standard" vs. "sequential") was similar except for an older age in the sequential arm (56 vs. 48, p=0.015). Complete remission (CR) rate was higher after sequential therapy (92% vs. 45%; p<0.05). Only 16 patients from the "standard" group received alloSCT after IDA-FLAG (34%) while 3 additional patients received autologous SCT in CR2. Non-relapse mortality at day +100 was 21% vs. 30% (p=ns) whereas survival at 1-year was 17.5% (±6) vs. 30% (±10; p=ns) after "standard" and "sequential" strategies, respectively (see figure) . Although this difference did not attain statistical significance, a survival plateau was observed only after the sequential approach. In conclusion, outcome of patients with ref or rel AML is dismal, although sequential salvage strategy provided durable responses in approximately one third of patients. Moreover, the sequential strategy yielded a higher response rate despite significant morbidity. Therefore, further studies aimed at investigating the benefits of this sequential treatment strategy are warranted. In vitro chemosensitivity of "non leukaemic" CFU-GM, in AML patients, is correlated with CD34+ mobilisation and may identify groups with different disease-free survival G. Milone*, B. Farsaci, G. Avola, S. Leotta, E. Mauro, E. Marturano, A. Di Marco, A. Cupri, A. Strano, S. Mercurio, M.G. Camuglia, M. Romeo, A. Triolo, R. Giustolisi Ospedale Ferrarotto (Catania, IT) Background: An high number of CD34+ cells in during mobilization has been associated in AML patients in CR to a high relapse rate and to greater amount of minimal residual disease (Keating, Feller 2003) . A different pharmacokinetics of chemotherapy drugs administered during induction or an intrinsic chemoresistence of normal bone marrow precursors have been hypothesized as possible explanations for the observed association between mobilization of non leukemic CD34+ cells and leukaemia residual disease. Methods: With this background we assessed in a group of AML in CR the in vitro chemosensitivity of non leukemic BM cells to Maphosphamide and Etoposide and correlated it to mobilization strength as well as to DFS. 37 patients affected by AML have been prospectively studied, all were treated using a same induction and consolidation chemotherapeutic regimen. Sensitivity to Maphosphamide and to Etoposide of CFU-GM, BFU-E, CFU-E, CFU-GEMM obtained from bone marrow in 1st CR was studied 2-4 weeks after PBSC mobilization. Results: Chemosensitivity of CFU-GM to ASTA-Z as well as to Etoposide and of CFU-GEMM to ASTA-Z, expressed as residual colony growth in comparison to untreated cells, was significantly correlated with peak of CD34+ cells in P.B. during mobilization (R=0.639, P=0.0001 at 75 mcg/ml of ASTA-Z, Fig 1) .To study relationship between chemosensitivity of non leukemic CFU-GM and survival we splitted patients in the 3 groups according to their chemosensitivity in respect to normal controls. Survival at 6 months was 0% for hyposensitive group, 60% in normosensitive group and 78% in hypersensitive group (log rank: 0.064). The survival of hyposensitive patients was significantly lower than the remaining patients (log rank: 0.01) (Fig. 2) . Sensitivity of CFU-GM to 100 mcg/ml to ASTA-Z was found important for DFS also when studied in Cox proportional hazard model (Likehood ratio P=0.03). In a stepwise selection, sensitivity to ASTA-Z but not CD34+ peak during mobilization was selected as important for DFS, moreover ASTA-Z sensitivity of normal non leukemic CFU-GM was found important for DFS also in group of AML pts having at diagnosis a normal cytogenetic. Conclusion: We have found that in AML patients sensitivity of normal non leukemic CFU-GM to maphosphamide and to Etoposide is highly variable and significantly correlated to CD34+ cells peak reached during mobilization. Chemosensitivity of normal non leukemic CFU-GM was also found to be related to DFS. Reduced-intensity conditioning and allogeneic stem cell transplantation is an effective treatment for high-risk acute myeloid leukemia patients in first complete remission P. Hemmati* (1), T. Terwey (1) , G. Massenkeil (2) , P. le Coutre (1), S. Neuburger (1) , L. Vuong (1) , B. Dörken (1) , R. Arnold (1) (1)Charite Medical University Berlin (Berlin, DE) ; (2) Ruhr-University Bochum (Bochum, DE) Conventional myeloablative conditioning is associated with a high treatment-related mortality (TRM), whereas less intensive conditioning regimens may be inefficient for long-term disease control in patients with high-risk AML. Here, we present a retrospective single-institution analysis of 90 patients (median age 39, range 17 -66 years) with high-risk AML who underwent alloHSCT in CR1 between 1994 and 2007 (median follow-up 26, range 1 -147 months). As stem cell source bone marrow (BM) was used in 17/90 patients (19%), whereas 73/90 patients (81%) received peripheral blood stem cells (PBSC). In 61/90 patients (68%) standard high-dose myeoloablative conditioning (12 Gy TBI + 120 mg/kg CY), (TBI group), was administered. 29/90 (32%) patients received reduced-intensity conditioning (FLUD/BU/ATG)(RIC). AlloHSCT was performed from a matched-related donor (MRD) in 62/90 patients (69%) or an unrelated donor (URD) in 28/90 patients (31%). Prevention of graft-versus-host disease (GvHD) consisted of CSA/MTX in the TBI group or CSA/MMF in the RIC group. 48/90 (53%) patients had a normal karyotype, whereas 39/90 patients (43%) had an aberrant karyotype. Projected overall survival (OS) and disease-free survival (DFS) of the whole cohort at 1, 3, and 5 years was 74%, 62%, and 57% and 72%, 58%, 54%, respectively. Causes of death were relapse (17/90 = 19%) or TRM (17/90 = 19%). There was no difference in the OS and DFS between patients who received BM versus PBSC. The OS in the TBI group versus the reduced-intensity conditioning (RIC) group was 72% vs. 79% at 1 year, 64% vs. 52% at 3 years, and 59% vs. 52% at 5 years (p = 0.78). The 1, 3, and 5-years DFS rates (72%, 62%, 57% vs. 75%, 50%, 49%) did not differ significantly between both groups. Relapse and TRM in the TBI group versus the RIC group were 21% vs. 14% and 16% vs. 24%. Again, there was no significant difference in the OS between the subgroups with a normal (n = 48) versus an aberrant (n = 39) karyotype at 1 year (83% vs. 65%), 3 years (70% vs. 47%), and 5 years (67% vs. 45%) (p = 0.06). Also, there was no significant difference in the incidence of chronic graft-versus-host disease (cGvHD) between the TBI group and the RIC group (46% vs. 52%). These results suggest that patients with AML in CR1 may achieve a robus long-term remission irrespective of the conditioning intensity (TBI vs. RIC) and the presence of an aberrant karyotype. Therefore, alloHSCT following RIC may represent a therapeutic option for all patients with high-risk AML in CR1. Inferior survival in patients with refractory acute myeloid leukaemia and extramedullary disease or high leukaemia burden after sequential treatment with chemotherapy and reduced-intensity conditioning for allogeneic stem cell transplantation S. von Harsdorf* (1), C. Schmid (2) Objectives: To analyze the influence of high leukemic burden (increased white blood cell count (WBC)) and extramedullary disease (ED) on outcome of patients with refractory acute myeloid leukemia (AML) after sequential treatment with chemotherapy and reduced-intensity conditioning (FLAMSA-RIC) for allogeneic stem cell transplantation (SCT). Patients and methods: In a retrospective single center study we have studied 48 pts with refractory AML from Februar 2003 to December 2007 in our institution. Refractory disease was defined as primary induction failure, early relapse, refractory relapse, or second relapse according to published criteria. The patient cohort comprised 25 males and 23 females with a median age of 47.6 years (y) (range 22. 6-63.3y) . At time of SCT 12 pts were in remission (n=12), 36 (75%) had refractory disease, 15 of them were primarily refractory, 14 had refractory relapse, 4 untreated relapse, 3 untreated relapse after allogeneic SCT. Fsm like tyrosin kinase internal tandem duplication (FLT3-ITD) was found in 9, complex karyotype in 8 pts. 17 pts had high WBC (n=10), extramedullary disease (ED) (n=14) or both (n=7). Conditioning consisted of fludarabine (4x30 mg/m 2 ), cytarabine (4x2g/m 2 ) and amsacrine (4x100 mg/m 2 ), followed 4 days later by RIC, comprising 4 Gy total body irradiation (TBI), cyclophosphamide 120 mg/kg and anti-thymocyte globulin. The stem cell source was peripheral blood stem cells (PBSC) in 47 pts and bone marrow in one. Donors were related in 10 and unrelated in 38 pts. Results: with a median follow up of 17.3 months (range 1.2-23.7 mo) the overall survival (OS) after 1, 2 and 4 years was 43.8%, 32.3% and 32.3% respectively. 9 of 14 evaluable pts (64.3%) with high WBC or ED relapsed after a median of 2.7 months after SCT. In the other group, relapse was seen in 11 of 29 pts (37.9%). Death due to relapse was 53% in the group with high WBC/ED versus 29% in the other group. The leukemia free survival after 2 years was 29% in the group with high WBC/ED versus 52% in the other group (p = 0.045). Especially unfavourable outcome with 100% mortality due to early relapse was seen in all 5 pts suffering from both high WBC and ED. Conclusions: with respect to a small patient cohort these results may indicate a significant worse outcome of pts with high disease activity and ED for this regimen. Especially in cases with evidence of both high disease activity and ED transplantation in this setting should be carefully considered. High-dose idarubicin and busulphan as conditioning regimen to autologous stem cell transplantation in elderly patients (age > 60 years) with acute myeloid leukaemia F. Ferrara*, S. Palmieri, C. Copia, F.P. Tambaro, M. Pedata, T. Izzo, F. Pollio, C. Falco, A. Viola, G. Mele Cardarelli Hospital (Naples, IT) Introduction: High relapse rate is a main reason accounting for adverse outcome in acute myeloid leukemia (AML) of the elderly. Different consolidation strategies, including autologous stem cell transplantation (ASCT) are currently under investigation. Here we report our results with a conditioning regimen based on the combination of idarubicin (IDA) and busulphan (BU) in a prospective study on 34 consecutive elderly AML patients eligible for ASCT in first complete remission (CR). Methods: The protocol consisted of high dose IDA, given at 20mg/sqm as 2 days continuous infusion (from day -11 to -10) and oral BU at 4 mg/kg from day -4 to -2. There were 22 males and 12 females with a median age of 66 years (range 61-77). Cytogenetics were intermediate, unfavorable or not available in 24, 7 and 3 patients, respectively, while 6 out of 22 evaluated patients (27%) were FLT3+. All patients received peripheral blood stem cells collected after consolidation plus G-CSF. The median interval between diagnosis and ASCT was 4 months (3) (4) (5) . The median number of CD34+ cells infused was 6,2x10 6 /kg (2, . Results: The median number of days to PMN >500/cmm and platelets >20000/cmm was 10 (8-18) and 12 , respectively. The median number of platelet and blood units transfused was 2 (1-7) and 2 (0-5), respectively. Extrahematological toxicity mainly consisted of grade WHO II-IV stomatitis (30/34 or 88%). More recently, a dramatic improvement of oral toxicity has been observed by replacing oral with intravenous BU. Furthermore, most patients had FUO, while two experienced documented infection. No transplant related death occurred. LVEF examination post-ASCT did not reveal any cardiac toxicity. Finally, median time of hospitalization was 27 days (20-37). At the time of writing, a total of 18 patients have relapsed [median disease free survival (DFS) duration for relapsed: 9 months, range 4-16]. Sixteen out of 34 patients (47%) are alive (13/16 in continuous CR) with a median follow up after ASCT of 25 months, range 3-96. Causes of death were AML in 15 patients, and gastric cancer, pulmonary cancer, and unknown in the remaining 3 non-relapsed cases. Median DFS and overall survival were 14 and 21 months as shown in the figure. Conclusion: ASCT with IBU regimen is feasible and effective in elderly AML, with extremely promising results in terms of DFS duration. These results compare favorably with data from patients undergoing reduced intensity allogeneic SCT. Follow-up of chimerism in myeloid cell fraction after allogeneic haematopoietic stem cell transplantation (HSCT) for patients with acute myeloid leukaemia I. Mollet (1) , D. Revez (2) , C. Martin (1) , C. Plesa (2) , C. Giannoli (1) , F. Nicolini (2) , G. Canas (2) , E. Nicolas (2) , X. Thomas (2) , V. Dubois (1) , M. Michallet (2) (1)HLA Laboratory (Lyon, FR); (2)Hopital Edouard Herriot (Lyon, FR) During the follow-up of allogeneic haematopoietic stem cell transplantation (HSCT), chimerism analysis is necessary to document engraftment, graft versus host disease (GVHD) and to adjust donor lymphocyte infusions. Although RQ-PCR technology (Real Time Quantitative polymerase Chain Reaction) allows a highly sensitive and reproducible detection. It is nevertheless usefull in some diseases to target selected sub-populations in order to have an earlier detection of recipient cell fractions. The principal aims of this study is to document engraftment and to detect as soon as possible relapse in patients with acute myeloid leukaemia (AML) who underwent allogeneic HSCT. We evaluated chimerism by using RQ-PCR technology after selection of myeloid fractions: CD33+ cells in peripheral blood samples and CD34 + cells in bone marrow samples., Twentyfive AML patients were studied, 13/26 (52%) underwent allogeneic HSCT after non myeloablative conditioning and 12/28 (48%) after standard conditioning. The chimerism study consisted of (1) peripheral blood samples studying whole cells (n=200); CD3+cells (n=128) in order to establish the state of engraftment by documentation of full donor chimerism conversion (FD); CD33+cells (n=77; 38.5% of blood samples) and (2) bone marrow samples: whole cells (n=92) and CD34+cells (n=47; 51% of bone marrow samples). After transplant, 40% of patients (10/25) relapsed. Chimerism analysis of the CD33+ and CD34+ fractions allowed an early detection of relapse in 70% of cases (7/10). Whereas the whole blood and bone marrow showed a FD profile (Table 1) for patients LY-1, LY-3, LY-5, LY-7, LY-10, the high sensitivity of RQ-PCR allowed the detection of recipient cells (mixed chimerism: MC) in the myeloid fraction. For the patients who did not converted to FD, even if a stable chimerism persisted in the whole blood, an increase of MC in the CD33+ population was detected. In parallel, we observed a continuous decrease of MC level in the CD3+ population (patients LY-2 and LY-8).These results demonstrate the importance of cellular sub-populations chimerism documentation enable to ascertain a stable engraftment and to detect early relapse after allogeneic transplantation allowing prompt therapy and/or cellular immunotherapy . Phenotypic analysis of primary CD34(high)+ CD38leukaemic blasts targeted by allogeneic cell therapy in acute myeloid leukaemia A. Jürchott*, E. Distler, A. Konur, E. Wagner, C. Huber, R. G. Meyer, W. Herr Johannes Gutenberg-University Mainz (Mainz, DE) Acute myeloid leukemia (AML) is thought to arise from a rare putative 'leukemic stem cell' that is capable of self-renewal and formation of leukemic blasts. Serial xeno-transplantation studies in immunodeficient mice have shown that this leukemia-initiating cell resides within CD34(high)+ CD38-AML cells. Thus, alloreactive T cells successfully eradicating this compartment after allogeneic stem-cell transplantation should result in cure from disease. Our study aimed at a detailed phenotypic characterization of the CD38-and CD38+ subsets of primary CD34(high)+ AML blasts ex vivo. We obtained therapeutic leukapheresis products from 11 AML patients with white blood cell counts exceeding 10 11 /L at primary diagnosis. These products were used to purify CD34+ cells by immunomagnetic microbeads. CD34(high)+ expressors were subsequently sorted by flow cytometry into CD38+ and CD38-subsets, respectively. Both fractions were then phenotyped for expression of surface markers previously described to differ between leukemic blasts, leukemic stem cells, and normal hematopoietic stem cells, i.e. CD71, CD90 (Thy-1), CD117 (c-kit), CD123 (IL3Ralpha), CD44, CD45RA, and CD11c. We also included markers relevant for recognition by NK and T cells, namely HLA class I, HLA-DR, CD80, CD86, CD40, CD54, and CD58. Our results demonstrated that the CD38+ and CD38-subsets of CD34(high)+ AML blasts differed significantly in the median proportion of cells expressing CD71 (81 versus 55%, p=0.0002), HLA-DR (31 versus 4%, p=0.03), and CD54 (74 versus 98%, p=0.04). Both subsets showed comparably intense staining for CD11c, CD44, CD45RA, CD58, CD86, CD117, and CD123. All CD38+ and CD38-cells expressed HLA class I. CD90, CD80, CD40, and the lineage markers CD2, CD3, CD4, CD7, CD8, CD10, CD14, CD19, CD20 and CD56 were negative in both fractions. We conclude that primary CD34(high)+ CD38-AML blasts containing rare leukemic stem cells express overall lower levels of the transferrin receptor CD71 compared to CD34(high)+ CD38+ counterparts. Decreased expression of HLA-DR in the CD38-fraction might impede direct recognition by alloreactive CD4+ T cells. However, all CD34(high)+ CD38cells expressed HLA class I making them accessible to alloreactive CD8+ T cells. Further functional studies will explore if the CD38-and CD38+ subsets of CD34(high)+ AML blasts differ in the immunogenicity for alloreactive CD4 and CD8 T cells, both in vitro as well as in immunodeficient mice in vivo. A survey of outcomes after autologous stem cell transplantation in adults patients with AML using intravenous busulfan-based conditioning regimen. A study on behalf of the ALWP A. Nagler, M. Labopin, C. Martin, D. Bunjes, A. Botelho Sousa, P. Colombat, C. Kenzey, B. Samey, N. Gorin, V. Rocha * on behalf of the ALWP EBMT Oral Busulfan (Bu) is used as a myeloablative drug in conditioning regimen prior to HSCT and it is associated with sinusoidal obstructive syndrome (SOS) in 3% to 10% of autologous HSCT (autoHSCT). In recent years, intravenous busulfan (IV Bu) instead of oral Bu has been frequently used since it has more predictable pharmacokinetics and less toxicity. However, very few data is available on outcomes and toxicity of IV Bu used as part of the conditioning regimen prior to autoHSCT for patients with AML. With this objective, the ALWP has performed a survey among European Centers in order to describe results of 49 patients with AML given and autoHSCT. The median age was 46 years (20-65) and the median transplant year was 2003 (2000-2006) . They were transplanted in CR1 (n=40, 82%), CR2 (n=7, 14%) and CR3 (n=2, 4%). Frequently, at diagnosis, AML FAB classification was M1, M2 , M3 or M4. Regarding the cytogenetic classification, 13% were good, 73% were intermediate and 14% were poor risk. Cumulative incidence of non-relapse mortality (NRM) at 2 years for all patients was 6% and it was 3% for patients transplanted in CR1 and 14% for those transplanted in CR2 or more. Only one patient had SOS (severe) at day 10. This patient was 65 years old and he was transplanted in CR3. He died of infection, pulmonary toxicity and SOS. The overall leukemia-free survival and relapse incidence at 2 years were 48% and 46% respectively; and it was 51% and 46 % for patients transplanted in CR1 and 43% and 43% for those transplanted in CR2, respectively. In conclusion, in this retrospective survey, incidence of NRM and SOS were low in this cohort of patients studied. LFS and relapse incidence seem to be not different compared to data in the literature using other conditioning regimen for autoHSCT for patients with AML. Outcome of patients with chemotherapy refractory acute myeloid leukaemia with a high pre-transplant associated mortality score after allogeneic stem cell transplantation N.K. Steckel*, R. Trenschel, M. Ditschkowski, L. Kordelas, H. Ottinger, M. Koldehoff, A.H. Elmaagacli, C. Schulte, D.W. Beelen Universityhospital Essen (Essen, DE) Recently, comorbidity assessment becomes more and more important as seen in new risk calculating scores like the HCT-CI (Sorror-Score) or the pretransplant associated mortality score (PAM-Score). We calculated the PAM-score in a cohort of 30 consecutive patients (pts) with refractory acute myeloid leukaemia (AML), who underwent allogeneic stem cell transplantation (SCT) after a sequential therapy of high-dose melphalan (140mg/m 2 24 pts, 200mg/m 2 6 pts) followed by myeloablative conditioning after a median interval of 11 (range 6-24) days. The clinical variables for the PAM-Score were as follows: age, donor type, disease risk, conditioning regimen, percentage of predicted FEV1, percentage of predicted DLCO, serum creatinine level, and serum ALT. The median PAM-Score was 29 points (range 24-36) and the median overall estimate of death within 2 years after transplant for this cohort was 67% (range 49%-85%). Cytogenetic risk stratification was as follows: 18 pts (61%) had an unfavorable, 11 pts (36%) an intermediate, and 1 patient (3%) a low risk karyotype, respectively. The median patient age was 58 years (range 19-67). After melphalan chemotherapy, marrow blast clearance was achieved in all pts (100%) as demonstrated by bone marrow examinations before the start of conditioning. A conditioning regimen with treosulfan was administered in 18 pts, 12 pts received total body irradiation (TBI), each combined with fludarabine. Nine pts (30%) were transplanted with an identical sibling donor, 3 pts (10%) with a haploidentical family donor, and 18 pts (60%) with an matched unrelated donor. All pts. surviving > 30 days post transplant (n=26) achieved complete remission with complete donor chimerism. The two-year posttransplant treatment related mortality of all 30 pts was 49% ±11%. This is lower than the predicted mortality by the PAM-Score. The two-years overall survival was 27% ± 14%. Six of 30 pts (20%) relapsed within in the first 14 months after allogeneic SCT, all relapsing pts died in association with relapse. With these data we confirm, that a sequential therapy with high dose melphalan followed by a myeloablative conditioning therapy for allogeneic SCT for patients with refractory AML who have a calculated high pretransplant associated mortality (PAM) score is a suitable therapy option with a comparable lower 2-years treatment related mortality. Graft-versus-leukaemia effect after unrelated donor haematopoietic stem cell transplantation for acute lymphoblastic leukaemia in first complete remission H. Huang*, X. Lai, Y. Luo, J. Shi, Z. Cai, M. Lin Zhejiang Uninversity School of Medicine (Hangzhou, CN) Allogeneic hematopoietic stem cell transplantation (Allo-SCT) has a better anti-leukemic effect than conventional chemotherapy or autologous hematopoietic stem cell transplantation (Auto-SCT) for acute lymphoblastic leukemia (ALL). Unrelated donor hematopoietic stem cell transplantation (URD-SCT) with stronger graft-versusleukemia (GVL) effect appears promising now, however, it was considered with higher transplant-related mortality (TRM). Until now, there were 37 patients with ALL in CR1 received URD-SCT in our bone marrow transplantation center between July 1999 and April 2007. Philadelphia chromosome occurred in 7 (18.9%) patients at diagnosis. The median age was 21 years (range 8~48 years). HLA high-resolution typing was used for donor-recipient matching with 18 cases of HLAmatched and 19 cases of HLA 1-2 alleles mismatched. All of the patients were received Bu/Cy2 regimen as conditioning with busulfan 16mg/kg plus cyclophosphamide 120mg/kg. Mycophenolate mofetil combined with CsA and short course MTX were performed to prevent aGVHD and 3 patients received additional anti-CD25 monoclonal antibody. All 37 patients achieved sustained engraftment by the analysis of cytogenetics and STR-DNA. MMF+CsA+MTX could be used as an effective and safe prophylaxis regimen for aGVHD, and incidence of aGVHD was 72.97%, aGVHD of grade I-II observed in 22 (59.46%) patients and the severe aGVHD of grade III-IV observed in 5 (13.51%) patients. The incidence of cGVHD was 58.82%. Early TRM was 8.11% at 100 days after transplant. With a median follow-up of 9.8 months (range 1.0~79.5 months), clinical relapse were detected in 6 (16.22%) patients and 22 (59.46%) patients achieved disease free survival. By Kaplan-Meier method, the accumulative probability of 3-year overall survival was 59.26±9.18%, and overall survival of Ph(+) ALL was 71.43±17.07%. A strong anti-leukemia effect of GVHD might occur in URD-SCT for ALL, and the 3-year overall survival was 70.16±11.78% vs 45.00±16.60% in patients with I-II aGVHD or without aGVHD (p=0.0085), which was 76.74±12.52% vs 34.62±14.40% in patients with or without cGVHD (p=0.0015). 4 of 5 patients who developed severe aGVHD died. In our experience of unrelated donor transplantation for ALL in CR1, I-II aGVHD and cGVHD are favorable factors for overall survival, and Bu/Cy2 conditioning regimen could be safely used. Unrelated donor transplantation with a greater GVL effect, appears promising for ALL in CR1. Stem cell transplantation for ALL in CR1 in children. A prospective single-centre study V. Mialou*, C. Galambrun, M.P. Goutagny, K. Kebaïli, J.M. André, C. Pondarre, N. Bleyzac, V. Dubois, O. Hequet, Y. Bertrand Hopital Debrousse (Lyon, FR) Objectives: The aim of this study was to evaluate the outcome of all our patients who received an allogenic Haematopoietic Stem Cell Transplantation (HSCT) for a Very High Risk (VHR) Acute Lymphoblastic Leukemia (ALL) in CR1 treated according to the EORTC 58951 trial. Methods: Two hundred and fifty patients with ALL were included prospectively from January 1999 to June 2007 in the EORTC 58951 protocol. Among those patients, we studied the children who received an allogenic HSCT because of the severity of their disease (VHR patients). Results: Nineteen out of 250 patients (7.6%) received an allogenic HSCT. Median age at diagnosis of the disease was 11 years [3 to 17] . There were 12 boys and 7 girls. They had B-ALL for 10, T-ALL for 7, biclonal or biphenotypic acute leukaemia for 2. Patients were classified as having VHR ALL according to several of the following criteria: poor corticosensitivity at day+8, presence of bad prognosis chromosomal abnormality (t(4,11), t(9;22), nearhaploidy or monosomy 7), non remission or positive minimal residual disease at the end of induction phase Ia(>10-2). Donors were of unrelated origin for 9 patients, familial HLA identical for 10. All patients had conditioning regimen with Total Body Irradiation (12gys) and cyclophosphamide for 9 or VP16 for 10. GVHD prophylaxis was ciclosporin A for all, with thymoglobulins (ATG) for unrelated donors. Among the 9 unrelated HSCT, 4 were matched unrelated (10/10), and 5 were mismatched unrelated (three 9/10 and two 8/10). Median follow-up is 97 months [3 to 243] . Four patients developed an acute GVHD > II (21%). Two patients died: one of relapse and one because of toxicity (endobronchial aspergillosis). Five patients developed a chronic GVHD , extensive only in 3. Long term follow-up showed sequelaes for several patients: two had endocrinologic dysfunction, two had cataract, one patient had mild chronic renal insufficiency, two patients had orthopaedic sequelae and one patient had a persistent severe humoral immunodeficiency. Conclusion: HSCT indications for children with VHR ALL have changed during the past few years. In 1999, at the beginning of the study, only HSCT with a familial HLA identical donor were performed. According to results of recent published studies, indications moved and unrelated HSCT were performed for the more severe diseases. Seventeen patients are disease free and survival for our VHR ALL patients is about 90 %. Sirolimus and mycophenolate mofetil as GVHD prophylaxis in allogeneic stem cell transplantation for high-risk leukaemia patients M. Schleuning*, D. Judith, T. Stuebig, Z. Jedlickova, F. Funk, M. Heshmat, H. Baurmann, R. Schwerdtfeger German Diagnostic Clinic Foundation (Wiesbaden, DE) Sirolimus, an immunosuppressant structurally related to the calcineurininhibitor (CNI) tacrolimus, exerts its action at a later stage of T-cell activation by inhibiting the mammalian target of rapamycin and thus arresting the cell cycle in G1. In contrast to CNIs sirolimus also promotes the generation of regulatory T-cells. In addition to its immunosuppressive effects sirolimus also has potent antineoplastic activity. Sirolimus has been used successfully in solid organ transplantation and in combination with CNI for graft versus host disease (GVHD) prophylaxis in hematopoietic stem cell transplantation (SCT). Within the FLAMSA-RIC protocol for high risk leukemia we enrolled 15 high risk patients (pts) in a pilot trial to test the efficacy of sirolimus in combination with MMF without CNI in allowing engraftment and preventing GVHD after allogeneic SCT from HLA-matched related (n=6) and unrelated donors (n=9). Pts with myeloid malignancies also received ATG during conditioning. The underlying diagnoses were relapsed or refractory T-ALL (n=3), AML with FLT3-ITD or MLL-PTD (n=10), CML in refractory myeloid blast crisis (n=2). One pt died in aplasia on d +6 from aspergillosis and pulmonary bleeding. The remaining pts engrafted after a median of 20 days. Four pts developed acute GVHD (grade I: n=2, grade II: n=1, grade IV: n=1). The pt (CML-BC) with grade IV (skin, gut) developed acute GVHD while already being outpatient und finally succumbed to it. Three patients developed chronic GVHD after cessation of immunosuppression which improved after resumption of sirolimus treatment. Despite the one death from aspergillosis peritransplantation toxicity was mild. One pt suffered from HHV6-associated erosive gastritis, which necessitated laser coagulation and her condition improved only after terminating sirolimus at day +48. None of the pts developed thrombotic microangiopathy or sinusoidal obstruction syndrome. Three pts with FLT3-ITD+ AML relapsed after a median of 112 days. At a median follow-up of 7 months after transplantation 10 pts are alive and in complete remission. Five of these patients received adjuvant donor lymphocyte transfusions. Although the follow-up is still short sirolimus in combination with MMF seems to be a promising alternative to CNI-based regimens for GVHD prophylaxis after allogeneic SCT. Engraftment is ensured and transplantrelated toxicity is modest. In conclusion, sirolimus-based GVHD prophylactic regimens deserve further investigation. High-dose melphalan in relapsed acute myeloid leukaemia -an update H. Martin (1) , R. Schwerdtfeger (2) , S. Mousset (1), D. Hoelzer (1) , H. Serve (1) , G. Bug (1) (1)J.W. Goethe-University Hospital (Frankfurt, DE) ; (2)DKD (Wiesbaden, DE) Background: Patients with AML in first relapse are reported to have a long-term survival ranging between 10-15%. To improve survival we introduced salvage therapy with highdose melphalan and autologous PBSC followed by a sequential allotransplant (Bug et al. Ann Hematol 2005; 84:748-54.) Objectives: (1) To improve second CR rate in relapsed AML; (2) to increase the proportion of relapsed AML patients achieving an allogeneic transplant; (3) to improve long-term survival in pts. with relapsed AML. Methods: AML patients in first relapse received salvage therapy with 200 mg/m² melphalan and autologous PBSC, which had been cryopreserved in early CR1. Subsequently an allogeneic donor was searched and a consolidating allogeneic transplant scheduled wthin 2 -3 months. Results: Twentytwo pts. with AML in first relapse and a median age of 48 (range 29-61) years are evaluable. Eighteen of 22 pts. (82%) achieved a second CR and 19/22 pts. (86%) achieved a consolidating allogeneic transplant in CR2 (n=14) or PR2 (n=2) from a related (n=3) or unrelated (n=16) donor within a median of 2,4 (1,9 -4,5) months after HD-Mel. Treatment-related mortality was 0/22 after high-dose melphalan (HD-Mel) and 4/19 (21%) after subsequent allograft; 5 pts. died due to relapse and 13 patients are alive in CR with a follow-up of median of 31 (range 5 -78) months and a projected long-term survival of 55%. Conclusions: By long-term follow up, HD-Mel remains superior to any other reported salvage regimen in relapsed AML. Allogeneic stem cell transplantation in adult acute lymphoblastic (ALL): long-term results after standard and after reduced-intensity conditioning R. Arnold (1) , P. Hemmati (1), S. Neuburger (1), T. Terwey (1) , P. le Coutre (1), L. Vuong (1), B. Dörken (1) , G. Massenkeil (2) (1)University Hospital Charité (Berlin, DE); (2)Ruhr University (Berlin, DE) In high risk ALL patients (Ph+, pro B ALL, B-lineage > 30.000 WBC at diagnosis or delayed complete remission and patients with complex aberrant karyotype) indication for allogeneic stem cell transplantation in CR 1 is given. Beyond CR 1 all patients are candidates for allogeneic stem cell transplantation (SCT). Donors are HLA identical siblings, HLA compatible family donors and matched unrelated donors. According to the GMALL protocol patients received conditioning regimes with 12 Gy TBI and etoposide (VP16) and cyclophosphamide (standard conditioning). For patients with contraindications against standard conditioning, i.e. active infections or impaired organ functions or age > 55 years, reduced intensity conditioning (RIC) with busulfan, fludarabine and ATG was given. We report here our single center data on 160 ALL patients. 139/160 patients received standard conditioning, 21/160 patients received RIC. Median follow up of the surviving patients is 40 months and 26 months, resp. For high risk patients transplanted in CR 1 probability of survival at 5 years is 0.51 and 0.60 in RIC patients, due to low transplant related mortality (TRM). In patients in CR 2 and beyond probability of survival at 5 years is 0.22 (0.37 at 2 years) in the standard conditioning group vs. 0.20 at 2 years in the RIC group. In patients with relapse probability of survival at 5 years is 0.20 versus 0.0. resp. In patients with relapse after standard conditioning and allogeneic SCT, allogeneic SCT after RIC is ineffective, all patients died due to relapse, as did the patients with relapse after RIC and retransplantation after TBI. In conclusion: in high risk ALL patients in CR 1 allogeneic SCT from a related or an unrelated donor is a curative treatment after standard conditioning or RIC. In the limited number of patients with RIC and transplantation in CR 1, the probability of survival is even better due to reduced TRM. In ALL patients with advanced disease RIC is insufficient. In patients with relapse after standard or reduced intensity conditioning and allogeneic SCT all patients relapsed after retransplantation independently of the conditioning regimen used. Sequential regimen of chemotherapy and reducedintensity conditioning for allogeneic haematopoietic stem cell transplantation in patients with advanced haematological malignancies M. Krejci*, J. Mayer, Y. Brychtova, M. Doubek, Z. Racil, Z. Koristek, M. Navratil, M. Tomiska, J. Vorlicek University Hospital Brno (Brno, CZ) Background: Recently reported sequential use of intensive chemotherapy and reduced-intensity conditioning (RIC) for allogeneic stem cell transplantation (SCT) in high-risk patients (pts) represents a promising approach (Schmid et al., JCO 23, 2005: 5675-87) . Here, we wanted to explore if these results are reproducible at another set of pts. Methods: We retrospectively analyzed 23 pts with hematological malignancies (AML, n=14; ALL, n=2; MDS, n=3; others, n=4) undergoing chemotherapy and RIC SCT. Fludarabine (30 mg/m 2 ), cytarabine (2 g/m 2 ), and amsacrine (100 mg/m 2 ) for 4 days (FLAMSA) were used for cytoreduction. After 3 days of rest, RIC consisting of 4 Gy TBI, ATG (Fresenius) 10-20 mg/kg/day for 3 days, and cyclophosphamide 40-60 mg/kg/day for 2 days followed. Median age of pts was 52 years (range 22-61). Disease status before SCT was: CR1, n=1; CR2, n=7; CR3, n=1; refractory disease or relapse, n=14, type of donors: HLA identical sibling, n=8; unrelated donor, n=15, and grafts used were: PBSCs, n=22; BM, n=1. The median follow up from SCT was 3 months (range 1-18). Results: The median time to platelet recovery (above 50x10E9/L) was 26 days, the neutrophil engraftment (above 1.0x10E9/L) was achieved at a median time of 21.0 days. No graft rejection was observed. Treatment response was evaluated in 17 pts: remission was achieved in 16 pts, only one patient had progression of leukemia. Five relapses occurred in a median follow-up of 3 months. Transplantrelated mortality (TRM) to the day +100 was evaluated in 16 pts. Six pts died (37%), causes of death were GVHD (n=1), septic shock (n=2), multiorgan failure (n=1), and brain hemorrhage (n=2). All early deaths were seen in very high risk pts only: relapse or refractory disease, n=5; CR3, n=1. Incidence of acute GVHD was evaluated in 19 pts: 55% (10/19) of pts had acute GVHD (grade I+II in 9 pts, grade III in 1 case). Incidence of chronic GVHD was evaluated in 9 pts, chronic limited GVHD developed in 5 pts, chronic extensive GVHD was presented in 1 case. With median follow-up 3 months, 15 from 24 pts were alive (13 pts in remission, 2 pts with relapse), 8 patients died (6 deaths from TRM, 2 deaths from relapse of leukemia). Conclusion: FLAMSA-RIC protocol seems to be effective salvage treatment in patients with advanced hematological malignancies. Response rate is high and the toxicity moderate. Early deaths in very high risk pts call for careful indication of this treatment. Haematopoietic stem cell transplantation in acute leukaemia: report of 17 years experience in Iran A. Ghavamzadeh*, F. Ashrafi, A. Mousavi, M. Irvani, K. Alimoghadam, M. Jahani Hematology, Oncology and BMT Resarch Center (Tehran, IR) Background: Hematopoietic stem cell transplantation (HSCT) remains the best therapy for the control of acute leukemia in adult patients in recent years. After the first HSCT had been done in Iran in 1991, acute leukemia encompassed the greatest proportion of HSCT in this center. This study concerns the results of HSCT in acute leukemia patients in shariati hospital, the first center of HSCT in Iran. Patients and methods: We included all acute leukemia patients referred for HSCT to shariati hospital from 1991 till 15th December 2007 and evaluated outcomes of them. Results: 810 patients (66% AML and 33% ALL) enrolled in this retrospective analysis. In AML group, allogeneic HSCT was performed in 64%, autologous HSCT in 35% and syngeneic HSCT in 1% of patients. In ALL group allogeneic HSCT was performed in 88%, autologous HSCT in 10% and syngeneic HSCT in 2% of patients. In allogeneic transplanted patients, donor was HLA matched sibling in 99% and 97% of AML and ALL groups respectively. Source of stem cells was peripheral stem cells in 88% and 89% of patients in AML and ALL groups respectively. In AML group, 77% of HSCTs was performed in CR1, 17% in CR≥ and 5%in relapsed or primary induction failure (PIF). In ALL group, 66% of HSCTs was performed in CR1, 26% in CR≥2 and8%in relapsed or PIF. Acute and chronic graft versus host disease (GVHD) occurred in 64% and23% of patients in AML group respectively. Acute and chronic GVHD occurred in 70% and22% of patients in ALL group respectively. With median follow up of 20 months, relapse and transplant related mortality was cause of death in 14% and 9%of AML group and in 22%and 13% of ALL group respectively. In AML group, 2 years disease free survival (DFS) and overall survival (OS) was 48% and 58% in autologous; 72% and 79% in allogeneic subgroups respectively. (P value <0.001) In ALL group, 2 years (DFS) and (OS) was 31% and 38% in autologous; 53% and 59% in allogeneic subgroups respectively. (P value >0.05) Conclusion: We conclude that HSCT is a major treatment of acute leukemia patients in Iran with comparable efficacy with other parts of the world. Efficacy and safety of thiotepa as part of the conditioning regimen for advanced haematological malignancies I. BATSIS, D. Mallouri*, D. Bartzoudis, P. Kaloyannidis, E. Yannaki, M. Papathanasiou, E. Bitzioni, I. Sakellari, A. Anagnostoulos George Papanicolaou Hospital (Thessaloniki, GR) Despite recent advances, the overall clinical outcome after transplantation for refractory disease remains poor and mortality rates high. We retrospectively analyzed the impact of intensified conditioning including Thiotepa in 21 patients (pts) with advanced hematologic malignancies undergoing hematopoietic stem cell transplantation (HCT) from 2004 to 2007. Grafts were donated from 17 matched and 3 unrelated donors whereas one patient underwent autologous HCT. Twenty patients were conditioned with Thiotepa (750mg/m²)/ S111 Busilvex (12.8mg/kg)/ Cyclophospamide (120mg/kg) and one with Thiotepa, Busilvex, Melphalan (100 mg/m²). Twelve pts were male and 9 female of median age 37(8-56) years. Thirteen pts had AML (secondary disease 3), ALL 4, HD 2, NHL 1 and secondary MDS 1. Primary refractory disease had 17 pts, and 4 pts were on 2nd or 3rd CR. The number of prior chemotherapy regimens was 4(2-7). Mucositis was the main regimen-related toxicity present in all pts (grade≥II 13/21 pts) for 12(4-65) days. Granulocyte colony stimulating factor was administered in 8/21 pts during hospitalization. All pts engrafted successfully and were discharged on day +22(16-62) after receiving transfusions with a median of 8 RBC and 13 PLT units. The incidence of grade ≥ acute graft versus host disease was 45% and of chronic 85%, in evaluable pts. Toxicity was associated with a high incidence of severe infections (CMV 7, EBV reactivations 3, fungal 4) and in 4 pts with thrombotic thrombopenic purpura. Ninety per cent were in CR following transplantation with median duration of CR 5(1-35) months. Relapse incidence was 42%. Currently, 9 pts (43%) are alive, 8 pts in continous CR1 and one in refractory relapse. The cause of death was disease-related in 7 and treatment-related in 5 pts. Non relapse mortality was attributed to CMV pneumonia (n=2), pulmonary aspergillosis (n=1), aGvHD (n=1) and encephalitis (n=1). With a median follow up of 15 months, the 3-year disease free survival and overall survival was 36%. The 3-year relapse and TRM rate was 60% and 29% respectively. This outcome didn't seem to differ significantly when compared to the outcome of a historical control of 20 pts with advanced acute leukemia who received the standard BUCY conditioning. These results suggest that the intensified conditioning with Thiotepa for the refractory disease treatment is a tolerable and effective regimen but it does not significantly improve HCT outcomes when compared to standard BUCY. We report our results of autologous stem cell transplantation (SCT) in patients with AML during the last 16 years. Between December 1991 and July 2007, 102 patients with Acute Myeloid Leukemia (AML) received an autologous SCT. The main characteristics were reported in Table 1 . The median patient age was 46 years (range17 -67 years). The conditioning regimen most used was Busulphan + Cyclophosfamide (90 pts). The Transplant Related Mortality (TRM) was 2/102 (2%) and the causes of death were infection and haemorrhage. The overall survival (OS) was 50% with a median follow up of 52 months (Figure1). We calculated OS for age stratified in two groups: 17-45 y vs 46-67 that was 47% versus 52%, respectively, without statistical difference. The majority of patients (90) was transplanted in first complete remission (CR), 11 in 2nd CR and 1 in 3th CR. Furthermore, all patients in 2nd CR, included in second group age, died. The OS of disease status at transplant was statistical significant: 53% vs 18%, p < 0,01 (3th CR were excluded from analysis).We also analyzed the OS distributed for sex and cell source without statistical difference. We have documented a statistical significant correlation between FAB group and survival. In fact, the patients with FAB M2 and M4 (excluded M3) had a superior OS than those with the other FAB (58% vs 35%, p < 0.01). We conclude that autologous bone marrow transplantation is an effective treatment in AML with the possibility of long survivorship, particularly in patients with FAB M2, M3 and M4, without difference for age. In our experience, the status of disease at transplant plays an important role and is correlate to the overall survival. Busulfan and fludarabine is a safe and effective conditioning regimen for allogenic bone marrow transplantation in adults with acute myeloid leukaemia T. Fagot* (1), J. Konopacky (1) Objectives. The determining in vitro drug resistance may reveal clinically relevant information for prognosis the efficacy of chemotherapy in childhood leukemia. Aims: of this study was to evaluate the correlation between blast cells sensitivity profile to chemotherapy ex vivo (according to range scale) and the probabilities of 7-year disease-free survival. Methods: The material of the study bone marrow from 169 children with newly diagnosed ALL. The blast cells sensitivity to chemotherapy (10 drugs) was examined in MTT-assay. Results: For each drug (Prednisolone, Dexamethasone, Vincristine, L-Asparaginase -PDVA) LC50 was determined. The degree of cytotoxity was estimated in ranks scale: each step of a drug dilution (from greatest to lowest) was corresponded to rank (from 1 up to 7). The high sensitivity to a drug -LC50 value from 1 to 4 ranks; low sensitivity -LC50 value from 5 to 7 ranks. The present data on analyze correlations between these potential prognostic factors and well-known favourable and unfavourable markers of prognostic in ALL. The probability of 7-year disease-free survival was significantly higher in patients with high sensitivity (1-4 ranks) to Prednisolone (p=0.036), Dexamethasone (p=0.043), Vincristine (p=0.041), L-Asparaginase (p=0.042), in comparison with the patients with low sensitivity (5-7 ranks). The probability of 7-year disease-free survival in patients with high simultaneous sensitivity to 3 of 4 drugs (PDVA) was 0.88 versus 0.62 in patients with low simultaneous sensitivity to three of these drugs (p=0.037). Conclusions: the initial sensitivity of leukemic cells to Prednisolone, Dexamethasone, Vincristine, and L-Asparaginase is very significant in prognosis of 7 -years disease-free survival in pediatric ALL. High simultaneous sensitivity to at least 3 of 4 above mentioned drugs appears to be good prognostic factor. Outcomes of allogeneic haematopoietic stem cell transplantation in relapsed good risk acute myelocytic leukaemia carrying t(8:21) or inv(16) J.H. Lim, K.H. Lim, K.W. Lee, B.S. Kim, S.M. Bang, I. Kim, D. Lee, S.S. Yoon, S. Park, B.K. Kim Seoul National Univ. Hospital (Seoul, KR) Objectives: Patients with acute myelocytic leukemia (AML) with favorable cytogenetics such as t(8;21) and inv(16) are classified as good risk with chemotherapy regimens and are not subject to hematopoietic stem cell transplantation (HSCT) front line. However there are limited data following HSCT in relapsed good risk AML. Since allogeneic HSCT has been regarded traditionally as the only curative option in this population, we evaluated the outcomes of the patients who were treated with allogeneic HSCT in relapsed good risk AML carrying inv(16) or t(8:21). Methods: We evaluated the outcomes of 22 consecutive patients who underwent allogeneic HSCT with either myeloablative (n=17) or reduced-intensity conditioning (RIC) regimen (n=5) between January 1997 and May 2007 at Seoul National University Hospital. Results: Among 22 patients (median age 33, 18-70), 13 patients were classified as AML with t(8;21), 9 patients as AML with inv(16). 13 patients received sibling donor transplantation and the remaining 9 received unrelated donor transplantation. Median follow up duration was 8.3 months (range: 5.9 -10.7 months). 20 patients remained in remission after HSCT and 10 patients relapsed after remission with HSCT, among them, 8 (61.5%) with t(8;21) and 2 (22.2%) with inv(16). At the time of analysis, only 7 patients are alive (3 (23%) with t(8;21), 4 (44%) with inv (16)). 2 patients with inv(16) were lost to follow up. 13 patients (61.5%) expired, 10 with t(8;21) and 3 with inv(16). 9 patients with myeloablative conditioning regimen and 4 patients with RIC regimen expired. There were no statistical difference between the myeloablative and RIC regimen groups (p=.14). The main causes of death were relapse (53.8%, 7 patients), infection (23%, 3 patients), Graft-versus-host disease (23%, 3 patients). 3-year survival rate was 43.8%. However, in patients with t(8;21) AML (n=13), 3-year survival rate was 28.7%. On the contrary, in patients with inv(16) AML (n=9), 3-year survival rate was 65.8%. Conclusion: Patients with relapsed favorable risk AML showed divergent outcomes after allogeneic HSCT according to cytogenetic abnormalities. Patients with relapsed t(8;21) AML particularly exhibited high relapse rate and poor prognosis after allogeneic HSCT which is similar to treatment outcome of poor risk group AML. New treatment approaches including first-line HSCT with either allogeneic or autologous approaches should be tried in a prospective setting in this population. The role of reduced-intensity allogeneic stem cell transplantation in patients with advanced acute myeloid leukaemia and myelodysplastic syndromes I. Espigado*, F. J. Marquez-Malaver, F. de la Cruz, J. Falantes, I. Montero, R. Parody, M. Carmona, J.M. Perez-Hurtado, A. Urbano-Ispizua UH Virgen del Rocío (Sevilla, ES) Background: Nonmyeloablative allogeneic stem cell transplantation (SCT) is an effective therapy for patients with high risk acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS) ineligible for conventional myeloablation. But it is unclear the clinical role of reduced-intensity conditioning (RIC) in patients with advance disease and there are very few data on long term results in this setting. Objective: To compare overall survival (OS), event-free survival (EFS) and transplant related mortality (TRM) following SCT with reduced-intensity conditioning in patients with high risk advanced disease versus patients in complete first remission. Patients/Methods: twenty six consecutive patients with AML or MDS, non eligible for myeloablative SCT were included from May 2004 through September 2006. Median age was 43.5 years (4-64) and 14 (54 %) were male. Seven patients were in complete first remission (CR1) and 19 had a more advanced disease (AD). Twelve patients in the AD group had undergone an autologous (n =5) or allogeneic (n =7) transplant previously. The graft source was peripheral blood, except for two bone marrow cases. The donor was an HLA-identical sibling in 17 cases, an haploidentical mother in one case and a matched unrelated donor in 8 cases. Conditioning consisted of fludarabine (30mg/m² x 5 days) followed by total body irradiation (200 cGy). Busulfan (4 mg/Kg x 2 days) was added in the AD group. Immunosupression was methotrexate (10 mg/m² on days: +1,+3,+6) and Cyclosporine (3mg/Kg/day until +100). Results: The probability of 4 years OS was 66% in patients in CR1 and 34% in patients with AD (p=NS). The EFS was 66% in CR1 and 23% with AD (p=0.05). Long term TRM occurred in 2/7 (28%) patients in CR1 and in 9/19 (41%) in AD (p=NS). Conclusions: 1. With this RIC approach, patients ineligibles for myeloablation with AML/MDS beyond first complete remission had lower OS, lower EFS and higher TRM than those in complete first remission at transplant. 2. Nevertheless, as the 4 years EFS of patients with advanced disease was 23%, this reduced-intensity conditioning approach should be explored in controlled prospective clinical trials in this very poor prognosis group of patients. Factors influencing relapse after allogeneic haematopoietic stem cell transplantation in leukaemia: a single-centre experience P. Bavaro (1) , P. Olioso (1) Allogeneic hematopoietic stem cell transplantation (HSCT) is considered a potential curative approach for patients with hematological malignancies. Unfortunately relapse remains the major cause of treatment failure. Disease relapse usually involves the bone marrow (BM), but some patients develop extramedullary (EM) relapse either alone or in association with marrow relapse. In this retrospective analysis we investigated the pattern of relapse and the risk factors in 227 consecutive patients who underwent HSCT in our institution between January 1997 and September 2007 for acute nonlymphoblastic leukemia (n=89), acute lymphoblastic leukemia (n=59), myelodysplastic syndromes (n=20), and chronic myeloprolipherative syndromes (n=59). After a median followup of 31 months (range, 1-130) 91 patients relapsed (40.1%) . Seventy-six patients (83.5%) relapsed in the BM only, 9 patients (9.9%) showed an EM relapse, and 6 patients (6.6%) had both EM and BM relapse. The median time to relapse was 7 months (range, 1-74). The following factors were analysed with regard to relapse: disease type, disease status at transplant (first CR or chronic phase vs subsequent CR vs relapsed or refractory disease), sex, age at transplant, conditioning regimen, donor source, HLA typing, ABO incompatibility, GvHD prophylaxis, grade of acute GvHD, stage of chronic GvHD. Multivariate logistic regression model was performed to estimate the odds ratio (OR) and relative 95% confidence intervals (95%CI) for each of the variables resulted statistically significant at the univariate analysis. The independent prognostic factors who significantly influenced the risk of relapse were: a) ALL diagnosis (p =0.003 OR=4.89; 95%CI 1.73-13.76); b) disease status at time of transplantation (p=0.02 OR 3.99; ); c) BUCY-based conditioning regimen (p=0.05 OR 3.14; 95%CI 1.00-9.81); d) chronic GvHD limited (p=0.05 OR 0.31; 95%CI 0.09-1.00) or extensive (p<0.001 OR 0.07; 95%CI 0.17-0.31). Cumulative overall survival is 54% at 5 years and 49% at 10 years. This study confirms that ALL diagnosis and relapsed or refractory disease at transplant are associated with higher risk of relapse. The incidence of relapse is reduced in patients with chronic GvHD due to associated graft-versus-leukemia effect, as previously well demonstrated. The risk of leukemia relapse is reduced in patients receiving the BUCY-based conditioning regimen as compared to the TBI-CY regimen. Treosulfan-based myeloablative conditioning for autologous stem cell transplantation in acute myeloid leukaemia and myelodysplastic syndrome patients: preliminary data of feasibility and efficacy M. Bernardi *, M. Tassara, A. Crotta, J. Peccatori, C. Messina, A. Assanelli, D. Clerici, S. Mastaglio, C. Corti, M. Carrabba, F. Ciceri San Raffaele Scientific Institute (Milan, IT) Background: consolidation of complete remission (CR) with intensified-dose chemotherapy and autologous (auto) stem cell transplantation (SCT) can prolong DFS in pts with AML or MDS, when allogeneic SCT is not feasible. Treosulfan is a bifunctional alkylating agent approved for the therapy of advanced ovarian carcinoma. A fractionated dose of treosulfan can induce a stem cell toxicity comparable to that of busulfan, which is registered in combination with cyclophosphamide for conditioning prior to autoSCT. Serious toxicities, known to occur with busulfan, were not observed in high-dose (HD) treosulfan studies. We have combined HD treosulfan with the fludarabine-cytarabine association, whose efficacy in treatment of acute myeloid leukaemia (AML) and myelodisplastic syndrome (MDS) has been well established, in a new conditioning regimen prior to autoSCT for AML and MDS pts. Aim: evaluation of preliminary data on toxicity and efficacy of a treosulfan-containing conditioning regimen prior to autoSCT in MDS and AML pts. Methods: period 7/2006 Methods: period 7/ to 8/2007 . Diagnosis (WHO): de novo AML 3, AMLMD 2, RAEB2 1, t-MDS 1. Cytogenetics: favourable 1, normal 3, complex 2, not evaluable 1. Status at transplant: CR 6 pts, t-MDS 1. Median number of chemo cycles before transplant: 2 (range 0-4). Conditioning regimen (FLAT): treosulfan 10 gr/sqm for 3 days, fludarabine 30 mg/sqm for 5 days, cytarabine 2 gr/sqm for 5 days, PEG filgrastim 1 s.c. vial after autoSCT (only pts 6 and 7). Results: all pts were evaluable for treatment-related early toxicities; no toxicities of grade > 2 (CTC) were observed. Six pts were evaluable for long term outcome, 1 patient received a haploidentical SCT about 40 days after autoSCT. Principal results are summarized in the table; at last follow up all pts are alive, 5 in first CR 471 days (median, range:105-498) after autoSCT. Conclusions: in these preliminary pts the FLAT regimen proved to be feasibile, as very low toxicities were observed, remarkably in the elderly. Duration of neutropenia seemed to be shortened after infusion of an adequate number of hematopoietic stem cells (≥ 10 6 ) and PEG filgrastim administration. Prolonged DFS from CR and EFS from transplant were documented, in poor risk pts too. A phase II study in pts older than 65 is ongoing in our Center to confirm these data. Second allogeneic stem cell transplantation as treatment for leukaemic relapse following a first allograft: a singlecentre experience C. Dobbelstein*, E. Dammann, S. Buchholz, M. Port, A. Trummer, H. Diedrich, M. Stadler, J. Krauter, B. Hertenstein, A. Ganser, M. Eder Hannover Medical School (Hannover, DE) Introduction: Allogeneic stem cell transplantation (SCT) is the most effective antileukemic therapy available so far. However, there is no standard therapy to treat leukemic relapse after allogeneic SCT. Current treatment options encompass chemotherapy (CT), donor lymphocyte transfusion (DLI), and 2nd SCT. Here we report the outcome of 2nd SCT in our center. Patients and methods: From 1996-2007, 20 patients (pts) underwent 2nd SCT from the same or an alternative donor for treatment of leukemic relapse after allogeneic SCT: 4 pts with CML, 8 and 5 pts with de novo and secondary AML, respectively, 2 pts with ALL, and 1 pt with acute undifferentiated leukemia. The cohort also includes 1 pt with donor cell AML developing after 1st SCT for CML. Median follow-up is 6.6 months (range 0.4 to 113.2). Results: Leukemic relapse occurred at a median of 480 days after 1st SCT (range: 104-2168) and was treated with CT alone (n=10, including tyrosine kinase inhibitors for Ph+ leukemia), CT + DLI (n=7), DLI without CT (n=2), and 1 pt received 2nd SCT as initial therapy. There were 4 and 6 pts with unfavorable karyotype at 1st and 2nd SCT, respectively, and 40% of pts were either in CR or chronic phase at 2nd SCT. Conditioning for 2nd SCT was either myeloablative (n=4) or of reduced intensity (n=16). 10 pts each received grafts from the same and alternative donors at 2nd SCT: 9 and 1 pts were transplanted from identical MRD and MMRD, respectively, whereas alternative donors included 8 MUD (3 MRD/MUD, 3 MUD/MUD, 1 MMRD/MUD, 1 MMUD/MUD) and 2 MMUD (MRD/MMUD). Engraftment occurred in 90% after 2nd SCT with a median time for neutrophil recovery of 15.5 days (range 12-49). Prophylactic DLI after 2nd SCT were given to 3 pts. Overall survival (OS) of all pts is 30%, all of them with AML: 5 pts are currently in CR and 1 in relapse. Median survival time after 2nd SCT is 238 days (238 and 63 days for identical and alternative donors, respectively). 2nd SCT from alternative donors was associated with reduced relapse rate (10% vs. 50%) but higher non-relapse mortality (60% vs. 30%) as compared to SCT from identical donors with identical OS. In addition, pts with response to DLI after 1st SCT and with cGvHD after 2nd SCT seem to have a better outcome. Discussion: 2nd SCT for leukemic relapse after allogeneic SCT can result in long-term survival for a minority of pts. Modifications in conditioning and prophylactic DLI may further improve survival after 2nd SCT in the future. The trials comparing AHCT vs. alloHCT or chemotherapy in general demonstrate advantage of AHCT vs. chemotherapy in disease free survival (DFS) and prove the feasibility of AHCT even in patients >60 y. The transplant related mortality is much lower compared to allografts but the relapse incidence is much higher. Therefore the place of AHCT in AML remains disputable. The goal of this study was to evaluate the efficacy of AHCT compared to alloHCT in adult AML. Among over 1700 patients treated in our department with HCT between 1991 and 2007 there were 335 AML patients;Tab.1. Results: The regeneration time of granulocytes and of platelets, the cumulative incidence of non relapse mortality -NRM and of relapse incidence -RI as well as the Kaplan Mayer estimates of DFS and of overall survival -OS at median observation time of 6.5 year are presented in Tab.2. The analysis of AML's transplanted before and after 2000 y revealed a decrease of AHCTs 61% to 29% in favor of allografts. Patients autotransplanted > 2000 were older 48 y (16-61) vs. 29 y (16-58) <0,000001, and obtained more frequently APBSCT than ABMT 83% vs. 23% p<0,00001. The results in 57 patients treated >2000 were in spite of older age better then in 84 patients autografted earlier; TRM 8.3% vs. 3.5%, and OS 37% vs. 57%, p = 0,03. Two independent factors influence OS: CR1 vs. CR2 (HR=2,1, p=0,02) and transplantation period 2000y (HR=0,6, p=0,04). Our observation demonstrates the superiority of allotransplantation particularly from sibling donor in terms of survival. The results obtained with URDHCT do not differ significantly from SibHCT in spite of the prevalence of high risk patients in this group. AHCT is an effective and save option for patients not eligible for allotransplantation and the results of this procedure improved in the last decade. Clinical trials of subgroups stratified according to new molecular risk criteria's, introduction of less toxic conditioning and posttransplant treatments preventing from relapses are keys for HCT to become still more effective treatment. Behenoyl cytarabine-based chemotherapy followed by haematopoietic stem cell transplantation for paediatric non-promyelocytic acute myelogenous leukaemia K.H. Yoo*, K.W. Sung, E.J. Cho, S.Y. Lee, J.Y. Kim, H.L. Jung, H.H. Koo Samsung Medical Center (Seoul, KR) Behenoyl cytarabine (BHAC) is a derivative of cytarabine, and has been used in Korea and Japan in combination therapy for acute myelogenous leukemia (AML) patients. Reported clinical trials using BHAC are mostly from adult series. Furthermore, data on hematopoietic stem cell transplantation (HSCT) following BHAC-containing chemotherapy in childhood AML is lacking. In our institution, between July 1998 and July 2006, 62 children who were newly diagnosed as nonpromyelocytic AML received induction therapy comprised with BHAC (300 mg/m² on days 0 to 6, 0-500 mg/m² on days 7 to 9), idarubicin (12 mg/m² on days 0 to 2), and 6-thioguanine (100 mg/m² on days 0 to 6). Patients' age at diagnosis was median 7.3 y (0.1-15.7 y). Unfavorable karyotypes were found in 25 patients (40.2%), intermediate in 22 (35.5%) , favorable in 14 (22.6%), and unknown in 1. Fifty-two of 62 patients (83.9%) achieved CR and there were 3 induction deaths (4.8%). Of 52 patients who achieved CR, 14 could not proceed to HSCT in CR1: 7 relapsed prematurely during consolidation therapy, 5 were refused by parents, 1 was lost to follow-up, and 1 was Down syndrome. As a result, 38 patients who achieved CR by BHAC induction therapy underwent HSCT in CR1 following 1 to 6 courses (median 3) of consolidation therapy (BHAC included in initial 2 courses). The time interval between the diagnosis and HSCT was median 6 mo (3-13 mo). The stem cell sources were autologous peripheral blood (n=8), matched-related sibling marrow or peripheral blood (n=5), matched-unrelated marrow (n=13), or unrelated cord blood (n=12). The 5-y event-free survival (EFS) of 38 patients was 73.7% (APBSCT 62.5%; matched-related BMT or PBSCT 80.0%; matched-unrelated BMT 92.3%; UCBT 58.3%) with a median follow-up of 41 mo (20-114 mo). The 5-y overall survival and EFS of all patients (n=62) was 58.1% and 54.2%, respectively. These results suggest that the efficacy of BHAC-based regimen is comparable to that of cytarabine-based combinations for remission induction in pediatric AML. Even the alternative donor HSCT yielded good outcomes for those who achieved sustained CR until the time of HSCT. Since our study has limitation in that it is not a comparative analysis, further studies are needed to determine the feasibility of BHAC in pediatric AML. High-dose melphalan as conditioning for second allogenic haematological stem cell transplantation in relapsed acute myeloid leukaemia G. Guillerm*, J.C. Ianotto, A. Tempescul, C. Berthou CHU Brest (Brest, FR) Early relapse of acute myeloid leukemia after allogenic haematological stem cell transplantation (HSCT) is a difficult issue. The use of donor lymphocyte injection (DLI) for strengthening the graft versus leukemia effect is effective only in patients with minimal-moderate aggressiveness of their leukaemia. High dose melphalan and autologous HSCT proved to be able to achieve complete remission in refractory or relapsed AML. In this study we described the cases of 6 patients with AML in which we performed a second allogenic BMT mostly after failure of DLI and chemotherapy(CT) . Characteristics of the six patients are presented in table 1. The AML patients relapsed within 1 year after first allogenic HSCT. Relapses were treated with chemotherapy then DLI infusion in 4 of 6 patients with failure of this treatment. The use of high dose melphalan followed by a second bone marrow infusion from the same sibling without graft-versushost disease (GvHD) prevention results in complete remission with an acceptable toxicity. Haematopoietic reconstitution was quick as neutrophil recovery (>0.5G/L) median is 13.5 days (range 9-15 days). Acute grade 2 skin GvHD occurred in three of five evaluable patients. Two of five evaluable patients had an extramedullary relapse at 6 and 9 months from second BMT, one died. Three of five evaluable patients are in complete remission after a follow up of 5, 10 and 11 months. Our experience confirmed the efficacy of high dose melphalan in relapsed AML without lethal treatment related toxicity. GVHD was not able to control the disease progression in extramedullary site in two of these advanced stages of AML. Further follow up and more patients are necessary to validate these data. Introduction: chemotherapy for acute myeloid leukaemia (AML) results in 65-80% complete remission, but only 20-30% of patients (pts) maintain a durable remission with standard consolidation therapies. The use of allogeneic hematopoietic stem cell transplantation (HSCT) increases the rate of long term survival although a substantial number of patients still relapse. We report the results of allogeneic HSCT from HLA identical sibling donors underwent in 177 pts with AML. Patients and methods: from september 1998 to november 2006, 177 pts with AML (M1 30; M2:71 ; M3:14; M4:30; M5:12;M6:2;M7:1;M0:7;NP:10) received 178 allogeneic HSCT and one boost from HLA identical sibling donors; median age 25 years (6 to 47);sex ratio 1,08 ; status disease at transplant, first complete remission (CR1):155 pts,CR2:15 pts,in relapse:8 pts; median interval from remission to allograft 5,6 months (1 to 24). All patients received conditioning regimen with chemotherapy alone: Tutshka with additional VP16 134 pts, Tutshka: 8 pts, Santos: 35 pts and Fludarabine based conditioning regimen:1 pt. The GVHD prophylaxis consisted of ciclosporine and methotrexate. Ten pts received bone marrow transplantation and 169 pts peripheral blood stem cell. At september 2007 maximal follow-up is 111 months and minimal 10,8 months. Results: the median time to engrafment was 15 days (9 to 24). One hundred and eleven pts (62,7%) are alive in remission after median follow-up 46,8 months (9,8 to 110). Acute GVHD occurred in 45 pts (27,6% )with 29 grade II-IV and chronique GVHD in 54 pts (37%), extensive:33. Sixty six pts died (37,3%):43 pts (24,3%) by transplant related mortality (TRM;infectious:18,AGVHD:11,CGVHD:2,VOD:8, cerebral hemorrhage:2, metabolic disorder:2). Twenty three pts (12,9%) relapsed and died except one pt is alive after successfully second transplant. Actuarial overall survival (OS) and event free survival (EFS) at 6 years are respectively 62% and 61%. When we compare the results between two groups of age: under 35 years old (139 pts) and up to 35 years old (38 pts), TRM is higher in second group (35,3% vs 44,7%;p= 0,02) . No difference between the two groups for OS (61% vs 53%;p=0,3)and EFS (62% vs 53%;p=0,4). Conclusion: our results are similar to literature but with lowest relapse rate. Because of higher TRM in group up to age 35 years old we should purpose for this pts Tutshka conditioning regime without VP16 or reduced intensity conditioning regimen. Outcomes of patients with high-risk acute myeloid leukaemia relapsed after allogeneic stem cell transplantation. Single-centre experience K. Steinerova*, V. Koza, P. Jindra, M. Karas, T. Svoboda, D. Lysak, S. Vokurka, V. Vozobulova Charles University Hospital (Pilsen, CZ) The prognosis of the patients (pts) with AML relapsed after stem cell transplantation (SCT) is dismal. We describe treatment and outcomes for patients who relapse following SCT and compare the outcome of the group of patients transplanted with myeloablative and with reduced intensity conditioning regimen. Materials and methods: in a retrospective single center study we analyzed 124 pts with high risk AML transplanted between the years 1993 and 2007. 36 pts (29%) 17 males and 19 females relapsed after SCT with a median of 3,5 months (range 1-49). 25 pts were transplanted with myeloablative conditioning (group I) and relapsed with median of 3 months (1-49) after SCT. 11 pts were transplanted with reduced intensity conditioning regimen (group II) and relapsed with median of 5 months after SCT (p=0,7887). Mean recipient age was 40 years (21-55) in the group I and 54 years (29-64) in the group II (p=0,0126). Both groups were comparable according to disease status, prognostic factors and the donor type (HLA identical sibling vs.unrelated donor, p=1,0). 16 pts (44%) received aggressive treatment for relapse (10/25 and 6/11, p=0,4834) including donor lymphocyte infusion (DLI, 62,5%), chemotherapy (25%), second transplantation (12,5%). Results: 2 pts archieved complete response (CR) after treatment for relapse and still remain in remission 4 and 5 months after relapse. 4 pts alive (1 from group I and 3 from group II) with median of overall suvival (OS) in the group I 5 months, the probability of 1 year OS 28% and with median of OS in the group II 7 months, the probability of 1 year OS 34% (p=0,3287). 32 pts died, most of them due to disease progression (30/32, 94%), 1 pts died due to GVHD after DLI and 1 pts due to respiratory failure. We did not observe difference in the outcome among pts treated with DLI, chemotherapy and second transplantation. Conclusion: salvage chemotherapy or cellular therapy for AML relapse after SCT is feasible with low treatment-related mortality, but optimal management of the therapy for AML relapsing after SCT remains to be defined. Comparing the group of pts transplanted with myeloablative regimen and pts transplanted with reduced intensity regimen no statistical significant difference (except of age) were found, however our results suggest better overall survival for patients relapsing after reduced intensity regimen SCT. Second allogeneic haematopoietic stem cells transplantation for acute myeloid leukaemia relapse after first transplant: single-centre experience S. Guidi, B. Bartolozzi, C. Nozzoli, L. Lombardini, A. Gozzini, R. Saccardi, I. Donnini, A.M. Vannucchi, A. Bosi BMT Unit (Florence, IT) Introduction: Relapse after allogeneic haematopoietic stem cell transplantation for acute myeloid leukaemia occurs in 30-50% of patients. Patient prognosis after relapse is usually poor. Chemotherapy, donor lymphocyte infusion or second transplantation from the original or from alternative donor are treatment options offered to relapsed patients. Salvage treatments are unsatisfactory and rarely cure patients. Controlled studies about the best treatment options are lacking, but retrospective studies about second transplantation in relapsed patients show interesting results. Objective: Our aim was to evaluate the impact of a second transplant or any other treatment options for patient relapsed after an allogeneic transplant. Patients: From January 1988 to October 2007 we treated 146 Acute Myeloid Leukaemia patients. 54 (37%) over 146 patients relapsed. 9 early and 45 advanced, donors were 44 HLA id-sibling and 10 alternative donors, 44 were treated with myeloablative and 10 reduced intensity conditioning regimen. Stem cell source was bone marrow in 25, peripheral blood in 28 and cord blood in 1. 17/54 (31%) patients underwent a second transplant from the original 16/17or from another donor 1/17. Methods: If not too ill, the relapsed patient was given at least a reinduction chemotherapy cycle. The choice of a second transplant was done on an individual basis depending on many factors: the transplant era, presence of comorbidity, age, the moment of relapse, chimerism, availability of an alternative donor, Graft versus Host Disease, a reduced intensity or a myeloablative conditioning regimen for the first transplant, chemotherapy alone or a total body irradiation plus chemotherapy conditioning, and finally, if the patient reached a subsequent remission or not. The survival functions are plotted starting from the relapse date. Results: 4/17 (24%) patients of the second transplant cohort are alive and in remission; three of them in cGvHD, 5/17 (29%) died transplant related, 8/17 (47%) patient relapsed after the second transplant and died. Of the patients that were not transplanted, only 3 (8%) over 37 patient are alive but still undergoing chemotherapy treatment. Median survival was 373 days in transplant cohort versus 117 days in the other group and the difference was highly significant(p= 0,000). Conclusion: We confirm the poor prognosis for relapsed patients, but in our experience the only chance to became a long term survivor is to undergo a second transplant. Outcomes of allogeneic haematopoietic stem cell transplantation in acute lymphoblastic leukaemia: a young program's experience R. Rihani*, O. Hlalah, F. Abdel-Rahman, A. Ahmed, H. Taani, T. Nserat, A. Elayan, M. Batheeb, M. Sarhan King Hussein Cancer Center (Amman, JO) Objectives: To determine outcomes of allogeneic hematopoietic stem cell transplantation (HSCT) for patients (pts) with acute lymphoblastic leukemia (ALL). Patients and methods: Between January 2003 and December 2007, 33 pts [24 (73%) male and 9 (27%)] female with high risk or relapsed ALL, received allogeneic HSCT. Twenty four (74%) pts were children and 9 (26%) were adults. Median recipient age was 9 years and 25 years, respectively. Precursor B ALL was found in 26 (79%) pts whereas T cell ALL was present in 7 (21%). Cytogenetic abnormalities included t (9; 22) n=9 (27%), t (1; 9) n=2 (6%) and hyperdiploidy n=1 .Indications for HSCT were high risk features in 14 (42%) pts in first complete remission (CR) and subsequent CR in 19 (58%) pts. Donor types were HLA matched family donors (MFD) (n= 29), unrelated cord blood (n=3) and one allele mismatched unrelated donor (n=1). Stem cell sources included bone marrow (n=9), peripheral stem cells (n=21), cord blood (n=3).Median infused CD34 dose for (MFD) was 4.7x10 6 . Donor-recipient sex mismatch was present in 19 (57%) HSCTs. Conditioning regimens consisted of total body irradiation and cyclophosphamide in 31 (94%) pts. Results: Acute graft versus host disease (GVHD) grade II to IV occurred in 18 (55%) while chronic (cGVHD) occurred in 14 (42%) pts. At median follow up of 17 months (range 1-59 months) after HSCT, 24 pts are alive and 9 patients are dead. Projected overall survival (OS) at 55 months after HSCT for the entire group is 67%.OS at 55 months was calculated according to several factors including: recepient's age, where OS for children is 71% versus 51% for adults; disease phase at HSCT revealed OS for pts in CR1 of 71% compared with 64% for pts in subsequent CR; OS in pts with cGVHD is 71% while in pts no cGVHD OS is 60% ;donor-recipient sex mismatch provided 72% OS compared to 64% in sex matched HSCT; infused CD34 dose ≥ 5x10 6 is associated with 73% OS versus 65% in CD34 dose <5x10 6 .Leukemia relapse occurred in 8 (24%) pts accounting for the majority of deaths (89%);one patient died of transplant related toxicities. Conclusions: We are encouraged as a young program with the results obtained which are comparable to published data. Peculiarities of SCT at our Institution will be discussed in the presentation. S118 P508 Outcome after autologous haematopoietic stem cell transplantation in adult acute leukaemia: a 14 year single-centre experience M. J. Uriz (1) , M.L. Antelo* (1), A. Valiente (2) , N. Ugalde (1) , A. Gorosquieta (1) , F. Sala (1) , M.T. Zudaire (1) , G. Hurtado (1) , I. Ceberio (1) , M.C Viguria (1) , E. Pena (1) , A. Corcoz (1) , S. Zalba (1) , M.T Orúe (1) (1)Hospital of Navarra (Pamplona, ES); (2)Hospital Virgen del Camino (Pamplona, ES) Objective: Perform a retrospective analysis of our singlecentre series of patients with acute leukaemia who underwent autologous hematopoietic stem cell transplantation (SCT), with a special emphasis on the results of the cytogenetics and analysis of molecular mutations (FLT3, NPM1) in apheresis products. Patients and Methods: We analysed retrospectively our whole series of 37 adult patients with de novo acute leukaemia and autologous SCT in the period 1993-2007.16 patients were female.25 patients had AML and 12 ALL. Mean age at SCT was 30.4 for ALL and 45.25 for AML. Cytogenetics and/or analysis of molecular mutations in apheresis products were directly available in 20 patients. Analyses were carried out from frozen aliquots in 12 additional patients for the present study. No karyotype or molecular data were available from the remaining 5 patients. Results: The median time from the end of the treatment up to the SCT was 3.1 months for AML and 2.5 for ALL patients. 21 AML and 10 ALL patients were in first CR at transplantation. Graft source was peripheral blood in 28 patients. From the 32 patients with cytogenetics and/or analysis of molecular mutations available: 7 had favourable findings (t (8, 21), t (15,17), inv(16) in AML; hyperdiploidy in ALL); 12 patients had intermediate prognosis (normal karyotype) and the remaining patients had disfavourable cytogenetics (complex k., BCR-ABL,E2A-PBX1 and others). Overall, 15/25 (60%) patients from the AML group and 5/12 (41.6%) from the ALL are alive. Mean disease-free survival was 43.4 months for ALL and 50.89 months for AML. Mean overall survival was 45.66 months for ALL and 54 months for AML (no statistically significant differences were found because of the small number of cases). We found minimal residual disease in the apheresis product from two patients with AML, one before transplantation and one retrospectively from a frozen aliquot. In the former, a second apheresis product was obtained before the SCT, and he is currently disease-free after 73 months. The latter patient (M4 type AML), with a normal karyotype (no inv.16) and a FLT3/ITD mutation detected from a frozen aliquot, died 7 months after transplantation. Conclusions: As other authors have described, the prognosis after autologous SCT in adult patients seems to be better for AML in our series. The molecular and cytogenetic analysis of the apheresis product is mandatory to detect minimal residual disease (6.25 % in our series) and prevent early relapses. Does autologous transplant have impact in better overall survival of patients with acute myeloid leukaemia in comparison with conventional therapy? M. Elez *, D. Stamatovic, L. Tukic, B. Balint, O. Tarabar, B. Todoric-Zivanovic, V. Skuletic, O. Tasic, S. Marjanovic, M. Malesevic Military Medical Academy (Belgrade, RS) Introduction: There are three approaches for consolidation in patients (pts) with acute myeloid leukemia (AML) in first remission (with exception of acute promyelocitic leukemia). Those approaches are either allogeneic or autologous stem cell transplantation (SCT) or conventional therapy. Superiority of allogeneic SCT is undoubted, so we put attention on whether addition of autologous SCT or conventional chemotherapy alone have better outcome. Aim: We have analyzed retrospectively 86 pts with AML treated either with autologous SCT or with conventional approach, with respect to disease free (DFS) and overall survival (OS). Material and methods: We have treated 149 pts with AML in our clinic from 1997. till 2007. Nine of them had acute promyelocytic leukemia, 29 were allocated for allogeneic transplant and 25 pts have showed primary resistance. We have enrolled 86 pts into this analysis, 37 pts in the "autologous transplant group" and 49 in the "conventional group". After one or two courses of standard induction chemotherapy ("7+3"), pts have received three more cycles of consolidation (MAE, MidAC and ICE) and last one was used for mobilization of autologous peripheral stem cells in pts with approved complete remission. "Autologous group" immediately undergone high-dose chemotherapy and "conventional group" have received maintenance therapy consisted of Thioguanin 160 mg daily per os and Cyto ARA 100 mg subcutaneously daily for 5 days (mostly due to their own rejection of autologous SCT or their age). In those pts who received autologous transplant, conditioning was up to standard BuCy2 regimen. Results: On intention-to-treat analysis number of relapses was significantly lower in autologous SCT group (15/37 -40,54% vs 29/49 -59,18%, p<0,05) . There are both DFS and OS advantage in autologous SCT group at 7 years (52% vs 39%, p<0,05 for DFS and 55%vs43%,p<0,05forOS). Discussion: Autologous SCT as an addition to conventional chemotherapy is connected with lower frequency of relapses a thus with better overall survival. Usage of mylotarg before treosulfan-containing conditioning regimen in children with resistant acute myeloid leukaemia Z. Dyshlevaya*, L. Shelikhova, Y. Skvortsova Russian Children Hospital (Moscow, RU) Despite of the impressive progress in the treatment of AML allogeneic hematopoietic stem cell transplantation (HSCT) remains the single curative approach to the treatment of resistant AML both in adults and children. These patients have extremely poor prognosis with the level of relapse 70-80% and the risk of TRM 45-50%. As known, MRD level to the moment of transplantation influences on DFS and OS. The addition of Mylotarg (anti-CD33 monoclonal antibodies) with the aim to reduce the level of blasts maximally and the usage of treosulfan in conditioning regimen in order to decrease the risk of TRM can increase both DFS and OS. Patients and methods: we reviewed the records of 7 refractory AML patients (6 m/1 f) who underwent HSCT at the Russian Children Research Hospital between June 2006 and November 2007. The median age was 7 (1-17) years. FABtype: M5 -3 patients, M6 -1 patients, M7 -1 patient, Mx -2 patients. Primary resistant AML was diagnosed in 4 patients and 1st-2nd refractory relapses -in 3 patients. Four kids were transplanted from MUD, 1 patient -from MMUD, 2 patientsfrom MMFD with CD3+/CD19±depletion of the graft. The median level of blasts in bone marrow prior to HSCT was 53,6% (10,4% -73,5%). Mylotarg (3-5 mg/m 2 ) alone (1 patient) or in combination with the course FLAG (4 patients) or HAM (2 patients) was administered 7-10 days before the start of the conditioning. In 5 patients the conditioning regimen included Treosulfan/ Melphalan/ ATG ± Fludara and 2 patients received Treosulfan/Thiothepa/ATG. A median dose of CD34+-cells was 8,5 (1, (1) (2) (3) (4) (5) (6) (7) (8) (9) 4) x 10 6 /kg. S119 Results: the achievement of engraftment was 100% with a median time to neutrophil recovery 15 days (12-33) and to platelets recovery -15 days (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) . The full donor chimerism was achieved in all patients to 30 day after HSCT. Acute GvHD developed in 1 patient (14%), chronic GvHD -in 1 (14%) from 4 patients who were alive to day 100 after HSCT. 5 patients had the liver toxicity grade 2-3 (WHO-classification). Three patients are alive in CR, 3 patients died from the relapse and 1 patient died from viral infection. Overall RFS depended on the presence of acute GvHD, the usage of Tdepletion. Conclusion. Our results show that the usage of Mylotarg in combination with treosulfan-containing conditioning regimen generally doesn't increase the level of liver toxicity and TRM and allows to achieve the long-term engraftment in patients with high risk AML Salvage therapy with a sequential high-dose regimen in a primary refractory acute myelogenous leukaemia elderly patient High-dose sequential (HDS) chemotherapy has been proved to be an effective therapy in lymphoma but its efficacy in acute leukaemia has not yet demonstrated. Here we report a case of a 65-year-old woman with relapsing AML treated with HDS. In December 2004, after AML M4 inv(16) diagnosis, she underwent conventional induction (ARA-C 100 mg/sm for 7 days, Daunorubicin 45 mg/sm for 3 days) and reduced (PS 65%) consolidation (ARA-C 100 mg/sm for 5 days, Daunorubicin 45 mg/sm for 2 days) therapy resulting refractory to therapy (bone marrow leukemic blasts>25%). In case of relapse of disease different schemes are actually available, we decided a salvage therapy with sequential high dose regimen. The patient was successively treated with etoposide 1 gr/sm (total dose 1,8 gr shared in 600 mg/die for 3 days) and Gentuzumab achieving hematological complete response and peripheral blood stem cells (PBSC) harvest (CD34 1074,4 x10 6 ; CNT 268,72 x 10 8 ). Gentuzumab was given for a total of 1 dose (day 14 ), after HD-Etoposide, as in vivo purging before CD34+ cell harvest. The patient underwent HD-ARA-C (2000 mg x 2 for 6 days) followed by ASCT (CD34 537,2 x 10 6 ; CNT 134 x10 8 ), and PBSC harvest (CD34 331,5 x 10 6 ; CNT 332 x 10 8 ) on March 2005 and the same scheme with HD-ARA-C, ASCT (CD34 165,2 x 10 6 ; CNT 161 x 10 8 ) and a second PBSC harvest (CD34 401 x 10 6 ;CNT 351 x 10 8 ) was performed on May 2005 maintaining complete remission (bone marrow blasts<0,5%). Following HDS chemotherapy and tandem ASCT a myeloablative transplantation program with BuCy-2 and ASCT (CD34 300 x 10 6 ; CNT 263,1 x 10 8 ) was planned. BuCy-2 is the conditioning regimen more used in BMT in AML patient because of its myeloablative and immunosuppressive features that favour the engraftment. Over two years from treatment the patient is still in CR. Although larger prospective randomized trials are needed to define the role of HDS regimen in AML relapsed patient our data suggest that HDS followed by ASCT might improve the CR and prolong the survival even in elderly patient. In addition, our data on PBSC harvests indicate the feasibility of sequential collection after high dose regimen. Role of autologous stem cell transplantation in patients with acute myeloid leukaemia M. Kwon, M. Ballesteros, D. Serrano, R. Carrion, M. Mayayo, I. Perez, J. Gayoso, J. Anguita, P. Balsalobre, C. Munoz, J. Sanchez, A. Pineda, J.L. Diez Martin Gregorio Marañon G. U. Hospital (Madrid, ES) In patients ( and median disease-free survival (DFS) was 53 months (95% CI, 50-56). Eleven pts relapsed (42%) after transplantation in a median time of 6 months, 82% relapsed within the first year. All of them died in spite of allogeneic SCT performed in two cases. An additional pt died due to second neoplasia. Transplant related mortality (TRM) at +100 days observed was 0%. Remarkably, all 4 pts with good risk showed 100% of OS and DFS at 5 years. Intermediate risk pts showed an OS of 50% at 5 years with a relapse rate of 43%. Pts with de novo AML showed a better OS and DFS compared to pts with secondary AML, although not statistically significant. After a median follow-up of 53 months, 14 pts (54%) are alive, in CR and without second neoplasias. Conclusions: Our data show that in AML pts with intermediate risk in CR1 and good risk in CR2 not candidates for allogeneic SCT, ASCT offers a safe alternative, providing a long-term DFS, with a 0% TRM and a low rate of second neoplasias. Mortality of relapsed pts after ASCT was 100%, which occurred mostly within the first year after transplantation. New molecular markers will define subgroups of patients who benefit by this strategy. S120 Experimental stem cell transplantation P513 Evaluation of engraftment and graft-versus-host reactivity of ex vivo modified human donor T-cell grafts in a chimeric NOD/SCID/IL2Rgcnull transplantation model M. Nonn* (1), M. Hörz (1), S.A. Khan (1) , L. Shultz (2) , R. Handgretinger (3) , C. Huber (1) , W. Herr (1) , U.F. Hartwig (1) (1)Gutenberg University Medical School (Mainz, DE) ; (2) The Jackson Laboratory (Bar Harbor, US); (3)Eberhard-Karls University School of Medicine (Tubingen, DE) Donor lymphocyte graft engineering to prevent graft-vs-host (GVH) reactivity while improving graft-vs-leukemia (GVL) immunity is of central interest in allogeneic hematopoietic stem cell transplantation (HSCT). According to a recently reported protocol (1) we generated human leukemia/tumorreactive T cell lines by in vitro stimulation of donor lymphocytes with primary acute myeloid leukemia (AML) or renal cell carcinoma (RCC) cells followed by CD137-mediated immunodepletion of alloreactivity using haploidentical fibroblasts. In order to investigate engraftment and residual GVH reactivity of these ex vivo modified T cell grafts in vivo we established a human-murin chimeric HSCT model using immunodeficient NOD/SCID/IL2R common g-chain null (gcnull) mice. Following adoptive transfer of unmodified naive or anti-CD3/CD28 stimulated T lymphocytes into NOD/SCID/gcnull mice, naive or primed CD3+ T cells engrafted to high levels in spleen and bone marrow and induced acute xenogeneic graftvs-host disease (xGVHD) within 3-4 weeks. In contrast, recipients of 5x10 6 -1x10 7 AML-or RCC-reactive cytotoxic T lymphocytes demonstrated engraftment of T cells in the absence of xGVHD for at least 8-10 weeks post injection. T cell receptor (TCR)-Vß analysis of T cells engrafted into the spleen of these mice revealed no significant difference when compared to the TCR-Vß diversity determined prior to transfer. In addition, T cells isolated from spleen retained their reactivity to leukemia/tumor blasts originally used as stimulators but did not recognize NOD/SCID/gcnull derived DC. To examine residual GVH reactivity of these modified donor CTL lines, we subcutanously implanted skin substitutes composed of human primary allogeneic fibroblasts embedded in a collagen-based matrix into NOD/SCID/IL2Rgcnull mice to examine alloreactivity in vivo. Upon adoptive transfer of undepleted AML-or RCC-reactive CTL lines, up to 75% of CD8+ T cells migrated into neovascularized skin substitutes explanted 49 days post transfer. In contrast, no human CD8+ T lymphocytes could be found in allogeneic skin substitutes following allodepletion. Studies on GVH-and GVL-immunity to haploidentical hematopoietic cells and AML-blasts are in progress. In summary, our NOD/SCID/IL2Rgcnull transplantation model may represent a valuable tool to study the biological significance of ex vivo modified human donor lymphocyte grafts in vivo. (1) Wehler T and Nonn M, et al. Blood 2007; 109:365-373 P514 Macrophage migration inhibitory factor as a new target in graft-versus-host disease after allogeneic stem cell transplantation G. Mueller (1), S. Miklos (1) , E. Holler (1) , P. Lindner (1) , L. Leng (2) , T. Schubert (1) , Y. Chang (1) , R. Andreesen (1) , R. Bucala (2) , G.C. Hildebrandt* (1) (1)University of Regensburg Medical School (Regensburg, DE) ; (2) Yale University School of Medicine (New Haven, US) Acute graft versus host disease (aGVHD) is the major complication after allogeneic (allo) stem cell transplantation (SCT). Its pathophysiology involves injury to host tissues by both inflammatory cytokines and donor-derived cellular effectors. Macrophage migration inhibitory factor (MIF) is produced by various cell types and has a broad range of proinflammatory properties. We tested the contribution of MIF to systemic inflammation and mortality after allo-SCT. Lethally (1300cGy) irradiated B6D2F1 (H-2bxd) mice received SCT either from syngeneic (syn) (B6D2F1) or allo (B6; H2-b) donors. One half of animals after either syn or allo SCT were treated with polyclonal antibodies against mouse MIF from day 0 until day 14, whereas the other halves of syn and allo recipients were treated with control IgG. The severity of GVHD was assessed by survival and clinical scores. Recipients after syn SCT all survived and were indistinguishable from naïve, untransplanted controls regardless their treatment with control or anti-MIF. Animals receiving allo-SCT plus control IgG developed severe aGVHD and none of the animals survived by day +28. By contrast, in allo recipients treated with anti-MIF IgG, GVHD scores (day 21: 4.4±0.4 vs. 5.7±0.5) and weight loss (24.6% vs. 45.4%) were decreased and translated into significantly improved survival (p<0.05). In addition, serum IFN-gamma and TNF-alpha levels of anti-MIF treated recipients were decreased when compared to allo controls and target organ injury to the gut was reduced (score: 4.3±0.7 vs. 8.0±2.6). Comparable results were seen when animals were treated after allo-SCT with ISO-1, a small molecule inhibiting MIF tautomerase activity. We next challenged syn and allo recipients with P815 tumor cells (H-2d) at time of transplantation to asses the effect of anti-MIF on graft versus leukaemia (GVL) responses. No tumor cell elimination was seen after syn SCT as demonstrated by FACS analysis of spleens (67.2% P815 cells/all splenic cells) and by histopathology of the liver. Also, no direct anti-tumor effect of anti-MIF antibodies was seen in syn mice (53% P815 cells/all splenic cells). In contrast, P815 cells were significantly cleared in allo recipients treated with either control or anti-MIF IgG (splenic residual disease: 0.81% and 12.1% P815 cells/all cells). In summary, our data show an important role for MIF in GVHD pathology and suggest MIF as a promising target in reducing graft versus host disease without the loss of GVL. Repair of iatrogenic sphincter damage and urinary incontinence by autologous skeletal muscle derived cells R. Karig* (1), J.W. Bagner (1) , H. Gerullis (1) , C. Eimer (1) , G. Heusch (2) , T. Otto (1) (1)Lukaskrankenhaus Neuss (Neuss, DE) ; (2)Clinical Hospital University of Essen (Essen, DE) Background: Urinary incontinence by iatrogenic damage of the external sphincter is not curable conservatively. We show initial results of succesful repair of sphincter function by implantation of muscle derived cells (MDC) (J. Urol. 177(4):488,2007) . Here we present data with a minimum follow up of 12 moths after implantation in a homogenous cohort of 43 patients. Methods: A biopsy tissue is obtained from the left deltoid muscle. Primary cell culture, expansion and processing for transplantation were performed in the local tissue engineering center according §20 pharmaceutical law (AMG). We investigated MDC by immunocytochemistry for the expression of different markers involved in muscle cell development and differentiation. 49.5% (standard error of mean SEM 2.8) were positive for a-sarcomeric actin, 7.3% (SEM 1.8) for a-smooth muscle actin, and 32.7% (SEM 10.1)for desmin. Coimmunostaining demonstrated that 2.8% (SEM 0.5) of the MDC were positive for both a-sarcomeric actin and a-smooth muscle actin. The myogenic transcription factor MyoD1 was present in up to 9.1% while no CD34 positive cells were detected. Cells injected into the sphincter were 50% of myogenic origin. Transplantation was performed 61 days (range 16-122) after biopsy. Results: Forty three male patients (mean age: 70 years, range 56-81) were enrolled. The iatrogenic sphincter defect had caused refractory grade III incontinence for 48. 6 months (range:12-192) . Endoscopic transplantation of 5.18x106 cells (range 0.21-19.17x106) was performed. After a minimum period of one year (range 12-60 months), 4 patients were completely continent and 19 patients had an improvement from incontinence grade III to I. The improvement was observed after 4.7 months (range: 2-9) and remained during follow up. In 20 patients no improvement was observed. Minor side effects (grade 1) were observed in 5/43 patients. Conclusions: Implantation of MDC is safe and succesful procedure for treatment of refractory urínary grade III incontinence caused by iatrogenic sphincter damage. T. J. Boeld*, J. Albrecht, K. Doser, R. Eder, J. Stahl, E. Typlt, A. Schuster, R. Andreesen, P. Hoffmann, M. Edinger Universityhospital of Regensburg (Regensburg, DE) The adoptive transfer of donor CD4+CD25+ regulatory T cells (Treg) prevents lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation in murine disease models. We recently described methods for the isolation and in vitro expansion of human Treg and found that only CD45RA+ but not CD45RA-CD4+CD25high Treg (RA+ and RA-Treg, respectively) maintain FOXP3 expression and their suppressive activity after prolonged culture (Hoffmann et al. Blood 108:4260-7; 2006) . We compared the immunoregulatory function of these Treg subpopulations in a xenogeneic GVHD (xGVHD) model. Transplantation of 20x10 6 human peripheral blood mononuclear cells (hPBMCs) into Rag2-/-gc-/-mice led to engraftment of human CD45+ cells (hCD45+) which was accompanied by clinical signs of xGVHD. Histological and flow cytometric evaluation revealed that mainly CD8+ T cells caused xGVHD and main targets are spleen, liver, lung and mouse hematopoiesis. When expanded CD45RA+ Treg were co-transplanted at a 1:1 ratio with CD4+ and CD8+ T cells contained in hPBMC, the frequency of hCD45+ cells at d14 was reduced to 12±4,9% (n=15), while animals that received expanded RA-Treg had 30±7,5% (n=15) and animals that received no Treg 40±8,3% (n=14) hCD45+ cells in peripheral blood (PB). The engraftment of conventional T cells was only delayed not prevented, as by d21 the percentage of hCD45+ cells in PB increased to 35±9,2% (n=15) when RA+ Treg were co-transplanted as compared to 40±9,3% (n=15) with RA-Treg and 50±6,4% (n=14) without Treg administration. All mice developed clinical signs of xGVHD and 26,7% of mice co-transplanted with RA+ Treg, 13,3% with RA-Treg and 21,4% of mice transplanted with hPBMCs alone survived for 100 days. As Treg mainly inhibited early engraftment of hPBMC, we next examined the splenic cellularity at d10 after transfer. While spleens of mice that received only hPBMC contained 20±3x10 6 hCD45+ cells (n=10), co-transfer of RA-Treg reduced hCD45+ cells to 14±4,8x10 6 (n=9) while animals that received RA+ Treg contained only 5±1,1x10 6 splenic hCD45+ cells (n=11). Furthermore, IL-2 and IFN-g production of conventional T cells re-isolated on d10 was reduced fivefold in mice cotransplanted with RA+ Treg as compared to recipients of RA-Treg or hPBMCs alone. Thus, in vitro expanded human RA+ Treg suppress the proliferation and cytokine production of conventional T cells in early phases of xGVHD, but ultimately do not prevent disease onset and mortality. Co-transplantation of autologous peripheral blood progenitor-cell and mesenchymal stem cells is associated with rapid haematopoietic recovery V. Sergeevicheva, I. Lisukov, A. Kulagin, I. Kruchkova, S. Sizikova, A. Gilevich, E. Shevela, E. Chernykch, V. Kozlov Institute of Clinical Immunology SB RAMS (Novosibirsk, RU) Objectives: High level of the serious implications, associated with pancytopenia after peripheral blood progenitor-cell transplantation PBPC followed by HDCT, is an ongoing problem. Multipotential mesenchymal stem cells (MSCs) are found in human bone marrow and are shown to secrete hematopoietic cytokines and support hematopoietic progenitors in vitro. We supposed that infusion of autologous MSCs after myeloablative therapy would facilitate engraftment by haematopoietic stem cells, and we investigated the feasibility, safety, and hematopoietic effects of cultureexpanded MSCs in patients with heamotological tumors receiving autologous peripheral blood progenitor-cell transplantation (PBPCT). Patients and Methods: We developed an efficient method of isolating and culture-expanding a purity population of MSCs from a small marrow-aspirate sample obtained from 39 patients with heterogenous haemotolgical tumors. Patients were given high-dose chemotherapy and autologous PBPCT with culture-expanded MSC infusion. Results: MSCs were isolated from a mean 40 mL of bone marrow aspirate from all patients during restaging evaluation. Expansion cultures generated over 20 to 50 days after maximum 3 passages, respectively. Thirty-nine patients were infused with 2 to 22.3x10 6 expanded autologous MSCs intravenously after PBPCT at once. There were no toxicities related to the infusion of MSCs. Median time to achieve a neutrophil count greater than 500/mL and platelets count > 50,000/mL untransfused was significantly shorter in MSC group: 10.7 days and 6.4 days versus 16.4 days (U-test, p=0.021) and 24.4 (U-test, p=0.05) days in control group (without MSC infusion), respectively. MCS supporting influence was revealed as particularly significance in patients with suboptimal count of mobilizied and transplanted PBSC (<3.5x10 6 /kg) Conclusion: This report is the one of few describing of infusion of autologous MSCs with therapeutic intent. We found that autologous MSC infusion at the time of PBPC transplantation is feasible and safe. The observed more rapid haematopoietic recovery suggests that MSC infusion after high-dose chemotherapy therapy may have a positive impact on haematopoiesis and should be tested in randomized trials. A cDNA-based assay to analyse donor chimerism in small samples using a nested PCR-approach T. Schmitt*, A. Konur, S. Bakhtiar, K. Bender, J. Hemmerling, C. Huber, W. Herr, R.G. Meyer University Clinic Mainz (Mainz, DE) The stimulation of donor T cells by residing host antigen presenting cells (APC) plays a central role for the induction of acute GvHD. In addition, donor T cells mediate the switch of tissue APC to donor origin. The detection of persisting host APC after T-cell depleted allogeneic stem cell transplantation is therefore of particular interest. Up to now, chimerism of tissue resident APC is determined by fluorescent in situ hybridisation of sex-chromosomes or analysis of tandem repeats in the non-coding regions of genomic DNA. Whereas the first method is limited to sex-mismatched transplants, the latter cannot be used to acquire any further information (e.g. tissue-specificity, activation status). We established a cDNAbased assay utilizing single nucleotide polymorphisms (SNPs) located within coding sequences and tested for the expression of SNP-encoding mRNAs in T-cells, keratinocytes, fibroblasts, and Langerhans cells (LC) . In addition, we tested the ability of the SNPs to differentiate between HLA-matched siblings. Subsequently, we combined primers for 6 SNP-encoding regions with langerin primers in a 7-plex PCR in which the langerin primers were located on different exons to exclude genomic DNA contamination. The 6 SNP were sufficient to differentiate between 7 out of 8 pairs of HLA-identical siblings. A nested multiplex PCR step was introduced that facilitated testing of very small cell numbers. As a final step, the SNPs as well as the presence or absence of langerin were analyzed in a capillary DNA sequencer by primer extension method in which specific sequencing probes are only elongated by one base (minisequencing). Using this approach, we were able to test the SNPs in parallel with langerin-expression in cell samples of less than 100 FACS-sorted LC, isolated from epidermal sheets. In first experiments, we could also demonstrate that this can be performed on a single cell level. However, this needs further improvement for general applicability. We have demonstrated that the use of cDNA-encoded SNPs can be used for chimerism-analysis and that in the same assay, information of tissue specificity can be obtained. The inclusion of a nested PCR step increases applicability to very low cell numbers. The inclusion of additional primers to this approach might allow obtaining information like tissuespecificity and activation status along with chimerism-analysis from different isolated cells ex vivo. Everolimus in combination with cyclosporin A as a potential alternative immunosuppressant in nonmyeloablative haematopoietic stem cell transplantation S. Rathsack*, S. Lange, S. Altmann, R. Wacke, B. Drewelow, M. Freund, C. Junghanss Universität Rostock (Rostock, DE) Everolimus (RAD) is an mTOR inhibitor and is successfully used as immunosuppressant in solid organ transplantation (SOT). In SOT combination of RAD with Cyclosporin A (CSA) resulted in synergistic immunosuppressive activity that allows CSA dose reduction and, therefore, mitigate renal toxicity. The role of RAD in hematopoietic stem cell transplantation (HSCT) is not yet investigated. Here we assessed pharmacokinetics of RAD and its efficacy and safety in a canine model of nonmyeloablative allogeneic HSCT. Methods: Dogs (n=9) received dog leukocyte antigen-identical bone marrow grafts. Five dogs were given unmodified grafts (UG) and 4 dogs received grafts from donors that were sensitized to the recipient by a previous HSCT in which rejection occurred (MG). Immunosuppression consisted of 15mg/kg cyclosporin A (CSA) BID PO (days -1 to +35) and 0.25mg RAD BID PO (days 0 to +27) initially. Subsequently doses were individualized to reach CSA C2 levels and RAD through levels of 1000-1500ng/ml and 3-8ng/ml, respectively. At days 5 and 21 post HSCT blood samples were collected before and 0.5, 1, 1.5, 2, 3, 4, 6, 12h after dosing for pharmacokinetic analyses. Peripheral blood chimerism was determined weekly and hematotoxicity, blood lipids and creatinine were monitored. Results: All dogs engrafted. Two dogs (1 UG, 1 MG) rejected the graft after 10 weeks, all other dogs remained mixed chimeras (median follow up >87d [63-315d] Pharmacokinetic analyses revealed a median RAD trough level of 6.9 (1.99-12.0) ng/ml, cmax was in median 13.6 (5.9-17.3) ng/ml and the corresponding AUC was 123 ng/ml*h. Tmax was 2 (1) (2) (3) (4) h and the elimination half-life amounted to 11.5 (5. 3-16.8) h. Compared to our historic MMF/CSA regimen CSA C2 levels were significantly enhanced and C0 and AUC tended to be increased, allowing CSA dose reduction. Conclusion: Our data suggest that RAD in combination with CSA is well tolerated and might be an alternative to MMF/CSA as pre-and posttransplantation immunosuppression in allogeneic HSCT. (1), R. Andreesen (1), J. Ermann (2) , M. Edinger (1) , P. Hoffmann (1) (1)University of Regensburg (Regensburg, DE) ; (2)Brigham and Women's Hospital (Boston, US) Natural CD4+CD25+ regulatory T cells (Treg) contribute to tolerance induction after transplantation. We, and others, previously showed that the adoptive transfer of donor-derived Treg cells prevents lethal graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) in mice. In contrast, host-type Treg cells failed to protect when cotransplanted under identical conditions. This raised the question whether MHC compatibility with conventional CD25-CD4+ and CD8+ T cells (Tconv) is a prerequisite for the suppression of alloresponses by Treg cells, or whether elimination of host-type Treg by allo-aggressive donor Tconv cells had occurred. To address this issue, mixed lymphocyte reactions were performed in which CFSE-labelled responder T cells (Tresp), Treg cells and antigen presenting cells (APC) were systematically varied with regard to their MHC haplotype. When BALB/c Thy1.1 (H-2d) Tresp cells were stimulated with mixed BALB/c (H-2d) and C57BL/6 (H-2b) APC, 26.0 ± 3.1% of the CD4+ cells and 86.2 ± 2.2% of the CD8+ cells had gone through at least one cell cycle after 6 d. In the presence of syngeneic BALB/c Treg cells, proliferation of CD4+ and CD8+ Tresp cells was decreased to 9.1 ± 4.7% and 25.1 ± 4.9%, respectively. In contrast, in cultures with allogeneic C57BL/6 Treg cells, proliferation remained at 22.1 ± 1.8% and 89.6 ± 0.4% for CD4+ and CD8+ Tresp cells, respectively. Stimulation with either F1 (C57BL/6 x BALB/c; H-2b/d) or third party (DBA/1; H-2q) APC yielded comparable results. Lack of suppression in co-cultures of MHCmismatched Tresp and Treg cells cannot be explained by an early elimination of allogeneic Treg cells, as they were still detectable after 6 d of allo-stimulation. In corresponding in vivo studies, CB6F1 as well as DBA/1 recipients were protected from lethal GVHD only when both T cell populations were from MHC-identical donors, but not when Tconv and Treg cells were derived from two MHC-disparate strains. In conclusion, these data indicate that MHC-identity between Tconv and Treg cells is required for maximum suppression of an alloresponse and that Treg cells isolated from a third party S123 donor might not be suited for the prevention of GVHD after allogeneic BMT. Treatment of HIV-1 infection by allogeneic CCR5-delta32 stem cell transplantation G. Hütter* (1), D. Nowak (1) , M. Mossner (1), S. Ganepola (1), K. Allers (1) , T. Schneider (1), J. Hofmann (2) , W.K. Hofmann (1) , E. Thiel (1) (1)Charité Campus Benjamin Franklin (Berlin, DE) ; (2)Charité Campus Mitte (Berlin, DE) Background: In the past, first approaches to decelerate the HIV-1 disease by allogeneic stem cell transplantation (allo SCT) failed. Here, we report from a 40-year-old man with HIV-1 infection since 1995 having a relapse of acute myeloid leukemia (AML) first diagnosed in 2006. He underwent allo SCT with a donor selected to be homozygous for CCR5-(d)elta32. Methods: The request at the Bone Marrow Donor Registry revealed 232 HLA-identical donors for this patient. Donors were screened for the deletion using a genomic PCR assay. The patient received peripheral stem cells from donor #61, identified to be homozygous for CCR5-d32. HAART was stopped from day of transplantation. To determine co-receptor usage of the patients` HIV-1 phenotype, the V3 region of the HIV-1 env gene was amplified. The susceptibility of peripheral blood mononuclear cells (PBMC) towards infection was determined by limiting dilution experiments using defined HIV-1 strains. Quantification of HIV-1 infection was measured by measurement of HIV-1 RNA and proviral cDNA using the env and LTR region in peripheral blood, bone marrow and rectal mucosa. Results: With ongoing engraftment, CCR5-PCR patterns of PBMCs transformed into a homozygous delta32 genotype. From rectal biopsies, taken on day +145, macrophages showed an expression of CCR5, whereas a remarkable CCR5-expressing population could not be found on the mucosal CD4+ T-cells. Analysis of HIV-1 co-receptor phenotype indicates a CCR5 usage of our patients` HIV-1 strain. After engraftment PBMCs were not susceptible for CCR5-tropic strains. During the whole follow-up period, measurement of serum HIV-1-RNA remained negative and the semi-quantitative proviral DNA assay was under the limit of detection since day +61. Conclusions: We could demonstrate the first successful performance of an allogeneic stem cell transplantation in an HIV+ patient with a donor selected to be homozygous for the CCR5-d32-allele. The switch of the patients CCR5 genotype was not associated with an increased risk regarding the transplant procedure. Although HAART is discontinued since more than 250 days, HIV-1-load could not be detected in peripheral blood, bone marrow, and rectal mucosa. Our data are highly suggestive that the postulated "gatekeeper" mechanism for HIV-1 infection preferring the CCR5-tropic strain, has been re-initiated during engraftment leading to a disruption of virus replication. This finding provides a possible therapeutic option for HIV-infected patients. The proliferation of infused donor lymphocytes following alemtuzumab-mediated T-cell depleted allogeneic stem cell transplantation can be monitored ex vivo by the expression of CD52 E. Wagner, S. Wenzel, T. Schmitt, U.F. Hartwig, C. Huber, W. Herr, R.G. Meyer Uniklinik Mainz (Mainz, DE) In vitro studies have demonstrated that the CD52-expression in lymphocytes is down-regulated after incubation with the anti CD52-antibody alemtuzumab and remains low in emerging lymphocytes after proliferation. The impact of in vivo applied alemtuzumab on the CD52-expression of reconstituting T cells after allogeneic hematopoietic stem cell transplantation (HSCT) has not yet been investigated. We have recently introduced a protocol combining a fludarabine/melphalan conditioning with T cell depletion by alemtuzumab (100mg) in allogeneic HSCT. In the absence of acute GVHD, prophylactic CD8 depleted donor lymphocyte infusions (DLI) were administered in escalating doses, starting with 1x10 6 CD3/CD4-positive cells/kg bodyweight after day +60. In the peripheral blood of 19 patients undergoing this protocol, the numbers of CD4 and CD8 T-cells as well as their expression of CD52 were monitored by flow-cytometry. Nine out of the 19 patients received CD8-depleted DLI and their application increased the absolute T-cell numbers (CD4>CD8) in all of these patients. However, since the clinical situation with regard to inflammation (infection and GVHD) and the need for immunosuppressive medication varied inter-individually, it was impossible to differentiate the effect of the DLI from that of other clinical events. Interestingly, the expression of CD52 on reconstituting T cells was markedly decreased in all 19 patients. This was independent of both donor T cell chimerism and the presence of GVHD, indicating a down-regulation of CD52 on proliferating donor and host T cells. Only in those patients who received CD8-depleted DLI, the percentage of CD52-expressing CD4 T cells increased after the application. The mean percentage of CD52-positive cells was significantly higher in the DLI group compared to the non-DLI group after day +170. In the latter cohort, the proportion of CD52expressing cells as well as the level of CD52-expression remained low throughout the first year after transplantation. This significant difference was not seen for CD8 T cells, were only a trend towards an increased number of CD52-positive cells was demonstrated in the DLI group. We provide evidence that the expression of CD52 is down regulated in reconstituting T cells following HSCT with an alemtuzumabbased conditioning regimen and that by the analysis of CD52 expression, a proliferation of DLI-derived CD4 T cells in vivo can be demonstrated. Kinetics of Langerhans cell chimerism in the skin of dogs following 2Gy TBI allogeneic haematopoietic stem cell transplantation S. Mueller*, S. Lange, S. Altmann, V. Weirich, H. Vogel, M. Freund, C. F. Junghanss Universität Rostock (Rostock, DE) Objectives: Langerhans cells (LC) are bone marrow (BM)derived dendritic cells in the epidermis that play a central role in skin immunity. It is assumed that kinetics of donor/recipient chimerism of the LC after hematopoietic stem cell transplantation (HSCT) influence the incidence and severity of graft-versus-host response. In nonmyeloablative (NM) HSCT the onset of acute graft-versus-host disease (aGVHD) is delayed compared with myeloablative conditioning regimen and signs and symptoms of aGVHD also arise beyond day 100. However, data regarding LC kinetics after NM-HSCT are rare. In this study we examined the development of LC chimerism in a canine HSCT model. Methods: Dogs (n=8) were conditioned with 2Gy of total body irradiation and received BM from dog leukocyte antigenidentical littermates at a median of 3.2 (1.9-6.2) total nucleated cells/kg. Immunosuppression consisted of 15mg/kg cyclosporin A (CSA) BID PO (days -1 to +35) and 0.25mg everolimus (RAD) BID PO (days 0 to +27). Skin biopsies of 8mm² diameters were obtained before and on various days after transplantation (d+28, d+56, d+105). LC were isolated using immunomagnetic separation and chimerism was measured with PCR for Variable-Short-Tandem-Repeat markers. Results: So far results of 4 dogs are evaluated. All dogs engrafted. One dog rejected its graft by day 70 and 3 animals remained mixed chimeras (weeks 31, 43, 45) . In none of the dogs LC donor chimerism could be detected at day +28, although donor chimerism in the granulocyte (Gran) and peripheral blood mononuclear cell (PBMC) compartments was median 54% (41-82%) and 26% (18-58%), respectively. By day +56 donor chimerism was 37 and 50% in the Gran compartment and 67 and 64% in the PBMC compartment in two animals that later developed full donor hematopoietic chimerism (d+91, d+77). LC donor chimerism at d+56 was 6 and 42% and at d+105 16 and 88%, respectively. In another dog that showed 10% Gran and 41% PBMC chimerism at d+112, LC chimerism was 0% at d+56 and 12% at d+112. In the fourth dog that rejected its graft donor LC were not detectable at any time. Conclusion: Our study indicate that LC chimerism kinetics are delayed following NM-HSCT compared to chimerism development in the peripheral blood. In comparison with published data on LC chimerism kinetics following myeloablative or reduced intensity conditioning, the 2Gy TBI regimen seems to results in the slowest LC chimerism development. Quantitative assessment of unrelated umbilical cord blood engraftment by real-time PCR D. Bories* (1), V. Beziat (2) , S. Lapusan (3), S. Nguyen (2), J.P. Marie (3) , V. Vieillard (2) , N. Dhedin (2) , B. Rio (3) ( Sequential analysis of chimerism was performed to monitor engraftment in 32 patients who received unrelated umbilical cord blood transplantation between November 2004 and October 2007. Patients: 9 females and 23 males, age 19 to 66, were transplanted for AML (18 cases), ALL (3 cases), NHL (3 cases), myeloma (3 cases), aplastic anaemia (2 cases), mylelofibrosis (1 case), HL (1 case). They all received 1 transplantation except for 5 who received 2 transplantations and for 1 who received 3 transplantations. They all received 1 CBU except for 8 who received 2 cord blood units. Nb of injected CD34+ cells ranged from 0.05 to 0.18/kg. Nb of injected CD3+ cells ranged from 0.06 to 0.89/kg. Conditioning regimen was reduced in all cases but 3. Chimerism analyses were performed using quantitative PCR of informative donor and host specific polymorphic sequences as published by Alizadeh et al (Blood 2002) . We used a LightCycler (Roche) and hydrolysis probes. In order to find an informative marker, host and donors DNAs were tested with a panel of 32 primers and probes. The task was rendered more difficult by the fact that some patients received several transplantations with several cord blood units (up to five possible different haematopoiesis origins in one person). Quantification was performed using the DeltaDeltaCT method with albumin as a reference gene. The sensitivity of the technique was 0.1% as previously reported. Cell separation was performed in certain cases to evaluate the CD3+ fraction origin after transplantation. Follow up has been from 4 to 36 months. After 1 month post transplantation: 18 patients haematopoiesis was more than 95% of donor origin, 1 was 74%, 1 was 67%, 1 was 59%, 1 was 22% and 1 had no detectable cells of donor origin. After 2 months, the one with 59 increased to 93, and is now around 72% at M6. The one with 22% increased up to 99% (M6), the one with 74% increased to 99 at M2. The one with 67% decreased to 11%, by M2. In cases of multiple (2) cord blood units which represented 8 of the studied transplantations, the haematopoiesis was rapidly of only one cord blood origin, the other cord blood hematopoiesis becoming rapidly undetectable. In conclusion, in the highly complex setting of unique or multiple cord bloob transplantation after reduced intensity conditioning regimen, quantitative real time PCR was well adapted to post transplantation monitoring of engraftment. Among 32 patients, only two have rejected to date. Clofarabine pre-conditioning prior to full intensity or reduced toxicity allogeneic stem cell transplantation: a promising new regimen for the treatment of high-risk acute myeloid leukaemia D.S. Richardson, K. Hill, C. Hurlock, J. Newman, N. McKeag, L. Groves, K.H. Orchard Southampton University Hospitals (Southampton, UK) The outcome for patients with high-risk AML, particularly those with leukaemia present at the time of transplant, remains poor even using full intensity conditioning schedules. A recently published approach is to use pre-conditioning chemotherapy prior to the delivery of a reduced intensity (RIC) transplant during the following period of cytopenia. Initial results indicate feasibility in poor risk disease. The bi-halogen purine analogue Clofarabine has significant activity as a single agent for the treatment of AML and is well tolerated, characteristics suggesting Clofarabine is an ideal agent to use in pre-conditioning. We have treated 5 patients with high-risk, refractory AML using Clofarabine pre-conditioning to reduce disease burden before full intensity or RIC allogeneic transplantation. In 4 of 5 patients, Clofarabine was administered as a single agent (40mg/m2/day for 5 days) and in 1 in combination with Cytarabine. 2 received RIC Busulphan-containing transplant schedules; 3 full intensity total body irradiation (TBI) based conditioning. Indications for using Clofarabine included severe allergy to Cytarabine and heavy prior exposure to anthracycline. All 5 patients had marrow involvement with AML prior to administration of Clofarabine. In this cohort we have not seen additional unexpected complications. 1 patient who received a RIC regimen, was treated for VOD on clinical suspicion but liver biopsy indicated grade 3-4 haemosiderosis only. 2 patients who received TBI-based transplants suffered grade 3-4 mucositis. The extended period of pancytopenia compared with conventional transplant schedules was manageable. All patients achieved >98% donor chimerism by day +30. 1 patient experienced grade 3 cutaneous acute GvHD which responded to additional immunosuppression. 4 of 5 patients achieved substantial bulk reduction or eradication of marrow leukaemic blasts following Clofarabine. The patient with Clofarabine-refractory disease achieved CR at day 30 following a TBI-based transplant but relapsed at 4 months and died at 5 months post transplant. One patient died in CR at 5 months from pneumonitis. 3 patients are currently in CR at 5-23 months follow up. The use of Clofarabine as a pre-conditioning agent prior to the administration of the transplant conditioning schedule is well tolerated with both full and reduced intensity protocols. This approach to allogeneic transplantation shows promise for the therapy of patients with high risk, refractory AML. Pre-clinical analysis of treosulfan in combination with total body irradiation as conditioning regimen prior to bone marrow transplantation in rats V. Sender* (1), K. Sievert (2) , N. Hofmeister-Mielke (1) (6) medac GmbH (Wedel, DE) Treosulfan (treo) is a derivative of busulfan with lower side effects compared to busulfan. The preclinical study was designed to determine the maximum tolerable dose of treo in combination with total body irradiation (TBI) prior to bone marrow transplantation (BMT) in rats. Methods: Female Lewis rats were treated with treo on 3 consecutive days followed by TBI with either 5Gy (n=8) or 7.5Gy (n=28) on day -1. Treo was administered i.p. with a dose of 0.25g/kg/day (n=4), 0.5g/kg/day (n=16), 0.6g/kg/day (n=4), 0.75g/kg/day (n=8) and 1g/kg/day (n=4) either on days -4,-3, -2 (n=16) or on days -5,-4,-3 (n=20). One day after TBI animals received 4x10E7 bone marrow cells (BM) from female Lewis rats. To confirm the maximum tolerated dose as well as immunosuppressive capacity additional 16 rats were transplanted with 4x10E7 BM and 1.5x10 7 spleen T-cells from female Brown-Norway (BN) rats one day after TBI with either 5Gy + 0.6g/kg/day treo (n=8) or 7.5Gy + 0.5g/kg/day treo (n=8) given on days -5,-4 and -3. Subsequently, all animals were examined daily for signs of toxicity. In addition toxicity was confirmed by autopsy and histology in all animals. Results: All animals receiving treo with a dose of 0.75g/kg/day and 1g/kg/day on days -4,-3,-2 followed by 7.5Gy TBI on day -1 died due to dominating gastrointestinal toxicity whereas 0.5g/kg/day treo resulted in survival in 1 out of 4 rats and 4 out of 4 animals survived after 0.25g/kg/day treo. In contrast all rats receiving 0.5 g/kg/day treo on days -5,-4 and -3 in combination with 7.5 Gy TBI on day -1 survived and 2 out of 4 animals receiving 0.6g/kg/day treo survived. The combination of 5Gy TBI given on day -1 and 0.75g/kg/day treo given on days -5,-4,-3 resulted in survival of 2 out of 4 rats whereas the same radiation dose combined with 3x0.5g/kg treo resulted in survival of 3 out of 4 animals. Allogeneic BMT from BN donors resulted in engraftment and survival in 7 of 8 animals after conditioning with 7.5Gy TBI and 3x0.5g/kg treo whereas 6 of 8 animals engrafted and survived after 5Gy TBI and 3x0.6g/kg treo. Conclusion: With gastrointestinal toxicity as the dose-limiting factor, the highest tolerable dose of treo in combination with 7.5Gy TBI is 3x0.5g/kg and the highest tolerable dose of treo in combination with 5Gy TBI is 3x0.6g/kg. Furthermore treo has features of a radiosensitizer since animals receiving TBI less than 36h after the last treo injection showed increased signs of enteric toxicity. The use of a 2 completely mismatched donors in murine bone marrow transplantation (multidonor transplantation) does not impede engraftment, significantly reduces graftversus-host disease and increases graft-versus-tumour effect E. Hirshfeld, Z. Yehtina, L. Weiss, M.Y. Shapira* Hadassah -Hebrew University Medical Center (Jerusalem, IL) Introduction: It has previously reported that the addition of a third party to murine stem cell transplantation improves engraftment. However, this was studied in a T cell depleted model. We have evaluated the effect of using 2 completely mismatched donors (multidonor transplantation -MDT) on graft-versus-host disease (GVHD) and graft-versus-tumour (GVT). Methods: BALB/c served as recipients and C57/BL and C3H were the donors. The recipients received nonmyeloablative TBI followed by MDT or by selective sensitization of anti donor-reactive host cells followed by MDT. These were compared to controls receiving the same conditioning followed by transplant from a single donor (either C57BL or C3H). Results: Hematological reconstitution was achieved in all animals and engraftment was stable. As expected, GVHD appeared in most of the animals transplanted from C57/BL (11/12 animals). However, MDT with both C57BL/6 and C3H, protected against GVHD with only 5/12 animals suffering from GVHD (figure 1A, p=0.02). Additionally, GVHD related weight loss was significantly higher in animal transplanted from C57/BL (figure 1B). MDT provided the strongest GVT effect following inoculation of BALB/c mice with murine breast cancer as compared to C3H and C57/BL alone (figure 3A and 3B respectively). Conclusions: MDT is feasible and safe. It is furthermore shown that MDT may reduce transplant toxicity (i.e. GVHD), increase GVT and accordingly may improve transplant success. The induction of bi-parental mixed chimerism in a murine model does not increase graft-versus-host disease despite T-cell repletion I. Shiber, E. Hirshfeld, L. Weiss, J. Kasir, M.Y. Shapira* Hadassah -Hebrew University Medical Center (Jerusalem, IL) Introduction: The paucity of stem cells in cord blood graft, which is a cause for delayed engraftment and increased rate of graft failure, initiated an interest in transplanting 2 or more cord blood units simultaneously. This approach results with shorter time to engraftment and perhaps improved graft vs. leukemia (GVL) effect. However, there is preliminary data that GVHD may be increased due to the combination of 2 mismatched grafts. We have established a murine model of combined bi-parental bone marrow transplantation without T cell depletion. Methods: (BALB/c x C57BL/6)F1 (F1) served as recipients while C57BL/6 and BALB/c were the donors. Animals were conditioned using total body irradiation in a myeloablative dose (9Gy) and non myeloablative (7 or 5 Gy) in a single fraction on day -1 and then received T cell repleted bone marrow from either F1, C57BL/6, BALB/c or both C57BL/6 and BALB/c simultaneously (MDT -multi donor transplantation). Animals were followed for the hematological reconstitution, development of clinical signs of GVHD including hunched back, diarrhea and weight loss, GVHD related mortality and donor(s)-recipient chimerism. Results: results were similar regardless of conditioning used. For example: despite bi-parental chimerism, in F1 animals conditioned with 9 Gy, the time to the clinical appearance of GVHD was not different in MDT as compared to the control groups (C57BL/6 or BALB/c; p= 0.29, figure 1A ). GVHD associated weight loss was again indifferent in the MDT animals (figure 1B). GVHD related mortality in the MDT group was similar to the allogeneic single donor control groups and was also similar to the syngeneic group mortality (2/10, 1/12, 1/9 and 1/12 respectively in 2 separate experiments, figure 2, p=0.68). Conclusion: we conclude that combined simultaneous biparental bone marrow transplantation without T cell depletion does not increase GVHD following either myeloablative or non myeloablative conditioning despite bi-parental chimerism. Background: Haplo-identical hematopoietic stem cell transplantation (haplo HSCT) is increasingly being employed when matched donors are unavailable. The profound in vitro T cell depletion necessary for successful haplo HSCT leads to an impaired immune reconstitution resulting in a high incidence of infection and disease relapse. To improve immune reconstitution, prophylactic or therapeutic donor lymphocyte infusions (haplo DLI) may be effective. However, in order to evaluate their potential role in haplo HSCT, questions about safety, efficacy, dose, and timing need to be addressed. Patients and methods: In a small, oligocentric, retrospective survey of the German Collaborative Transplant Group, we attempted to evaluate safety and efficacy of unmodified haplo DLI. 7 female and 7 male patients aged 17 to 51 (median: 31) years with advanced hematologic diseases (9 AML, 1 ALL, 2 CML, 1 NHL, 1 myeloma) had received prophylactic (n = 6) or therapeutic (n = 8) haplo DLI. In the prophylactic group, 1 or 2 haplo DLI were administered during the first 1 to 3 (median: 2) months after haplo HSCT, with careful dosing (2.5 x 10 4 CD3/kg) or with suicide gene transduced donor lymphocytes. In the therapeutic group, interval from haplo HSCT to the first of 1 to 7 (median: 3) haplo DLI was at a median of 8 (1 to 24) months, reflecting relapse kinetics; haplo DLI doses ranged from 1 x 10 4 to 5 x 10 7 CD3/kg. Results: Paradoxically, in the prophylactic haplo DLI group, safety (the main concern) was not completely satisfactory with grade 2 to 4 graft-versus-host disease (GvHD) in 2/6 patients, but efficacy was excellent with long-term leukemia-free survival in 5/6 patients. Conversely, in the therapeutic group, administration of even high haplo DLI doses was safe with only one grade 2 skin GvHD. Achievement of a complete response was significantly associated with the use of chemotherapy or gemtuzumab ozogamicin prior to haplo DLI (p = 0.018, odds ratio 770). However, all patients (except one rescued by second transplantation) eventually succumbed to malignant relapse, questioning the long-term efficacy of this approach. Conclusions: Unmodified haplo DLI were effective, but not completely safe in the prophylactic setting, whereas in the therapeutic situation, they appeared safe, but not effective. Further studies might examine the combination of re-induction therapy and even higher doses (beyond 10 7 CD3/kg) of therapeutic haplo DLI. Factors that influence early and late haemopoietic reconstitution after autotransplantation with peripheral blood stem cells L. Falchi (1) Factors affecting the time of early (≥1000 granulocytes/µl and ≥ 20000 PLT/µl on three consecutive tests) or late (≥4000 granulocytes/µl, ≥ 120000 PLT /µl and ≥ 12 g/µl on three consecutive tests ) hematological recovery after myeloablative therapy and peripheral blood stem cells (PBSC) support were evaluated in 187 pts (98/89 M/F, 47, 12-70 yr old) suffering from multiple myeloma (MM, 69 pts), clinical indolent or aggressive non-Hodgkin lymphoma (I-NHL, 17 pts; A-NHL, 70 pts) or classical Hodgkin lymphoma (HL, 31 pts). The factors possibly affecting haematological recovery were divided into host-related (age, disease histology and stage), therapeutic history-related that is the time when autotransplant was performed (during first line or as salvage therapy, number of chemotherapy cycles and antitumor drugs used and finally exposure to alkylating agents or radiotherapy), myeloablative scheme-related (type of combination chemotherapy and type and dose of hemopoietic growth factor employed) and number PBSC inoculum-related. The myeloablative treatments were BEAM (BCNU 300mg/m², ARA-C 400mg/m², VP-16 200mg/m², L-PAM 140mg/m²) L-PAM (60 mg/m²)+ Mitho (mithoxantrone, 8 mg/m²), L-PAM (60-140 mg/m²)+Bu (busulfan, 16 mg/kg), L-PAM (200 mg/m²),L-PAM (60-100 mg/m²) or miniBEAM (BCNU 200mg/m², ARA-C 400mg/m², VP-16 200mg/m², L-PAM 140mg/m²). The median number/Kg of unfractioned mononuclear cells (MNC) CFU-GM and CD34+ cells reinfused was 302x10 6 , 42,3x10 4 , 5,42x10 6 , respectively. Linear regression analysis indicated that the time of early recovery of granulocytes was significantly affected by the histological type of disease (increasing time from MM to I-NHL) and the type of myeloablative therapy employed (increasing time from L-PAM 100mg/m² to BEAM), while that of thrombocytes was significantly influenced only by the number of MNC reinfused. In contrast, the time of late recovery of thrombocytes (≥ 120000µl) was negatively influenced by previous exposure to mechlorethamine. No further other elements were found significantly affecting the early or late haematological recovery. Thus, the optimal amount of PBSC reinfused overcomes the influence of all other factors which may exert any effect on haematological recovery. Donor lymphocyte transfusion (DLT) has been shown to control leukemia in patients after allogeneic stem cell transplantation. Graft-versus-host disease (GVHD) represents the major risk factor of DLT. In DLA-identical littermate dogs DLT converted mixed into complete chimerism without producing GVHD, if DLT was delayed for 2 months and more after transplantation. Here we studied GVHD and chimerism in DLA-haploidentical recipients after CD6-depleted marrow transplantation. Marrow recipients were DLA-heterozygous and DLA-haploidentical to their DLA-homozygous donors. T cells were depleted from the marrow transplant by treatment with CD6 antibody (MT-606) and rabbit complement. DLTs were administered on days 3, 7, 14 and 20. Chimerism was studied by cytogenetics in marrow and blood on days 20, 50, 100 and yearly thereafter. GVHD was diagnosed by clinical signs and histology. CD6 antibody used with rabbit complement depleted T cells effectively without affecting hematopoietic progenitor cells. In the control group 7 dogs given unmanipulated marrow died of GVHD within 28 days of transplantation. After T cell depletion with CD6 antibody two dogs had sustained engraftment without occurrence of GvHD. The establishment of tolerance to the bone marrow donor was demonstrated by the survival of a skin transplant from the donor to the host. Transfusion of donor lymphocytes (1.0 x 10 8 MNC/kg body weight) on days 3, 7 or 14 produced severe GvHD in the host. However, after DLT on day 20, only 2 out of 4 dogs developed GvHD. Nevertheless mixed chimerism was converted into complete chimerism. We conclude from this study that CD6 antibody MT-606 effectively depletes T cells in the presence of rabbit complement and prevents GVHD without jeopardizing engraftment. DLT early after transplantation abrogates tolerance, whereas tolerance is not abrogated by DLT at a later time in all animals. Evaluation of safety and efficacy of POL6326 for mobilisation of haematopoietic progenitor cells C. Ludin* (1), S. DeMarco (1), J. Chiesa (2) , K. Kornelissen (2) , A. Brooks (2) , L. Heyes (2) (1)Polyphor AG (Allschwil, CH); (2) Covance (Leeds, UK) The trafficking and homing of human CD34(+) haematopoietic stem and precursor cells to the bone marrow compartment is largely dependent on stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4. Antagonists of the SDF-1/CXCR4 axis can mobilise CD34(+) cells to the peripheral blood. POL6326, a cyclic peptide antagonist of CXCR4, selectively competes for SDF-1 binding to the receptor in radioligand, calcium mobilisation and chemotactic assays. The compound POL6326 mobilizes haematopoietic precursors to the peripheral blood in mice. The intended use of POL6326 is stem cell mobilization and collection of haematopoietic stem cells (HSC) for subsequent clinical transplantation. We are currently investigating the safety, tolerability, pharmacodynamic and pharmacokinetic data of ascending intravenous infusion doses of POL6326 in a single blind, randomised, placebo controlled Phase I study. In addition we want to define the potential time window for apheresis allowing the optimal collection of haematopoietic stem cells. In the current single-blind dose escalation study, comprising of 6 subjects /cohort (4 active: 2 placebo), each subject receives a single dose (10, 20, 40, 80, 160 µg/kg POL6326 or placebo) over a 3 h infusion time. A rapid increase in peripheral blood CD34(+) cells was observed, with a clear dose dependent effect. After a 3 h infusion of 160 µg/kg, up to a 9 fold increase in peripheral blood CD34(+) cells was observed 5 hours after the infusion commenced. No adverse events of note have been reported, indicating that POL6326 is safe and well tolerated up to a dose of 160 µg/kg during 3 hour slow infusion. Continued dose escalation studies at higher doses and shorter infusion times are planned. Introduction: The most commonly used therapeutic targets in nephrology are the reduction of injury, the delay of progression, or renal replacement therapy. Many animal and human studies demonstrated the role of stem cells in repair and regenerations of kidney. Mesenchymal stem cells (MSCs) have shown to improve outcome of acute renal injury models. It is controversial whether MSCs can reduce injury following a toxic/ ischemic event and delay renal failure in chronic kidney disease. We evaluated the hypothesis that the treatment with MSCs could improve renal function and attenuate injury in chronic renal failure (CRF). Materials and methods: Sprague-Dawley female rats (8 weeks old, 182.2 ± 7.2g) were underwent modified 5/6 nephrectomy. Rats in the MSC group received an injection of MSCs (1 x 10 6 cells) via tail vein 1 day after nephrectomy. Blood and urine samples were collected after 7 days and every month thereafter. The kidneys of rats were removed for histologic evaluation after 24-hr urine collection and blood sampling. The Y-chromosome stain using fluorescent in situ hybridization was performed to verify the presence of male MSCs in the kidney of female recipients. Results: No significant differences in blood urea nitrogen and creatinine concentration were observed between MSC group and untreated CRF group. However, the weight gain and creatinine clearance in the MSC group were greater than those of the CRF group. Proteinuria in the MSC group were less after 4 months. Y chromosome was detected in the kidney of MSC group. Although no significances were observed between two groups, the histologic analysis suggest that MSCs have positive effect against glomerulosclerosis. Conclusions: These results suggest that MSCs help preserve renal function and attenuate renal injury in CRF. Further studies are required to determine when MSC treatment should be initiated and what amount of MSCs is most effective. Human platelet-derived factors regulate gene expression profile and function of human multipotent mesenchymal stromal cells A. Reinisch*, C. Bartmann, K. Schallmoser, E. Rohde, G. Lanzer, W. Linkesch, D. Strunk Medical University of Graz (Graz, AT) Objectives: The use of animal-derived products during human stem cell (SC) processing bears the risk of xenogeneic immunization and prion, virus, or zoonose infection. Pooled human platelet lysate (pHPL) has recently been recognized as a rich and stable source of cytokines and growth factors with the potential to replace fetal bovine serum (FBS) during ex vivo SC manipulation. Umbilical cord blood (UCB) is an alternative source for multipotent mesenchymal stromal cells (MSC) and is believed to provide MSC with a higher proliferative potential compared to adult bone marrow (BM). Limitations in cell number and strict dependence of expansion procedures from selected lots of (FBS) have hampered the progress of clinical applications with UCB-derived MSC. Methods: In this study we investigated gene expression profile and function of human MSCs during clinical scale ex vivo expansion under the aegis of either FBS or pHPL. The AB1700 Expression Array System was used for full genome profiling of MSCs in an optimized clonal density expansion system. Data have been obtained from biological as well as technical replicates. Attribution of regulated genes to biological processes and pathways was done using the PANTHER® db analysis software. MSC function was tested in hematopoiesis regulation, vascular-like network formation and immune modulation potency assays. Results: MSCs could be propagated in clinical quantity with pHPL-as well as FBS-supplemented medium from BM and UCB. We identified 45 genes that are significantly regulated (p<0.01; >2 fold up) upon culture of MSCs in pHPL compared to FBS-driven expansion. Biological processes specifically activated by pHPL included mesoderm development, hematopoiesis and immunological processes correspond to a considerable proportion of the regenerative potential of MSCs. Functional data indicate intact hematopoiesis regulation, immune modulation and vascular-like network formation by MSCs propagated in pHPL-supplemented medium. Conclusion: Replacing FBS with pHPL definitively avoids xenoimmunization and bovine prion, viral and zoonose contamination of MSC for clinical use. New insights into the tightly regulated gene expression under the aegis of human growth factors and cytokines may further improve our understanding of stromal cell biology in a native environment provided by pHPL and may help to develop new therapeutic strategies. The effect of bone marrow-derived mesenchymal stromal cells on the ex vivo expansion of cord blood haematopoietic stem cells I. Pelagiadis, H. Dimitriou, E. Stiakaki, C. Perdikogianni, E. Hatzidaki, M. Kalmanti* University of Crete (Heraklion, GR) A major limitation of umbilical cord blood (UCB) hematopoietic stem cells (HSC) for allogeneic transplantation is the insufficient number of cells available in the graft especially for administration in adult patients. In order to overcome the restriction, ex vivo expansion of HSC has been considered. Mesenchymal stromal cells (MSC) constitute an essential element of normal bone marrow (BM) microenvironment, participating in the regulation of hematopoiesis. The aim of this study was the evaluation of the interaction between UCB CD34+ cells and MSC used as feeder layer and the assessment of the effect of different growth factor (GF) combinations on the expansion of HSC. CD34+ HSC from the UCB (n=10) of full term parturitions and MSC isolated and expanded from BM of children with Autoimmune Idiopathic Neutropenia were cocultured under different conditions in the presence or not of two GF combinations (GFmix1: SCF 10ng/ml, IL-3 5ng/ml, G-CSF 100ng/ml, GM-CSF 1ng/ml, GFmix2: SCF 20ng/ml, TPO 25ng/ml, IL-6 20ng/ml, Flt-3 ligand 50ng/ml). After 10 days of liquid culture, cells were collected, immunophenotypically characterized, CFU-GM, BFU-E development was assessed and total cell as well as CD34+ expansion were evaluated. CD34+ UCB cells cultured in the presence of both MSC and either GF combination (conditions B/C) resulted in a higher expansion of both total cell number and CD34+ cells. There was no difference between the two GF combinations in any of the parameters examined. The CD34+ cultured only on MSCs feeder layer (condition A) yielded a higher percentage of CD34+, CD34+CD38-and CD34+CD33-subpopulations. Additionally under these conditions the development of CFU-GM and BFU-E colonies was enhanced. BM-derived MSC feeder layer supports the ex vivo expansion and preserves the 'stemness' of CD34+ HSC in co-culture conditions, providing a population to overcome problems caused by limited HSC number for CB transplants. Mesenchymal stem cells (MSCs) are largely studied for their potential clinical use in patients with ischemic heart disease, tissue injury; graft vs host disease and autoimmune diseases. The umbilical cord (UC) has been shown to contain MSCs that can be easily isolated from Wharton's jelly and cultured. The aim of this study is to determine whether cryopreservation of UC derived MSCs (UC-MSCs) influenced their phenotypic and functional characteristics as well as proliferation and differentiation capacity. Blood vessels were removed from Wharton's jelly, which had been digested using collagenase, hyaluronidase and trypsin. The cultures were maintained at 37ºC in a 5% CO2 atmosphere and non-adherent cells were removed after 24 hours. To assess the presence of primitive cells, colony-forming unit fibroblast (CFU-F) assays had been performed and the immunophenotype of MSCs was verified with 3-colour flow cytometry. Gene expression was examined with reverse-transcriptase polymerase chain reaction (RT-PCR). To induce osteogenic, chondrogenic and adipogenic differentiation cells were cultured into the appropriate media. On isolation cells were counted and separated into 4 equal parts: ¾ of the cells had been frozen and stored at -80°C and ¼ put into culture. Upon reaching confluence half of the cultured cells were frozen (split into 2 equal parts) and the rest of the cells were used for controls. Frozen cultured cells were thawed after 1 week and put into culture. In these cultures expansion of MSCs was observed. Cells frozen on isolation were thawed after 1 week and put into culture. Cells that were cultured immediately after isolation and those frozen and thawed after having been cultivated had similar proliferation and differentiation capacity and immunophenotype. In the contrary, cells that were cryopreserved immediately after isolation were unable to proliferate and their culture was terminated one month later. The proliferation, differentiation capacity as well as the expression of MSC characteristic markers was identical in cultivated MSCs derived from freshly isolated UC-MNCs and in MSCs derived after freezing and storage of ex vivo cultivated MSCs. This highlights the potential clinical use of MSCs in patients with cardiac and degenerative diseases, as it would be possible to inject MSCs obtained from the same UC aspiration at different time points. Aging mechanisms in neonatale USSC and adult MSC M. Aktas, A. Houben, R. Siaj, P. Wernet, G. Kögler University of Dusseldorf Medical School (Dusseldorf, DE) Stem cells are interesting subjects for studies in the context of aging since they are known to have higher replicative capacities than usual somatic cells. Our group showed that neonatal unrestricted somatic stem cells (USSCs) could be expanded for up to 20 passages (P20) equivalent to 46 population doublings (PD) [Koegler et al. in J Exp Med. 2004; Exp Hematol. 2005; Exp Hematol. 2006; Cytotherapy. 2007 ]. In the analysis here different aspects of cellular aging in cord blood-derived USSC to adult mesenchymal stromal cells (MSC) from bone marrow are compared. For this purpose, USSC and MSC lines were held in long-term cultures to determine PD. To measure the proliferation status a BrdU labeling and detection kit (Roche) was used for quantifying DNA synthesis during cell proliferation. A senescence-associated b-galactosidase (SA-b-Gal) assay was also performed that is only expressed in senescent cells which entered growth arrest. Furthermore, the telomere length (TL) was checked by Southern analysis of terminal restriction fragments as well as telomerase activities by a sensitive photometric enzyme immunoassay based on the Telomeric Repeat Amplification Protocol (TRAP; both Roche). To assess hTERT mRNA, coding for the catalytic subunit of telomerase, we performed RT-PCR with primers capturing all splicing variants including full length transcript. In the experiments here, USSC were expanded up to passage 14 (P14) with a max. PD of 23. The replicative lifespan of MSC was much shorter, already terminating in P11 correlating with 16 PD. The better growth kinetic of USSC was also reflected in shorter PD time (1 doubling every 3,32±0,88 days) when compared to MSC (1 doubling every 18,95±10,73 days). In both cell types, loss of TL during cultivation occurred (USSC 130±46bp and MSC 172bp per PD), whereas TL of USSC were in general longer (7, (8) (9) (10) (11) (12) (13) 9kbp) . In USSC as well as in MSC long-term cultivation led to decelerated proliferation. Senescence was already detectable in low passages of MSC (e.g. 79% SA-b-Gal-positive cells in P6) but only detectable in high passages of USSC (e.g. 58% in P11). No telomerase activity or hTERT mRNA was found in any passage, neither in USSC nor in MSC. The data presented here shows that USSC is a much younger and easier expandable stem cell type than MSC. D. Josefsen*, A. Kuechler, M. Wang, G. Haraldsen, G. Kvalheim Rikshospitalet Medical Centre (Oslo, NO) The clinical results of using bone marrow-derived progenitor cells to repair ischemic tissue in coronary disease are controversial since some studies demonstrate improvement of cardiac function, whereas others do not. In these studies both unmanipulated mononuclear bone marrow (MNC) cells and highly enriched stem cells such as CD34+, CD133+ or mesenchymal cells (MSC) were used. In this study we have examined the in vitro and in vivo endothelial progenitor properties of these populations. MNC were obtained from whole BM. CD34+ cells and CD133+ cells were isolated using magnetic bead technology (MACS, Miltenyi). MSC was established from BM, and only second passages from MSC were used. Cytokine profiles of MSC were measured on a Bio-Plex system (BIO-RAD Laboratories). In vitro studies were performed by culturing cells in either complete EGM-2 medium to identify EPC, or in M199 with endothelial growth factors to investigate endothelial differentiation. We observed little or no endothelial differentiation when culturing CD133+ or CD34+ cells in M199 media. Furthermore, we did not observe EPC colony formation of MNC, CD133+ cells, or CD34+ cells cultured in EGM-2 medium. To study the in vivo effect of MNC, CD34+, CD133+ as well as MSC, we adoptively transferred human cells into immunodeficient mice and examined their endothelial potential assessing the formation of blood vessels as readout (Skovseth et al Am J of Pathol 2002 , Blood 2005 . Human umbilical cord-derived endothelial cells (HUVEC) served as positive controls. MNC only grew as round shaped clustered cells with no signs of vessel formation. Neither CD34+ cells nor CD133+ cells demonstrated signs of endothelial development in vivo. MSCs were found to ex vivo secrete endothelial growth factors, including VEGF, bFGF and EGF. When Matrigel plugs with MSCs were examined, an accumulation of smooth muscle actin-positive cells could be detected but no vessel like endothelial structures derived from human cells could be found. If these putative smooth muscle cells are differentiated from the MSCs, or are murine host cells attracted to the human MSCs due to the cytokine secretion effects of the MSCs is not clear. In the current experiments we did not observe endothelial cell differentiation or vessel formation from BM-MNC, CD34+ cells or CD133+ cells suggesting that such cells have a limited vasculogenic effect. Our findings with the MSCs are interesting and the mechanism of actions is under further investigation. Serum matrix metalloproteinase 9 levels are not predictive for complications after stem cell transplantation B. Kircher*, P. Schumacher, J. Clausen, D. Nachbaur Immunobiology & Stem Cell Laboratory (Innsbruck, AT) The matrix metalloproteinases (MMP) represent a family of more than 15 zinc-depending endopeptidases with extracellular activity. MMPs are involved in various processes such as tissue remodeling, tumor invasion and immune regulation mainly due to activation of cytokines. Therefore, MMPs may play a role in stem cell transplantation (SCT) as well. Indeed, MMP-2 is essential for the invasive capacity of human mesenchymal stem cells. MMP-1 and MMP-19 are expressed in intestinal graft-versus-host disease (GvHD) lesions, and the synthetic MMP inhibitor KB-R7785 prevents acute GvHD in mice undergoing bone marrow transplantation. Furthermore, a significant decrease of MMP-9 serum and mRNA levels was observed up to three years after autologous SCT for multiple sclerosis. Data on MMP-9 serum levels after allogeneic SCT, however, are lacking. Aim of this study was the evaluation of a possible correlation between in vivo MMP-9 serum levels and the occurrence of complications after SCT. Therefore, MMP-9 levels were determined by commercially available enzyme-linked immunoabsorbant assays in sera of 48 patients at various time points before and up to day +90 after allogeneic, HLAidentical SCT. The median MMP-9 serum levels of the 48 patients were lower (257,8 ± 31,6 ng/ml) than those of the two normal controls (1476,9 ± 42,9 ng/ml) and varied during transplant procedure. The median MMP-9 levels at day -4/-5 (251,3 ± 40,2 ng/ml) dropped to 1,2 ± 2,0 ng/ml on day +7/+8 and rose from day +10/+12 (2,6 ± 18,9 ng/ml) to 262,4 ± 29,4 ng/ml on day +90. A similar fall and rise was observed for leukocyte counts. Therefore, MMP-9 levels were divided by leukocyte counts for each time point. As consequence the highest median MMP-9 serum levels were detected two days before and at the day of SCT (103,3 ± 73,7 and 98,4 ± 33,9 µg/G leukocytes, respectively). Patients developing acute GvHD and patients without cytomegalovirus infection displayed higher pretransplant MMP-9 levels, however, this was statistically not significant. The lowest serum levels were again measured on day +7/+8 post transplant. Thereafter, serum levels increased up to one month after SCT and remained unchanged until day +90, irrespective if the patient developed a complication or not. In conclusion, MMP-9 levels measured in sera of SCT patients are not predictive for the occurrence of complications after SCT. Introduction: Mesenchymal stem cells (MSC) are multipotent cells that are considered one of the most promising product for cellular therapy in regenerative medicine. Efforts are ongoing to find the better culture conditions to obtain optimal expansion. Routinely, fetal bovine serum (FBS) is used for expansion protocols; however, concerns might be raised for the intrinsic risks of transmitting unknown animal diseases or for the potential immune responses to animal antigens, when utilised in the clinical settings. S131 Methods: We tested the effects of human platelet lysate in comparison to fetal bovine serum (FBS) on MSC expansion. Bone marrow aspirates (3-5 ml) were separated by negative lineage-depletion immunoselection (RosetteSep). Selected cells were seeded in multi-well plates (2 x 105/cm²) in MesenCult Basal Medium with the addition of either 5% platelet lysate or fetal bovine serum for human MSC (StemCell Technologies). Non-adherent cells were removed after 2-3 days and fresh medium changed twice weekly thereafter. On reaching confluence, adherent cells were detached by 0.25% trypsin-EDTA treatment and subsequently replated at low density (5 x 103/cm²) in the same medium conditions. At each passage, surface antigen expression was analyzed by flowcytometry (CD45, CD34, CD90, CD105, CD73, CD166, HLA-DR). Expanded MSC were analyzed for differentiative capacities by using specific differentiating medium for osteogenic (MesenCult Human Osteogenic Supplement) and adipogenic (MesenCult Human Adipogenic Supplement) differentiation. Moreover, expanded cells were tested for viability, bacterial, mycoplasma and viral contamination. Results: 2-3 x 106 cells were recovered after negative immunoselection. Confluent growth was obtained at first passage after 12-14 days, irrespective of medium used, while subsequent passages were done 7-10 days earlier for cells grown in platelet lysate containing medium than in FBS containing medium. Immunophenotyping showed negativity for CD45 and CD34 antigens, and positivity > 90% for CD90, CD73, CD105 and CD166 for cells grown in both culture conditions. Median fold expansion was 756 for platelet lysate cultures and 143 for FBS cultures, respectively. Conclusion: No differences were found for antigens expression or differentiation capability of expanded MSC with respect to the cultures media used. However the addition of platelet lysate resulted in a more rapid growth and a superior degree of MSC expansion. Introduction: Mesenchymal stem cells (MSCs) are derived from human bone marrow, umbilical cord blood (UCB) and other tissues. Normally, human embyonic stem cells(hESCs) are maintained on the feeder layers and these feeders have been derived from mouse or human sources. In our previous study, human placenta-derived mesenchymal stem cell (MSC) were used for feeder of human embryonic stem cells (hESCs), and these cells also showed good feeder for maintenance of hESCs. Placental MSC is more efficient as feeder than STO for co-culture with hESCs, so it could be useful for clinical application. However, their limited lifespan is a limitation, thus we immortalized human placenta-derived mesenchymal stem cells by using the simian virus 40 large T antigen (SV40T). We used immortalized placental MSCs (SVPC) as feeder of hESC and we are undergoing characterization of SVPC compared with parental placental MSCs . Method: Primary placenta-derived MSC at passage 3 were transfected with SV40T, called SVPC. To examined morphology, population doubling, SV40 T expression, gene expression and telomerase activity of these cells, we performed Western blot analysis, reverse transcription (RT)-PCR, and TRAP assay. Results: SVPC showed more spindle-like morphology than parental MSC. Growth rate of SVPC were remarkably different from the parental placenta MSC. A proliferation rate curve was similar to the parental placenta MSC's. However, the lifespan of SVPC was increased. SV40 T antigen was overexpressed in the SVPC as expected. Human ESs were cultured on immortalized feeder layers. However, The colonies of hES on immortalized feeder were undergoing differentiation. Immortalized MSC had lost the ability to support the expansion of undifferentiated hES. We examined Oct-4 gene expression and telomerase activity to find difference between normal placental MSC and SVPC. It was observed that Oct-4 expression in SVPC was higher than in normal PC, slightly. However, Telomerase activity showed no difference between normal PC and SVPC. Conclusion: We immortalized human placenta derived MSCs by using the simian virus 40 large T antigen (SV40T) for applied to feeder. High proliferative capacity of immortalized placental MSC is able to provide a large number of MSC. Further study is warranted to conmfirm characteristics of immortalized SVPC. Mesenchymal stromal cells in patients with haematologic disorders E. Shevela*, Y. Petrovsky, E. Kurganova, M. Tihonova, L. Sakhno, V. Sergeevicheva, A. Kulagin, I. Lisukov, A. Ostanin, E. Chernykh Institute of Clinical Immunology (Novosibirsk, RU) Objectives: Despite extensive functional characterization of mesenchymal stromal cells (MSCs) from normal sources, only a few studies have focused on MSCs from the bone marrow of patients affected by haematologic disorders. Taking into account that the using of autologous MSCs is favorable in clinical applications for ethical and practical reasons, we compared the characteristics of MSCs derived from the bone marrow of healthy volunteers (HV) and patients with haematologic malignancies (HM). Methods and Materials: MSCs were obtained from bone marrow of 24 healthy volunteers and 34 patients with HM (35 ± 2,9 years; 12 males; 22 females) including 11 patients with Hodgkin's lymphoma, 13 -non-Hodgkin's lymphoma, 5acute lymphoblastic leukemia, 5 -multiple myeloma. MSCs were cultured in alpha-MEM/20% FCS and evaluated at 1-3 passages for immunophenotype, morphology, clonogenety, immunosuppressive and haemopoietic activities. Results: MSCs of HV satisfied the minimal criteria stated by the International Society of Cellular Therapy and additionally were characterized by marked immunosuppressive activity. MSCs of patients with HM also displayed adhesiveness, fibroblast-like morphology and specific immunophenotype (Lyn-, CD34-, HLA-DR-, CD73+, CD90+, and CD105+). Furthermore patient's MSCs have been shown to suppress proliferation of activated T-cells and to induce haemopoietic precursor growth (assessed by CFU-E and CFU-GM). On the other hand, the quantity of relatively small cells (<20 micrometers) and CD90+cells in S/G2/M-phase in patients' MCSs population was higher than in HV, that obviously allows in vitro MSCs expansion despite of initial deficiency of clonogeneic precursors (CFU-F) in patients with HM. Conclusions: Intact haemopoietic stimulatory activity of patients' MSCs coupled with their safe potential for in vitro expansion prove the clinical use of autologous HSCs/MSCs co-transplantation in management of patients with haematologic malignancies with the view of reducing the period of haemopoiesis recovery. Hepatocytes of donor origin in recipient liver after haematopoietic stem cell transplantation in betathalassaemia major patients A. Ghavamzadeh, M. Mirzania*, N. Sedighi, M. Yaghmaie, N. Kamalian, K. Alimoghaddam, S.H. Ghaffari, P. Azimi Tehran University Shariati Hospital (Tehran, IR) Background: Bone marrow and circulating stem cells contains stem cells with the potential to differentiate into mature cells of various organs. We determined whether stem cells transformed to hepathcytes. Methods: Biopsy specimens from the liver were obtained from 11 patients who had undergone transplantation of hematopoietic stem cells from peripheral blood (8 patients) or bone marrow (3 patients). Four female patients had received transplants from a male donor and seven male patients had received transplants from a female donor. All patients had beta thalassemia major and fibrosis in biopsy specimens from the liver before hematopoietic stem-cell transplantation. Hematopoietic stem-cell engraftment was verified by short tandem repeat analysis. The biopsies were studied for the presence of donor-derived hepatocytes with the use of fluorescence in situ hybridization of interphase nuclei and immunohistochemical staining for CD45 (leukocyte common antigen), and a hepatocyte-specific antigen. Results: All 11 recipients of sex-mismatched transplants showed evidence of complete hematopoietic donor chimerism. XY-positive hepatocytes accounted for 4 to 6.7 percent of the cells in histologic sections of the biopsy specimens of female patients. These cells were detected in liver tissue as early as 1 year and as late as 8.5 year after the hematopoietic stem cell transplantation. Conclusions: Bone marrow and circulating stem cells can differentiate into mature hepatocytes in beta thalassemia major patients who undergone hematopoietic stem cell transplantation. Introduction: Oocyte production in females of most mammalian species is thought to cease before birth. Recently, it has been suggested that germline stem cells maintain oogenesis in postnatal mouse ovaries, and in considering the possibility of an extragonadal source of germ cells, the expression of germline markers in bone marrow (BM) was also showed. Materials and methods: We aimed to evaluate the presence of germline stem cells markers in the bone marrow of 12 healthy subjects, through magnetic isolation of CD34+ cells and RT-PCR study for the expression of the transcription factor Oct4, which plays a key role in stem cell pluripotency and early germ cell development. Characteristics of patients were: M/F 5/7, median age of 21 years, (range 15-46), median parity of 0 (range 1-3), CD34+% median value of 82.5 (range 50-96), CD34+/ ìL median value of 64.5 (range 17-240). Results: Oct4 was expressed in 10 out of 12 studied samples (83%). We didn't find any evident correlation between the expression of Oct4 and sex. The main expression of Oct4 was determined in youngest subjects (19, 17 , 15 e 20 years old respectively), while signal was absent in 34 and 36 years old subjects respectively. Oct4 was mainly expressed in nulliparous subjects, as 8/10 subjects (80%) expressing Oct4 didn't have offsprings, and only 2/10 subjects (20%) expressing Oct4 have 1 and 2 sons respectively, while the signal was absent in two subjects having 3 and 2 sons respectively. No statistical significance was evident for the limited number of samples. No correlation was evident between level of purity of the sample and expression of Oct4, and between CD34+/ìl and age. Conclusions: Our findings demonstrate that 2 different somatic tissues express the pluripotent state-specific transcription factor Oct4. We showed an inverse correlation between age and Oct4 expression, apparently independently from sex, and an inverse correlation between parity and Oct4 expression. Recent findings indicate that adult BM contains cells which can differentiate into epithelial cells of the liver, kidney, lung, skin, gastrointestinal tract, myocytes of heart and skeletal muscle. Oct4 may maintain the potency of stem and germline cells, but may also regulate the molecular differentiation of cells in the germ lineage. After a better understanding of the mechanisms involved in cellular differentiation, we would be able to apply adult stem cell plasticity for clinical purposes. In patients receiving allogeneic HSCT(hematopoietic stem cells transplantation) mononuclear cells(MNC) were examined during hematological recovery for parameters reflecting differentiation of lymphocytes, which includes NOTCH1 transcript measurement. We want to verify hypothesis on recapitulation of ontogeny during immune system recovery. 10 patients in 4 consecutive time points after transplantation (10-14; 21-25;28-31; 31-40 days) were analyzed for NOTCH1 relative expression using QPCR( CybrGreen chemistry) in mononuclear cells . 7 out of 10 patients showed NOTCH1 transcripts at least in 2 out of 4 time points examination. In two patients NOTCH1 transcripts were found in one observation. Only in one case we didn't observe any NOTCH1 transcript in MNC. 21 out of 36 MNC samples of alloHSCT patients were positive for NOTCH1. In contrast ten normal individuals MNC samples did not show NOTCH1 transcripts. Importantly NOTCH1 transcripts expression independently on time of examination were negatively correlated with CD4(+) cells contribution to the lymphocyte pool. It appears that NOTCH1 takes a part in the process of mononuclear cells differentiation early post hematopoetic stem cell transplantation. Objectives: We have recently proposed that the hematopoietic stem cell niche is a specialised metabolic environment, serving on the one hand to support and to contain HSC, and on the other hand to limit the long term accumulation of potentially oncogenic mutations in the stem cell pool. We are therefore characterising the metabolic relationship between HSC and stroma in order to determine the role of stem cell metabolism in the control of self-renewal and differentiation decisions, and to develop media which support the ex vivo expansion of HSC for transplantation, gene therapy or tissue engineering purposes. S133 Methods: We have assessed the effects of varying nutrient levels and osmolarity on survival and proliferation and on the maintenance of colony forming activity in cultures of i) primary CD133+ progenitors from umbilical cord blood (UCB) and ii) the murine multipotential progenitor cell line FDCPmix. Metabolic profiling of FDCPmix cells held under various conditions was carried out by gas chromatography / mass spectrometry of whole cell extracts. Results: Low glucose levels support the stem/progenitor activity of both CD133+ and FDCPmix cells, while high glucose supports differentiation. High osmolarity was found to decrease both the use of glucose and the expansion of differentiating cells. Furthermore, high osmolarity supported the maintenance of cobblestone area forming cell (CAFC) activity in cultured CD133+ cells and increased the frequency of clonogenic cells in FDCPmix cultures. Metabolic profiling of FDCPmix cells suggests that differentiation is accompanied by a shift from an amino acid-based metabolism in the multipotent population to a more familiar glucose-based metabolism in the differentiating progeny. Particularly striking is the increased usage of alanine, glycine and proline under low glucose conditions. These 3 amino acids are taken up via a single transporter known to be activated by high osmolarity. Conclusions: Our ongoing observations of metabolic requirements and metabolic composition support the metabolic niche hypothesis and suggest that stem cell maintenance involves an amino-acid based metabolism potentiated by high osmolarity. This should present new opportunities for the selective enrichment of stem cells based on functional parameters rather than surface markers, and provide a basis for the development of media conducive to the ex-vivo expansion of HSC for therapeutic purposes. Objectives: MSCs are multipotent cells with many potential clinical applications. However, the lack of a precise definition of the cells preparation and the heterogeneity of the obtained product render validation criteria of the final product of the expansion. Aims: to estimate dynamics of growth, immunophenotype and genetic stability (karyotype and aneuploid cell frequency) of MSC from healthy donors on early (2-4) and late (10-12) passages. Methods: MSC (n=13) were prepared by seeding bone marrow MNC in 75 cm² culture flasks in low glucose DMEM with 20% FCS. After 72 hours the non-adherent cells were discarded and cells were passed every 7 days by trypsinization and replanting at the density of 0.5x10 6 cells/75 cm² flask until the end of the culture. Chromosomal preparations of MSC were prepared according to a standard protocol. The karyotype was analyzed by Gbanding technique; 15-30 metaphase cells for each culture were analyzed. To analyze the level aneuploidy, fluorescent in situ hybridization (FISH) with chromosome enumeration probes (CEP) studies was performed. For each hybridized probe at least 1000 interphase nucleus were analyzed. Results: We found that in vitro expanded bone marrow MSC demonstrate higher proliferative activity on 2-4-th in comparison with 10-12-th passages (5.2-and 2.1-fold increase accordingly, p=0.049). After the 4-th passage the cells were positive for CD90, CD105, CD69, CD166, CD44 (median 91,3%; 97,6%; 99,5%; 94,9%; 85,6% accordingly) and negative for CD45, CD34, CD133. The phenotype of MSC on early and late passages did not differ. It was studied karyotypically 5 MSC cultures on early stages of culturing. In all cases the karyotype were normal (46,XY). It was observed the same picture on the later passages XY) . Aneuploidy rate for sex chromosomes (X and Y) in the MSC cultures was studied on the early and late passages of cultivation. In one individum, which was determined at the 2 passage as 46,XY, we found at the 12 passage 75,6% cells with two signals from chromosome X, 22% -XY, 2% -XO, 0,2% -XXX, 0,2% -XXXX cells. Conclusions: During the culturing procedures chromosome abnormalities can arise which can lead to long term consequences of cell therapy of complications of bone marrow transplantation . Further research is required concerning different passaging techniques and culture conditions to better understand their effects on MSC chromosomal stability. Autologous haematopoietic stem cell transplantation with busulfan and etoposide as conditioning regimen for acute myelogenous leukaemia patients A. Ghavamzadeh*, K. Alimogaddam, F. Khatami, F. Gaffari, A. Ashuri, A. Mousavi, M. Iravani, B. Bahar, M. Jahani, A. Khodabande Hematology-Oncology & Bone Marrow Transplantation Research Center (Tehran, IR) Objective: Acute Myelogenous Leukemia (AML) is a potentially lethal disease. Hematopoietic Stem Cell Transplantation (HSCT) has increased disease free survival (DFS) and overall survival (OS) of patients more than conventional treatment. Allogeneic HSCT for standard and high risk patients in first complete remission is standard of care. The result of autologous HSCT in patients without suitable donor is near to allogeneic transplantation. We performed autologous transplantation with busulfan and etoposide as conditioning regimen for patients who didn't have suitable donor. Methods: Since January 2003 till Jun 2007, 90 patients received autologous transplantation. We included all children and adult with AML in first or second complete remission without suitable donor and end organ failure who can tolerate high dose chemotherapy. Mobilization regimen was cyclophosphamide 2g/m² for one day and G-CSF 10µg/kg for 7 days. We have done stem cell harvesting when patient's white blood cell (WBC) count raised to 1000/µl then the patients received oral Busulfan 4mg/kg for 4 days (from -4 to-1) and Etoposide 15mg/kg (IV) for 2 days (from -4 to -3) as conditioning regimen. After that patients have transplanted with their peripheral blood stem cells. Conclusion: We conclude this conditioning regimen has low 100-days post transplant mortality and manageable complications and acceptable DFS and OS. Autologous haematopoietic stem cell transplantation in newly diagnosed type I diabetes mellitus: longer follow-up of 21 patients M.C. Oliveira, D.A. Moraes, A.B. Stracieri, F. Pieroni, G.M. Barros, M.A. Coutinho, M.I. Ayrosa, M.C. Favarin, C.E. Couri, B.P. Simões, J.C.. Voltarelli Ribeirão Preto School of Medicine (Ribeirão Preto, BR) Introduction: Type I diabetes mellitus (DM1) is an autoimmune disease in which insulin -producing pancreatic beta cells are destroyed, leading to insulin dependence and chronic complications. High dose immunosuppressive therapy increases beta cell function and induces insulin independence, as it is described in this longer follow-up of 21 patients. Objectives: to determine the safety and efficacy (phases I/II) of high-dose immunosuppression followed by autologous stem cell transplantation in newly diagnosed DM1. Materials and Methods: autologous stem cells were mobilized from the bone marrow, collected by leukapheresis and cryopreserved. Patients were then conditioned with 200mg/kg Cyclophosphamide and 4.5 mg/kg rabbit antithymocyte globulin, followed by stem cell infusion. Results: Since December 2003, 21 patients were enrolled in this study. The medium age was 17.3 years, (13 -31 years), and the time between diagnosis and mobilization was 29.3 days (16 to 49 days). The mean hospitalization period was 19.4 days (14-57 days). There were no deaths. All patients used exogenous insulin therapy before mobilization, with an average dose of 0,47 IU/kg (0,13 to 0,84 IU/kg). In a mean follow-up of 24 months (1-48 months), insulin therapy was resumed in 17 patients from D-7 to D+39 (average +24), three patients decreased insulin dose compared to the pretransplantation period and one patient remains using high dose insulin (1.7 IU/kg/d).Two patients relapsed 170 and 362 days after transplantation and exogenous insulin was restarted. Two patients presented ketoacidosis before AHSCT. In the first one high levels of exogenous insulin are still needed, 48 months from transplantation. The second patient was recently transplanted (60 days), but insulin requirements are progressively decreasing. C-peptide levels considerably increased in 13 evaluated patients with clinical response and serum levels of hemoglobin A1C were maintained less than 7%. These results indicate that high-dose immunosuppression associated with autologous stem cell rescue may induce prolonged clinical remission in newly-onset DM1, with acceptable toxicity. This longer follow-up shows sustained remission in most patients, although some relapses have been detected. The therapy seems to also improve beta cell pancreatic function in patients with previous ketoacidosis. Haemopoietic stem cell transplantation for severe autoimmune diseases: a 12-year experience F. Gualandi *, B. Bruno, M.T. van Lint, S. Luchetti, A. Uccelli, E. Capello, G.L. Mancardi, A. Bacigalupo, A. Marmont S.Martino's Hospital (Genoa, IT) Since the first autologous hematopoietic stem cell transplantation (ASCT) for a non-coincidental patient with SLE1, 45 patients (by intention to treat) were included in the HSCT program for severe autoimmune diseases (SADs) in Genova. Four of them were only mobilized (CY 4 g/m²), of them (SLE) because of complete remission (CR) following double mobilization, 1 (SSc) not mobilizing, and 1 (SSc) because of early death following toxicity and progression of disease. Autologous transplants were performed in 36 patients, including 22 with multiple sclerosis (MS), 7 with SLE, 2 with SSc, and 1 each for autoimmune thrombocytopenia (AITP), rheumatoid arthritis (RA) Behcet's disease (BD), vasculitis and autoimmune encephalomyelitis. Conditioning regimens included BEAM+ATG (for MS), CY 120 mg/kg (for MS), CY-Thio-TEPA (for SLE). The OS at 180 months was 95% by intention to treat. One patient with MS died 70 days post-transplant of thromboembolic disease. Complete durable neuroimaging remissions were achieved in all MS patients, and malignant forms were neutralized2. Very good remissions were achieved in the SLE patients3,. Four patients received allogeneic SCT (Evans syndrome-ES, BD, pure white cell aplasia-PWCA, SLE with polyneuritis): the patient with ES relapsed 5 years post transplant4, and the others experienced attenuation of disease3. One patient with RA received a syngeneic transplant after conditioning with CY: the anti-cyclic citrullinated peptide (CCP) antibodies dropped from 270 to 2 U/ml, but the rheumatoid symptoms reappeared. The questions and problems arising from this and the worldwide experience about the utilization of SCT (autologous/allogeneic/syngeneic) for SADs are being addressed and will be published shortly. Introduction: Fibrosing alveolitis (FA) is the most frequent cause of death after cardiac involvement and pulmonary hypertension in systemic sclerosis (SSc). Cyclophosphamide (CY) was demonstrated in retrospective studies and, recently, in a randomised, placebo-controlled prospective study, to have significant, albeit modest, effect on FA-SSc and lung function. Since 1996, the use of high doses of CY followed by autologous hematopoietic stem cell transplantation (ASCT) allowed impressive clinical responses in severe diffuse SSc in several phase I-II studies. Data on the evolution of FA associated SSc and especially high-resolution CT scan (HRCT) patterns are lacking. The aim of our study is to describe the evolution of FA-SSc after ASCT with a special focus on serial HRCT changes. S135 Patients and methods: 9 diffuse SSc patients (6F/3M), diagnosed according to the ACR criteria, median age 41 (17-61) yrs, who had been treated for severe disease and early visceral involvement with CD34+-selected ASCT in the ISAMAIR phase I-II trial according to previously published criteria (Farge et al. Br J Haematol 2002; 119:726-39) were evaluated just before (T0) and then at 6 (M6) and 12 mths (M12) after ASCT for FA-SSc. Two independent observers reviewed all the HRCT to measure the extent and severity of fibrosis according to the Wells score (Desai SR et al. Radiology 2004; 232:560-7) , blindly to clinical results. Results (median, range) were analysed in light of the evolution of the modified Rodnan skin score (mRSS) and pulmonary function tests measured within the same week as HRCT evaluation using the non parametric paired Wilcoxon test to compare patients individual values. Results: Table 1 Conclusion: This study first reports improvement or stabilisation of FA-SSc in 9 SSc patients treated by ASCT, with significant regression of its extent at 6 months on serial HRCT evaluation. HRCT improvement was predominantly associated with attenuation of some ground-glass areas, meanwhile significant regression of the mRSS was found at 6 and 12 months after ASCT as previously reported. New increase in FA-SSc 1 year after ASCT raised the question of potential relapse after immune reconstitution in some patients (Farge et al. Arthritis Rheum 2005; 52:1555-63) . Over the last few years, the role of autologous haematopoietic stem cell transplantation (HSCT) in refractory autoimmune diseases have seen a growing interest. Experience in Crohn disease (CD) is very limited and entirely based on transplantation of CD34+-selected peripheral stem cells. In our Institution, between 11/2005 and 12/2007 seven pts with active moderate-severe refractory CD were enrolled in a phase I-II pilot study to investigate feasibility and efficacy of autologous unselected HSCT. 3 were male and 4 female, with a median age of 36 years (range 27-46). All had previously received multiple conventional treatments, including corticosteroids and at least 2 immunosuppressant agents. Median CD activity index (CDAI) was 333, ranging from 272 to 395; four had perianal disease at the time of transplant. PBSCs were collected after mobilization with CTX 1.5 g/m² and G-CSF 10 mcg/kg. Conditioning regimen included CTX 50 mg/kg on days -5 to -2 and rabbit ATG 2.5 mg/kg on days -4 to -2. Toxicity, clinical remission (CDAI <150), endoscopic remission (SES-CD), extramucosal response (ultrasound sonography [US] ) and quality of life (IBD-Q) were assessed after mobilization and at 3, 6, 9, 12 and 24 months after stem cells reinfusion. While no improvement was observed after mobilization (median CDAI 345, range 258-404), six pts were in clinical remission already at the first month after transplantation (median CDAI 125, . After 3 months, all evaluable pts (6/7) were in clinical remission (median CDAI 100, range 56-132) despite interruption of all medications. After a median follow-up of 16 months (range 1-25) only one patient relapsed (+4 months) while all other pts remained in clinical remission. Complete mucosal healing was achieved in 4/6 pts, with complete endoscopic remission in 2/3 pts with a follow-up > 20 months. Bowel thickness as assessed by ultrasonography improved slowly in all pts, reaching normal characteristic in 3. Complete perianal fistulas resolution was observed in 3/4 pts. Quality of life improved in all subjects. No deaths or life-threatening infections occurred. Unexpected adverse events included perianal abscess after mobilization (1 pt), pleural and pericardial effusions (1 pt) and BK virusrelated macroscopic hematuria (1 pt), all rapidly resolved with conservative treatment. In conlusion, autologous HSCT with unselected PBSC is feasible and can induce and maintain both clinical and endoscopic remission in refractory CD Background: Clinical trials have indicated that immunoablation followed by autologous haematopoietic stem cell transplantation (ASCT) has the potential to induce clinical remission in patients with refractory systemic lupus erythematosus. To elucidate the mechanisms mediating the beneficial long-term clinical responses we investigated the immune reconstitution in systemic lupus erythematosus (SLE) patients receiving ASCT as part of a monocentric phase I/II clinical trial. Methods: Seven patients with SLE were evaluated during a long-term follow-up (median follow-up period 60 months) who underwent immunoablation with cyclophosphamide and rabbit antithymocyte globulin (ATG), followed by transplantation of purified autologous CD34+ haematopoietic stem cells. Humoral immunity was evaluated and peripheral B lymphocytes were immunophenotyped in detail. Findings: In all patients clinical remission was observed, accompanied by disappearance of dsDNA-specific autoantibodies. In addition, a significant decline in serum antibody levels for measles, mumps, tetanus toxoid and diphtheria (all p=0.031) at time-points ranging from one to two years after ASCT was observed. The serological data point to an effective depletion of long-lived plasma cells from bone marrow. We were able to stain plasma cells ex vivo with the polyclonal rabbit ATG used for immunoablation. In a sample of bone marrow aspiration from one of the patients (p#7), obtained early after ASCT (+1 mo) an almost complete depletion of CD38+ CD138+ plasma cells was evident, with only 0.03% among BM-MNC as compared to a normal control bone marrow with 1.24% of such cells. Immunophenotypic analyses revealed the normalisation of the B cell compartment within 12 months after therapy, as compared to the preexisting B cell deficiencies, which had included naive (IgD+) B cell lymphopenia (p=0.031), relative predominance of memory (IgD-) B cells (p=0.016) and expansion of CD27high CD20plasma blasts. Interpretation: Our data demonstrate that the long-term therapy-free clinical remissions observed in SLE patients after immunoablation and ASCT are accompanied by the disappearance of pathogenic and protective serum antibodies and the complete reconfiguration of the peripheral B cell compartment. Apparently, ATG directly targets the (long-lived) plasma cells providing these serum antibodies. We propose that the depletion of the autoreactive memory a prerequisite for the regeneration of self-tolerance and induction of clinical remissions. Objective: To assess the long period of efficacy and safety of autologous peripheral blood stem cell transplantation (APBSCT) on multiple sclerosis (MS) in China. Methods: From July 2000 to May 2007, twenty-five patients with progressive multiple sclerosis (PMS) were enrolled. All patients (6 male, 19 female) were diagnosed as MS according to the Poser criteria. The average age was 37 years (range, 15 -64). The average EDSS of all patients was 7.3 (range, 3.0 -9.5) Peripheral blood stem cells were mobilized with cyclophosphamide (CY) and granulocyte colony-stimulating factor (G-CSF). CD34+ cells were purged by Clinic MACS (AmCell GmbH) in 19 patients. Two of the patients who without CD34+ cell purged used antithymocyte globulin (ATG) during conditioning. The CY/TBI and BEAC (BCNU, etoposide, Ara-C and CY) regimens were used in one and two cases respectively; and the BEAM was chosen in other 22 cases for conditioning. The median amount of transfused CD34+ cell was 3.38 x 10 6 /kg (range,0.88 -18.76). Results: All 25 patients got hematopoietic reconstitution after transplantation. The average follow-up period is 38.1 months (range, 6 -77) . Of the 25 patients who treated with APBSCT, 10 patients (40%) got better, 11 patients (44%) were stable and 4 patients (16%) got worse. The rate of efficacy was 84%. The confirmed progression-free survival and the disease activity-free survival in different follow-up can be seen in the table below. There was no treatment-related death. Five patients died during the follow-up. Of the 5 patients, one was died of severe pneumonia at 4.5 months post transplant; another was died of liver failure at 15 months post transplant; other two patients ware died of disease development at 18 and 36 months post transplant respectively; and the rest one was died of accident. Conclusion: HSCT seems beneficial in treating MS. Confirmed progression-free survival was 71%, and disease activity-free survival was 86% at 5 years. Multi-center random clinical research would be required. Autologous haematopoietic stem cell transplantation in patients with refractory inflammatory bowel disease: a report of 10 cases from China L.D. Chen*, J. Ouyang, X.Q. Zhang, Y. Yang, B. Chen, J.Y. Xu Drum Tower Hospital (Nanjing, CN) Objectives: To investigate the safety and efficacy of HSCT in patients with refractory inflammatory bowel disease (IBD). Methods: Ten patients with active moderate-severe IBD, including nine CD and one UC, were enrolled through January 2004 to August 2006. The group comprised of 7 male and 3 female, aging from 24 to 46 years. The mean CDAI for 9 CD patients was 352.3 (range 175-492), and one patient with severe UC(whole colon). Patients signed informed consent before stem cell mobilization and the regimen was CTX (2.0g/m² intravenously) and G-CSF(5ug/kg/d) beginning at 5 days later. Leukapheresis was initiated for all patients when WBC counts reached 5.0x109/L or the number of CD34+ cells reached 20 cells/µl. The CliniMACS cell separation system was used for CD34+ cells selection in 6 cases. The number of CD34+ cells exceeded 2 x10 6 /kg in all patients. The CTX(50mg/kg/d on days -5, -4, -3 and -2) was used as conditioning regimen. In the patients without CD34+ cell selection, the ATG(5mg/kg/d on -5, -4, -3 and -2) was used. The HSC graft was reinfused intravenously on day 0, 48 hours following the last dose of cyclophosphamide. Results: The patients were followed up at a median time period of 16.1 months (range 10-33 months). In 9 patients with CD, clinical complete remission (CDAI<150) was achieved in 5 (55.5%) at 3 months post-HSCT. The CDAI decreased in other 2 patients (2/9, 22.2%). At 12 months post-HSCT, one patient gained clinical complete remission after surgical remove of the abdominal mass, while one patient remained with active disease post-HSCT. The disease relapsed in 4 patients, at 3, 5, 8 and 30 months post-HSCT, respectively. However, no improvements were observed under repeated colonoscopy in 7 patients (6 CD and 1 UC). HSCT-related side effects included neutropenia caused fever, which was resolved with antibiotics and supportive therapy. One HBVcarrier prior to HSCT developed asymptomatic increases in liver enzymes and HBV-DNA copies after HSCT, and was resolved with anti-virus treatment. Conclusions: Autologous HSCT can be conducted safely and is well tolerated in patients with refractory IBD. It can induce clinical remission in most of these patients, although endoscopic remission may not be achieved, and relapses could not be avoided in some patients. Key words: Refractory Inflammatory Bowel Disease; Autologous haematopoietic stem cell transplantation. Thiotepa-cyclophosphamide high-dose immunsuppression with autologous stem cell transplantation in refractory or relapsed multiple sclerosis T. Roccia*, F. Ciceri, M. Bernardi, J. Peccatori, C. Corti, R. Greco, M. Radaelli, L. Moiola, G. Martino, G. Comi San Raffaele Scientific Institute (Milan, IT) Autologous Stem Cell Transplantation (ASCT) has been proposed as rescue therapy in patients with different courses of Multiple Sclerosis (MS), either refractory or relapsed after first and second line standard therapies. Various conditioning regimens have been used until now; unfortunately, myeloablation with BEAM ± immunesuppression with ATG has shown relevant toxicity and mortality. In our study, we evaluated a immunesuppressive regimen based on thiotepa (thio) and cyclophosphamide (cy) to assess efficacy and safety in refractory or relapsed MS. Between December 05 and December 07 nine patients affected by MS (secondary progressive and relapsingremitting) underwent ASCT at our centre. The median age at transplantation was 27 years with a median disease duration of 96 months. Six patients had a relapsing remitting course of MS and three a secondary progressive course. One patient showed an iperacute course of MS. In the previous two years before ASCT the median number of relapses was 2 and the median baseline EDSS was 6. The hematopoietic stem cells were mobilized with cy 4 gr/mq at day 0 followed by granulocyte colony stimulating factor 5mcg/Kg from day +2 to stem cell harvest. Harvest was not manipulated. The conditioning regimen consisted of thio 5mg/Kg bid on day -5 and cy 50 mg/Kg on days -3 and -2. The mobilization and PBSC collection was successful in all cases and no major side effects nor disease flares were observed. Following ASCT all patients developed grade IV neutropenia and all but one grade III piastrinopenia which lasted for a median of 12 and 9 days respectively. Fever was observed in 6 patients. The median duration of hospitalisation was 24 days. Transplant related mortality was 0%. At the last follow-up 7 out of 9 patients were clinically and radiologically stable or improved. Relapses were observed in 2 patients at 1 year after ASCT. A brain MRI at 6 months was performed in 8 patients: in all cases except one there was neither increase of lesional load nor enhancing lesions. Seven patients underwent the one year brain MRI: five patients were completely stable or improved while 2 patients showed new active lesions. In this series, autologous ASCT after thio-cy as conditioning resulted a safe and feasible treatment to arrest clinical and radiological signs of previously refractory or relapsed MS, with no extra-hematological toxicity. A larger number of patients and a longer follow up is warranted to better assess the value of this regimen. Introduction: Clinical trials have indicated that immunoablation followed by autologous haematopoietic stem cell transplantation (ASCT) has the potential to induce long-term clinical remissions in systemic lupus erythematosus (SLE) that are not reliable on further immunosuppression. However, relapses of disease may occur in a fraction of these patients. Methods: Since 1998, seven patients with refractory SLE underwent immunoablation with cyclophosphamide and rabbit antithymocyte globulin, followed by transplantation of purified CD34+ haematopoietic stem cells as part of a monocentric phase I/II clinical trial. While clinical remission could be achieved in all patients, one patient relapsed 18 months after receiving ASCT. We describe here immunologic and serologic changes during the lupus flare using immunophenotypic analysis of T-and B cell subsets and monitoring of humoral immunity. Results: After ASCT, clinical remission was achieved in the patient within 3 months, accompanied by the disappearance of antinuclear antibodies and pathogenic anti-dsDNA antibodies. In addition, protective serum antibodies specific for mumps, measles, tetanus and diphtheria were largely extinguished. Immunophenotypic analyses revealed the recurrence of IgD+ naïve B cells and CD45RA+ naïve CD4+ T cells. Two months before clinical signs of lupus activity we observed a significant expansion of circulating B cells with memory (IgD-) and plasma cell precursor phenotype (CD27high CD20-), and increase in CD45RO+ memory T cells. Serologic analysis revealed increase of ANA titres increased from 1:80 to 1:320 during that time. At the timepoint of lupus flare, dot-plot analysis of recurring serum ANA showed changes in the ENA pattern. While the patient displayed specificities for anti-Ro/SSA and anti-La/SSB before ASCT, the development of antibodies directed against nucleosomes and Sm-antigen was observed during the flare while anti-Ro/SSA and anti-La/SSB antibodies disappeared. Discussion: The complete disappearance of both pathogenic and protective serum antibodies points to the efficient depletion of the immunological memory early after ASCT while the recurrence of B-and T cells with naïve phenotype suggest a complete reconfiguration of the adaptive immune system. The development of lupus activity 18 months after ASCT was accompanied by changes in the antinuclear autoantibody pattern that implicates the de novo development of SLE in a genetically susceptible individual rather than a relapse of disease. High dose chemotherapy (HDCT) + autologous stem cell transplantation (ASCT) is a new and promising therapy for multiple sclerosis (MS) patients. Among a number of unclear questions is the term of conducting HDCT+ASCT. We verify 3 strategies of HDCT+ASCT depending on the terms of disease process: early (EDSS 1.5-3.0), conventional (3.5-6.5 ) and salvage (7.0-8.0). We aimed to study the clinical and QoL outcomes in MS patients after early, conventional and salvage HDCT +ASCT. Fifty patients with MS (secondary progressive -27 patients, primary progressive -11, progressive-relapsing -11, relapsing-remitting -1) were included in the study (mean age -32.0; male/female -22/28). Thirty seven patients underwent conventional HDCT +ASCT; 9 patients -early HDCT +ASCT and 4 patients -salvage HDCT +ASCT. Median EDSS at base-line was 5.0 (range 1.5 -8.0). The mean follow-up duration was 19 months (range 6 -90 months). All of the patients had previously undergone conventional treatment. Neurological and QoL evaluation was provided at baseline, at discharge, 3, 6, 9, 12 months, and then every 6 months after HDCT+ ASCT. MRI was conducted at baseline, at 6, 12 months, and at the end of follow-up. All of 45 patients included in the efficacy analysis experienced improvement (n=28) or clinical stabilization (n=17). Among the patients with improvement there were 20 patients after conventional HDCT +ASCT, 6 -after early HDCT +ASCT and 2 -after salvage HDCT +ASCT. Among the patients with stabilization there were 15 patients after conventional HDCT +ASCT and 2 -after salvage HDCT +ASCT. Two patients deteriorated to a worse score after 18 months of stabilization (in both cases conventional transplantation); 2 others progressed after 12 and 30 months of improvement (early and conventional transplantation), respectively. The progressionfree survival at 6 years after HDCT+ASCT was 72%. All the patients with clinical stabilization and improvement exhibited improved QoL. All the patients who did not have disease progression were off therapy throughout the post-transplant period. In conclusion, the results demonstrate high efficacy of HDCT+ASCT in MS. Notably, patients undergoing early, conventional and salvage HDCT+ASCT responded to treatment. The data obtained point to feasibility of early, conventional and salvage HDCT+ASCT in MS patients. Further studies should be done to investigate clinical and QoL response in MS patients receiving early, conventional and salvage HDCT+ASCT to better define treatment success. Objectives: Systemic lupus erythematosus (SLE) is characterised by polyclonal B-cell activation and autoantibody production by short-and long-lived plasma cells. New Zealand Black/White mice (NZB/W) develop high autoantibody titres and lupus nephritis resembling SLE. The cytotoxic drug cyclophosphamide depletes short-lived plasmablasts (SPCs) from spleens of these mice, but not long-lived plasma cells (LPCs). LPCs secrete huge quantities of (auto)antibodies and maintain plasma cell memory. Dexamethasone induces apoptosis in malignant plasma cells of multiple myeloma, and is used for treating SLE. We therefore investigated if dexamethasone alone or in combination with cyclophosphamide depletes LPCs in NZB/W mice. Methods: NZB/W mice were fed bromodeoxyuridine (BrdU) in drinking water for 3 weeks to label proliferating SPCs. During the third week, the mice received high doses of dexamethasone and/or cyclophosphamide. The SPC and LPC populations in the spleen, bone marrow and kidney were analysed by FACS and ELISPOT. Results: We show that only BrdU+ SPCs, but not BrdU-LPCs are depleted in spleen after dexamethasone and cyclophosphamide treatment, while both BrdU+ and BrdUplasma cells in bone marrow remain refractory. More intriguingly, anti-DNA antibody-secreting cells (ASCs) in bone marrow remain stable despite treatment. We also observed a preferential accumulation of IgG ASCs in the inflamed kidneys, in contrast to spleen and bone marrow which contained lower proportions of IgG ASCs. Conclusions: Bone marrow plasma cells, including autoreactive anti-DNA ASCs, are dexamethasone and/or cyclophosphamide resistant. Bone marrow could thereby contribute to maintenance of autoreactive plasma cell memory which is immunosuppressive drug resistant. These cells pose a challenge to treatment of autoimmunity. Immunoablation with antithymocyte globulin followed by transplantation of autologous CD34+ stem cells is currently the only therapeutic option capable of depleting autoreactive memory plasma cells. Specific targeting of autoreactive memory plasma cells remains a challenge of the future. Complete resolution by autologous stem cell transplantation of aquired inhibitor of the prothrombin time system associated with relapsing lymphoma A. Berrebi*, M. Haran, I. Lichman, L. Shvidel, M. Shtalrid Kaplan Medical Center (Rehovot, IL) A 41 year old woman was diagnosed with stage I lymphoplasmacytic lymphoma involving left axillary lymph nodes, in July 2003. She was treated with four cycles of CHOP chemotherapy and involved field irradiation with achievement of complete remission. In November 2006 she presented with enlarged left inguinal lymph node and referred to diagnostic biopsy. The biological parameters revealed repeatedly abnormal coagulation tests involving the prothrombin time (uncoagulable PT) and normal activated thromboplastin time. Lupus anticoagulant was not found. The mixing tests with normal plasma according to the TTI tromboplastin titration index didn't correct the prothropmbin time which revealed a considerable decrease of Factor X 10% and Factor VII 35%, thus suggesting the existence of inhibitors in the prothrombin system. Incision biopsy was performed without excessive bleeding. The histopathology revealed a lymphoplasmacytic lymphoma with areas of transformation to diffuse large cell lymphoma. Chemotherapy with CHOP-Rituximab was started and resulted in clinical remission and mild improvement in PT which remained prolonged 13%, INR 4.5. Soon after completion of chemotherapy, peripheral stem cell harvest was performed and followed by autologous stem cell transplant using the CBV conditioning regimen. The transplantation was uneventful. During follow-up repeated analyzes revealed total normalization of the coagulation tests. The acquired circulating anticoagulants have been rarely described during the course of relapsing lymphomas. Activity of these inhibitors induces hypercoaguability and immunosuppressive treatment usually is unsuccessful. Autologous stem cell transplantation was applied to treat variety of autoimmune disorders (Tyndal, Semin Hematol, 2007) , but to our knowledge, no reports have been described with coagulation inhibitors. We believe that in our case the high dose chemotherapy followed by autologous stem cell transplantation played a major role in disappearance of the coagulation inhibitors. Concept of autologous stem cell transplantation (ASCT) in autoimmune disease is resetting of the immune system. Immunoablation by high dose chemotherapy and ATG is followed by T cell depleted ASCT with the aim of development of a tolerant immune system. So far, 14 patients with refractory autoimmune diseases were treated. Inclusion criteria were life threatening disease with inadequate response to standard treatment, adequate organ function to tolerate conditioning and transplantation, patients aged 18-60 years. Patients included had systemic lupus erythematosus (SLE n = 7). Systemic sclerosis (SSc n = 4), polychondritis (n = 1), panniculitis (n = 1), Takayasu's arteritis (n = 1). For mobilisation of stem cells patients received cyclophosphamide (CY) 2 g/m² and G-CSF 5 µg/kg BID from d +5. The peripheral blood transplant underwent CD34+ selection (CliniMACS). In vivo immunoablation of the patient consisted of CY 4 x 50 mg/kg (d -5 to -2) and ATG 3 x 30 mg/kg (d -4 to -2). On day 0 patients received the purified CD34+ cells (> 2 x 106 CD34+ cells /kg and < 104 CD3+ cells/kg bw). The patients were evaluated during a long term follow up (median follow up period > 60 months). Clinical status, disease activity, immunosuppressive therapy and humoral and cellular immunity were evaluated. In all seven SLE patients clinical and serological remission was observed, accompanied by disappearance of dsDNAspecific autoantibodies. One patient died early due to cerebral aspergillosis. One patient developed a relapse 17 months after ASCT. The relapse was therapy refractory and the patient died due to fulminant SLE. In the remission a fundamental reset of the immune system could be demonstrated. Open questions are: firstly, is TRM associated with stage of disease at ASCT or intensive pre-treatment. Secondly, why do some patients relapse? To answer these questions, we have designed a controlled phase II multicenter trial of immunoablation with CY and ATG and transplantation of autologous CD34+-enriched haemopoietic stem cells versus currently available immunosuppressive therapy in refractory SLE. This trial that includes eight centres in Germany will start in the beginning of 2008. Efficacy, safety and tolerability of high-dose chemotherapy with autologous stem cell rescue as the treatment option for advanced multiple sclerosis stages V. Vavilov*, N. Osipova, S. Bondarenko, E. Babenko, E. Darskaya, B. Afanasyev I. Pavlov State Medical University (Saint Petersburg, RU) Multiple sclerosis is one of the mostly worldwide speeded inflammatory autoimmune diseases which often lead to the severe physical and psychological impairment of the patients because of high rate of insufficient immunosuppressive therapy. The possible way to ameliorate the immunosuppressive effect of cytostatic agents is high dose chemotherapy with hematopoietic stem cell support. Fifteen adult patients (26-44 years, median -33 years) with confirmed diagnosis of multiple sclerosis were transplanted in our clinic since Year 2000 till 2007. Disease duration before transplantation was 12 to 240 months with median of duration 87 months. Of these 15 patients, 5 (33%) have primaryprogressive, 6 (40%) secondary-progressive, and 4 (27%)relapse-remitting form of the disease. Median EDSS-score before transplantation was 6,0 (range 2,0-6,5). All patients were transplanted with G-CSF mobilized peripheral blood stem cells collected during two days with mean CD34+ cells dose 4,2x10 8 /kg (range 2,9-5,7x10 8 /kg). As conditioning regimen we provide BEAM (BCNU, etoposide, cytarabin, melphalan) in 11 (73%) cases and FM (fludarabine, melphalan) in 4 (27%) cases. All patients received 60 mg/kg ATG in three doses after transplantation. Among the main toxic effects of conditioning regimens were nausea and womiting (100%), transient neurologic deterioration (87%), mucositis (grade I-II -66%, grade III -20%), and serum sickness (60%). Patients (n=13) were observed for 12-60 months period (median -18 months). There was 100% overall survival, and with 60% of patients free from progression 2 years after transplantation. The efficacy of HSCT was estimated as improvement in 3 cases (23%) and stabilisation in 6 cases (46%) patients. The EDSS-score in this group of patient has improved to 4,5 (range 1,5-6,0). In 4 patients with primaryprogressive multiple sclerosis the disease progression was observed during 1-2 years after transplantation. High-dose chemotherapy with autologous stem cells transplantation is a safe and effective second line treatment option in patients with different forms of multiple sclerosis. It could provide disease stabilization and prevent relapses of the disease, and as well improve the quality of life. Aplastic anaemia and other autoimmune diseases. A link or just coincidence? M.P. Stalder*, A. Rovó, J. Halter, D. Heim, C. Arber, A. Buser, S. Meyer-Monard, J.R. Rischewski, M. Stern, A. Tichelli, A. Gratwohl Stem Cell Transplant Team (Basel, CH) Acquired aplastic anemia (AA) is considered a T-cell mediated autoimmune disorder (AID). Its association with other AID has been described. The link between both types of AID and the impact of the AA treatment on the concomitant AID remain unclear. We conducted a retrospective single centre cohort study of 243 patients with severe AA (SAA) treated between 1974 and 2006 at the University Hospital of Basel. We looked for other AID, diagnosed before and after SAA, its prevalence and possible risk factors. Only clinical AID defined according to international diagnostic criteria were included. Patients with isolated elevation of antibody titers without further clinical signs of a disease were not included. Treatment strategy was uniform throughout the observation period. Patients below the age of 40 years (y) with a matched sibling donor (57 patients, 23%) received allogeneic hematopoietic stem cell transplantation (HSCT), all others, 186 patients (77%) were treated with antithymocyte globulin (ATG) with or without Cyclosporin A as primary therapy. Due to refractoriness or relapse, 30% needed further therapies. The median age was 22 y (2-80 y). 115 patients (47%) were female. The median follow up time was 9.4 y (0-33 y). AID was diagnosed in 24/243 patients (10%), 9 of them (37.5%) were female. Four patients had more than one AID. Autoimmune gastritis and autoimmune thyroiditis were the most frequently found AID (6 patients each). Amongst the factors analyzed for an association with AID (age at diagnosis of SAA, sex, severity of the AA, presence of HLA-DR15, type of first-line therapy, splenectomy and follow up time) only age showed a significant association: median age 51.5 y (9-75 y) in patients with, versus 20 y (1-80 y) (p=0.00013) in patients without AID. All 13 patients with AID before SAA were treated with ATG. 2/12 showed remission of both diseases, AID and SAA, whereas in 10 patients the AID persisted. Concomitant AID did not impair outcome of SAA. After ATG therapy, an AID was diagnosed in 11 patients, with a median time to occurrence of 7 y (0.03-27.5 y). Two patients developed AID after HSCT, in both the donors did not have an AID. In conclusion, simultaneous AID is seen in 10% of patients with SAA. Coincidence is unlikely to be sufficient explanation. Response to therapy in contrast, is independent and AID can persist despite full remission of SAA. This indicates that SAA and AID most likely are triggered by independent immune mechanisms. Should haematopoietic growth factors be included in the immunosuppressive treatment for severe aplastic anaemia -a systematic review and meta-analysis R. Gurion* (1), A. Gafter-Gvili (1), M. Paul (1) , L. Vidal (1) , I. Ben-Bassat (2) , M. Yeshurun (1) , O. Shpilberg (1) , P. Raanani (1) (1)Rabin Medical Center (Petah-Tikva, IL); IL) Background: Immunosuppressive therapy (IST) is the alternative treatment for patients with severe aplastic anemia (SAA) not eligible for transplantation. The role of hematopoietic growth factors (HGF) as adjunct to IST in these patients is unclear. Objectives: To evaluate the role of the addition of HGF to IST in patients with SAA. Methods: Systematic review and meta-analysis of randomized controlled trials comparing treatment with IST and HGF to IST alone in patients with SAA. The Cochrane Library, MEDLINE, conference proceedings and references were searched until 2007. Outcomes assessed were: all-cause mortality, overall and complete hematologic response, infections and clonal evolution (transformation to myelodysplastic syndrome or leukemia). Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled. Results: Our search yielded 6 trials. The IST regimen for most trials consisted of anti-thymocyte globulin, cyclosporine and steroids. The HGF in 5 trials was G-SCF and in 1 trial GM-CSF and erythropoietin. The addition of HGF to IST, compared with IST alone yielded no difference in all cause mortality at 100 days and 1 year (RR 1.09, 95% CI 0.39-3.55; RR 0.69, 95% CI 0.34-1.38, respectively, Fig.1 ). There was a significant advantage in terms of complete hematologic response in favor of HGF at 3 months (RR 1.44, 95% CI 1.12-1.85), which was not statistically significant after 1 year (RR 1.41, 95% CI 0.88-2.25). There was a statistically significant decrease in relapse rate with HGF (RR 0.45, 95% CI 0.30-0.68). The number of patients with clonal evolution was higher, though not statistically significant, in the HGF arm (RR 2.8, 95% CI 0.98-8.05, Fig.2 ). There was no difference in the number of infections between the 2 arms (RR 1.07, 95%CI 0.87-1.32). No heterogeneity was observed for the presented analyses. Conclusions: The addition of HGF to IST in SAA improves hematologic response in the short term and reduces relapse rate. However, it does not influence all-cause mortality, long term response, or the incidence of infections. In addition, it is associated with a trend for an increased rate of clonal evolution. Therefore, it should not be routinely used for SAA. Double cord blood transplantation in bone marrow failure syndromes R. Peffault de Latour*, A. Ruggeri, V. Rocha, M. Robin, C. Arrais, J. Larghero, D. Rea, R. Traineau , P. Ribaud, C. Ferry, A. Devergie, E. Gluckman, G. Sociè Hôpital Saint Louis (Paris, FR) The outcome of severe aplastic anemia (SAA), refractory to immunosuppressive therapy or related to Fanconi anemia (FA), is usually poor in the absence of hematopoietic stem cell transplantation. Single cord blood is an alternative stem cell source for patients without matched related or unrelated donors, but is associated with high transplant related mortality due to the low cell dose infused. We thus performed double cord blood transplantation (dCBT) in 14 patients (6 male and 8 female) with a median age of 16 years , diagnosed with bone marrow failure syndromes (8 FA, 5 SAA and 1 PNH) from 2004 to 2007. Median disease duration before dCBT was 31 months (5-240). At transplant median neutrophil and platelet count were 0.3 x 10 9 /L (0.0-1.1) and 21 x 10 9 /L (3-93), respectively. All patients were highly transfused before transplant. Six patients (43%) received a dCBT as a rescue of previous rejected transplants (2 SAA and 4 FA). All patients received a fludarabine-based regimen with TBI (2 Gy) for 4 patients. Cord blood units were 4/6 or 5/6, HLA A, B and DR match with the patients (one was 3/6). Graft versus host disease (GVHD) prophylaxis consisted in cyclosporin + mycophenolate mophetile for 5 patients. Steroids were given from day 7 to day 14 and stopped in case of no GVHD. Median cell doses infused were 4.8 x 10 7 NC/Kg (1.8-9.7) and 2.9 x 10 5 CD34+cells/Kg (0.5-7.46). Graft rejection was observed in 6 patients (3 previously allotransplanted). Among the remaining 8 patients, the median time to achieve an absolute neutrophils count >500 was 28 days (14-42) and median time to a platelet count > 20,000 was 83 days. All engrafted patients obtained a full donor chimerism (one cord blood unit) at 100 days after dCBT. Acute GVHD grade II-III was scored in 9 patients (70%) (6 grade II, 3 grade III). One patient presented acute GVHD grade IV. Six patients out of 8 developed chronic GVHD (3 limited and 3 extensive). Six patients died (2 GVHD, 2 fungal infections, 1 thrombotic microangiopathy, 1 sepsis). With a median follow-up of 13 months (5-22), the overall survival (OS) was 57% (±13%) for all patients. The median survival of patients who were transplanted twice was 50% (±13%). dCBT seems to be an option to treat patients with bone marrow failure syndromes and without matched HLA donors. Those results need to be confirmed in a prospective study to warrant the inclusion of dCBT in the treatment strategy of diseases with high risk of rejection. Pre-transplant serum ferritin impacts on outcome in patients undergoing allogeneic haematopoietic stem cell transplantation for aplastic anaemia E. Grey-Davies*, P. Datta-Nemdharry, S. Ball, J. Marsh St George's Hospital (London, UK) Background: Iron overload has been identified as an adverse prognostic factor for haematopoietic stem cell transplantation (HSCT) and a risk factor for infection, hepatic dysfunction and veno-occlusive disease. No studies have investigated the contribution of iron overload to morbidity and mortality after HSCT in the aplastic anaemia patient group. Objectives: To investigate the impact of ferritin on transplant related mortality (TRM), overall survival (OS), frequency of infection, liver function and cardiac complications of HSCT. Patients and methods: A single centre retrospective study of all HSCTs carried out for aplastic anaemia (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) in whom a baseline ferritin within 3 months of transplant was available. Patient characteristics: 26 male, 15 female; median age 23 years (range 7 to 53); 36 acquired and 5 congenital; 27 matched sibling and 14 matched unrelated donors and median time from diagnosis to transplant 146 days. For acquired aplastic anaemia a non-irradiation based regimen was used. Analysis was done using univariate, Kaplan-Meier and Cox-Regression using the following co-variates; age, sex, graft source, donor type, CMV seropositivity and time from diagnosis to transplant. Results: Median pre-transplant ferritin was 1270 ug/l (range 63-7442) and ferritin level correlated with number of previous red cell transfusions (p=0.01, r=0.412). There was a significant relationship between baseline ferritin and both TRM (p=0.021) and OS (p=0.038, see figure) , with a higher ferritin associated with a poorer outcome. In multivariate analysis hazard ratios for patients with ferritin values in the highest quartile, as opposed to the other 3 quartiles, of 5.2 (p= 0.02) for TRM and 3.7 (p=0.044) for OS were observed. There was no association between ferritin and number of bacterial or fungal infections or peak liver function tests (bilirubin, alanine transaminase (ALT), alkaline phosphatase (ALP) and albumin) from the onset of conditioning to 100 days post transplant. Pre-transplant ferritin was positively correlated with ALT (p=0.047) but not with any other liver function parameter. No veno-occlusive disease or cardiac events were reported. Conclusion: This data suggests an elevated serum ferritin prior to HSCT for aplastic anaemia confers an increased risk of TRM and reduces OS. Myelosuppressive cytokine profile in acquired aplastic anaemia may potentiate immunosuppression with targeted anti-cytokine treatment C. Dufour *, E. Ferretti, O. Burlando, J. Svahn, M.T. van Lint, D. Longoni, M. Pillon, A.P. Iori, F. Bagnasco, U. Ramenghi, A. Bacigalupo, A. Locasciulli, A. Misuraca, M. Lanciotti, S. Pigullo, V. Pistoia, A. Corcione for the Marrow Failure Study Group of the AIEOP Background: TNF-a and IFN-g are final effectors of death of haematopoietic cells in Acquired Aplastic Anemia (AAA). Combined Immunosuppression (IS) with ATG and CyA improves haematopoiesis in most patients but has a relapse rate of about 30%. Aims: To characterize the role of TNF-a, IFN-g and IL-4 in the clinical course of AAA treated with combined IS. Methods: A first group of 72 subjects, 41 in Active Disease (AD) (onset, relapse, never responding to IS) and 31 in Non Active Disease (NAD) (responding to IS), median age 11.7 years (range 0.4-56 yrs) was tested for TNF-a, IFN-g and IL-4 expression in marrow CD3+. Thirty-six subjects (18 AD and 18 NAD) were also tested for BFU-e growth after in vitro block of TNF-a and/or IFN-g. A second group of 21 patients, median age 10 years (range 2-32) was prospectively tested for TNF-a, IFN-g and IL-4 expression in CD3+ marrow cells at diagnosis and at response evaluation time (RET) after IS. Results: First group: TNF-a and IFN-g were equally expressed in CD3+ of AD and NAD but more than in normal controls (p<0.0001 and 0.001 respectively). IL4 was equally expressed in AD, NAD and controls. Transfusions and CsA, did not influence cytokine expression. In NAD group in vitro TNF-a block equally increased BFU-e vs AD but more than in normal controls (p0.029). The same did the double TNF-a and IFN-g block (p0.014). Single IFN-g block did not increase BFU-e in NAD vs AD vs controls. In AD IFN-g and double block increased BFU-e over controls (p 0.011, 0.007 respectively) whereas TNF-a block did not. Second group: TNF-a and IFN-g at diagnosis did not differ in patients who at RET were Responders vs Non Responders to IS. Both TNF-a and IFN-g (median absolute number of CD3+ marrow cells expressing cytokine in the cytoplasm) were significantly lower at RET vs Diagnosis in Responders (p 0.02). No significant difference was found at RET vs Diagnosis in Non Responders. IL4 was significantly lower at RET vs Diagnosis (p 0.003) in Non Responders but not in Responders. Conclusion: 1.TNF-a and IFN-g are over-expressed in marrow of both AD and NAD AAA patients. 2.TNF-a is still present in the marrow of patients who respond to IS and impairs their haematopoiesis. 3. TNF-a and IFN-g decline more in patients Responding vs Non responding to IS. Overall these data point to need to strengthen IS with targeted anti TNF-a treatments that may reduce the risk of relapse after IS. Background: Telomeres are nucleoprotein structures that protect chromosomes ends. The ribonucleoprotein complex is responsible for de novo synthesis and maintenance of telomere, mutations in complex genes lead to premature telomere shortening and are responsible for different forms of dyskeratosis congenital: X-linked (DKC1), autosomal dominant (TERC) and autosomal recessive (NOP10, TERT). Heterozygous mutations in TERC gene were previously found by our group also in patients with apparently acquired aplastic anemia (AA). The aim of this work is to analyze the possible involvement of the telomerase complex gene GAR1, in a population of Italian AA patients. Methods: DNA of 108 AA patients (74 pediatrics and 34 adults) and 170 normal controls (94 pediatrics and 76 adults) was amplified by PCR analyzed by DHPLC. For each abnormal elution profile PCR products were directly sequenced using ABI prism 3100 genetic analyzer. Results: No mutation of GAR1 (GenBank NM_018983) was found in AA patients nor in controls. However, DHPLC analysis revealed the presence of the following polymorphisms already reported in GenBank: S142 Exon1: 5' UTR -148 -/T (refSNP ID rs11433030) Exon 2: c.376 T>C synonimus (refSNP ID: rs2276326). Exon 3: IVS3 -46C>T. Exon 7: 3'UTR + 1228C>G (refSNP ID: rs10365). Frequencies of these polymorphisms in our sample are similar to that reported in Genbank. Moreover, in exon 2, we identified, in two patients and one control, the new c.386A>T variation, which is not reported in GenBank, and leads to p.H28L. amminoacidic change. Also the frequency of this variation was similar in AA patients and controls (1.8% vs 0.6%). Telomere analysis shows that the subjects carrying the change have a telomere length comparable to that of healthy controls thus suggesting that this variation has no effect on telomerase complex activity. Conclusions: We did not found any clear disruptive mutation in GAR1 gene. The non conservative variation identified in our sample has no effect on telomeres length. This result suggests that heterozygous point mutations in GAR1 gene are not responsible for AA in our patients at least acting via telomere. However, in our experience, molecular analysis of other telomerase complex gene (TERC, TERT) is very important, for AA patients and familial, in order to set up an adequate therapeutic or surveillance program, to early identify mutation carrier and exclude them as potential bone marrow donor. Management of paroxysmal nocturnal haemoglobinuria in the eculizumab era: the bedside and beyond A Risitano* (1), L Marando (1) Eculizumab (Soliris®, EC) is a terminal complement inhibitor which proved to be very effective in controlling the complement-mediated intravascular hemolysis typical of PNH. We have collected clinical and experimental data from 31 transfusion-dependent Italian PNH patients, 22 of them initially enrolled within the EC-based international trials, and 9 receiving EC according to the Italian early access program. All patients showed blockade of intravascular hemolysis, pointed out by a striking LDH level reduction; this led to cessation (75%) or reduction (25%) in transfusion requirement, improvement in QoL measures and fewer thromboses. In order to maximize clinical response, some patients received ancillary medications, such as iron and/or erythropoietin supplementation or low dose steroids. Two patients with concomitant aplastic anemia received successful immunosuppression (IS) by alemtuzumab (while on EC). After the initial observation of positive C3d Coombs test in some patients, we systematically investigated C3 coating on RBC by flow cytometry, using anti-C3 polyclonal Abs in combination with anti-CD59. RBCs from healthy controls were CD59+/C3while RBCs from patients with cold agglutinin disease were mostly CD59+/C3+ (positive control). Untreated PNH patients had both CD59+/C3-and CD59-/C3-but not C3+ RBCs. During EC treatment all 31 patients showed a substantial proportion of CD59-/C3+ RBCs but never CD59+/C3+ RBCs; the kinetic of C3 opsonization was demonstrated in the 6 patients receiving EC de novo, showing the first C3+ RBCs after one week of therapy, with progressive increase after the subsequent administrations. We hypothesized that C3-coating of RBCs due to the lack of CD55 may lead to residual extravascular hemolysis through the reticulo-endothelial system via complement receptors, which is an undescribed mechanism of disease in PNH. As proof of principle, one patient still requiring transfusions underwent splenectomy, with excellent clinical response and no infectious complication, while some others obtained benefit by low dose steroids. In conclusion, we confirm that EC is the first safe and effective treatment for intravascular hemolysis of PNH patients and suggest that some patients may further benefit from ancillary therapies including iron, erythropoietin and IS. Our laboratory findings suggest that C3 accumulation on RBC surface may occur during EC therapy, which may contribute to residual extravascular hemolysis in PNH patients. Aplastic anemia (AA) has been rarely described in patients with previous autoimmune diseases (AIDs). We collected retrospectively the cases of AA recorded in the EBMT registry in patients previously diagnosed of AIDs. 46 patients from 12 countries and 18 centres were eligible. Considering the number of patients at risk in those centres (1374) the prevalence was 3.3%. Data collection was completed for 43 of 46 patients (93%). They were 20 male and 23 female with median age of 49 years (range 10-75) at AIDs and of 53 years (range 12-77) at AA. At last f-up, 25 patients were alive, 15 were died for several causes (infection 6, COPD 2, bleeding 2, other 5) whilst 2 patients were lost at f-up (data missing for 1 patient). The survival rate was 63% (25/40). Several diagnosis of AIDs were reported, rheumatoid arthritis being the most frequent (10). AA was considered an adverse effect of drugs administered for AIDs in 18 of 34 patients (53%) for whom the datum was reported by local investigator. The severity of AA was: NSAA or PRCA 16; SAA 12; VSAA 11; unknown 4. 40 patients were treated for AA whilst 2 patients had a spontaneous recovery. The treatment was unknown for 1 patient. 32 patient received IS therapy and 8 underwent HSCT. For patients treated with IS, 13 achieved a CR and 6 a PR (response rate of 59%). ATG/ALG were used in 30 of 32 patients treated with IS mainly associated to CSA. 12 patients received other treatments in addition or after ATG/ALG/CSA. 7 of 8 patients who performed HSCT were alive whilst 1 died of viral infection. Only 6 patients were reported as still on therapy for AA mainly with low doses of steroids or androgens. In 11/26 patients (42%) for whom the datum was reported, the local investigators judged that therapy for AA contributed to achieve the CR of previous AIDs whilst in 13 and 2 patients, respectively, no effect on or a flare of AIDs were observed with recovery of AA. We conclude that AA following AIDs needs in most cases a standard approach (IS ± HSCT), the response rate figures being superimposable to those of idiopathic AA. This treatment, in turn, may contribute to a better control of underlying AIDs. Comparable long-term results after related and unrelated stem cell transplantation for severe aplastic anaemia -a single-centre analysis K. Kolbe*, J. Beck, A. Ullmann, C. Huber Johannes-Gutenberg University (Mainz, DE) Severe aplastic anaemia (SAA) is a rare, potentially fatal bone marrow failure syndrome. Immunosuppressive combination regimens and allogeneic stem cell transplantation are the only curative treatment options. We report on fifteen patients with SAA transplanted at our centre between 1996 and 2005. Nine of them (aged 14-55 years) had HLA-identical related donors and were transplanted at time of diagnosis (n=4) or after failure on immunosuppressive therapy (n=5). Six patients (aged 31-49 years) with refractory/relapsed disease got stem cells from an unrelated donor (4 matched for 10/10 loci, 2 mismatched for 1 C-antigen). Molecular HLA-typing in the unrelated setting followed low resolution for class-I-antigens and high resolution for class-II-antigens since 2000. Peripheral blood stem cells had been predominently used, in 7/9 related and 5/6 unrelated transplants. Pre-transplant conditioning consisted of cyclophosphamide and ATG(rabbit, Fresenius). Three patients additionally received TLI 6 Gy or busulfan (2 patients because of HLA-differences, 1 patient had coexisting paroxysmal nocturnal haemoglobinuria [PNH] ). Cyclosporin A and short course of methotrexate were used for prophylaxis against graft-versus-host disease (GvHD). Fourteen patients are alive, one patient transplanted with related stem cells died of fungal infection before engraftment. All fourteen patients engrafted with a granulocyte count > 0,5 x 10h(9)/l and a platelet count > 20 x 10h(9)/l after a median of 21 days (range 16-48) and 23 days (range 13-51), respectively. Two patients experienced acute GvHD grade II or III after sibling transplantation. In the unrelated group only two patients had mild acute GvHD grade I, with 5/5 tested patients having complete donor chimaerism. No chronic GvHD has been observed. After a median follow up of 64 months (range 1-122) for the related and 29 months (range 24-100) for the unrelated group the 5-year probability of survival is 89% and 100%, respectively. We conclude that stem cell transplantation from unrelated donors provides excellent results for patients with refractory or relapsed aplastic anaemia with respect to morbidity and mortality, comparable to those after related transplantation. Outcome of adult patients with severe aplastic anaemia treated with immunosuppressive treatment: a singlecentre experience E. Bojtarova *, M. Mistrik University Hospital (Bratislava, SK) Objectives: Immunosuppression (IS) is the current treatment of choice for patients with acquired severe aplastic anaemia (SAA) who do not have matched sibling donors (MSD) or who are otherwise ineligible for allogeneic bone marrow transplantation (BMT). The aim of this analysis was to evaluate 19 years of local experience in treating SAA with IS with emphasis on long-term outcomes. Patients and methods: 32 adult SAA patients, aged 15 to 57 years, were treated with IS therapy, either horse antilymphocyte (ALG, 20 patients) or rabbit antithymocyte globulin (ATG, 12 patients) with ciclosporin (CsA) from 01/1988 to 01/2007. 22 patients had SAA a 10 very severe AA. The median interval from diagnosis to IS was 27 days (range, 2-3314). The median follow-up for surviving patients was 67 months (range, . Results: 5 patients died early after start of treatment because of sepsis and hemorrhage. 23 of 27 evaluable patients (85%) showed complete (CR) or partial response (PR). The median time to CR was 6.4 months (range, 1.1-13.9) and the median time to PR was 2.3 months (range, 0.03-16.6). Relapse of SAA was observed in 4 of 17 complete responders in median of 15.5 months after ATG therapy. 2 cases of acute leukemia developed in non-responders in 8.4 and 19.8 months after the first ATG therapy. 3 of 4 patients with no response (NR) and 3 of 4 patients with relapse were treated with second course of IS, 1 patient received a BMT from MSD. Unrelated BMT was performed in 1 patient with NR after 2 cycles of IS. The probability of 5-year survival was 70% and 10-year survival was 62%. The probability of disease-free survival was 55%. Probability of long-term survival was significantly influenced by the absolute neutrophil count (ANC) prior to ATG treatment (43% vs. 75%, for ANC ≤ 0.2 10 9 /L vs. ≥ 0.2 10 9 /L; P = 0.03) and by response to IS at 6 months after ATG treatment (88% vs. 63%, for response to IS at 6 months vs. no response; P = 0.04). Patient age, disease severity at diagnosis, disease duration, relapse and treatment regimen (horse ALG/rabbit ATG or number of days of ALG administration) did not significantly affect survival. Conclusion: These results suggest that combined immunosuppressive therapy can achieve a high response rate and long-term survival among adult patients with SAA. Long-term outcome of bone marrow versus peripheral blood stem cell transplantation: a retrospective singlecentre analysis D. Nachbaur*, J. Auberger, B. Kircher, B. Lindner, J. Clausen Hematology and Oncology (Innsbruck, AT) Background: The administration of hematopoietic cells from the peripheral blood provides an alternative to transplantation using bone marrow-derived hematopoietic cells. Patients and Methods: Between Sept 1983 and Jun 2007 329 adult patients (median age 40, range 18-76) received a first allogeneic SCT from either sibling (n=203) or volunteer unrelated donors (n=126). The source of stem cells was BM in 177 pts. and PB in 152 pts. Indications for SCT were malignant (AL, 181 pts.; CML, 66 pts.; Lymphoma/Myeloma, 49 pts.; MDS/MPD, 21 pts.) and non-malignant hematological diseases (AA, 9 pts.; others, 3 pts.). Conditioning was myeloablative in 245 pts. and of reduced intensity in 84 pts. Results: After a median survival for patients alive of 12.6 years for BM and 3.5 years for PB SCT recipients OS for the entire cohort was 37% (31%-43%, 95% CI), the relapse incidence was 30% (25%-36%, 95% CI, and the NRM was 43% (38%-49%, 95% CI). OS and relapse were identical for both groups. There was a trend for a higher NRM in BM vs. PB SCT recipients. Regarding GVHD there was a trend for more aGVHD II-IV in the PB group (59% vs. 53%) but aGVHD III-IV was significantly more frequent in the BM group (24% vs. 14%). In contrast cGHVD was significantly more frequent in the PB group (48% vs. 24%, p=0.0001). When only myeloablative transplants were considered for analysis there S144 was a trend for better OS (52% vs. 38%, p=0.1) in the PB group due to significantly lower NRM (32% vs. 46%, p=0.05). The trend for better survival was only observed in standardrisk pts. (AL in CR1 and CML in CP1) but not high-risk disease (data not shown). By multivariate analysis including recipient and donor age as continuous variables, and donor type, stem cell source, conditioning, risk by the underlying disease, HLA-mismatch, sex mismatch, CMV status of donor and recipient, and transplant >1995 as categorical variables peripheral blood stem cell transplantation was only significantly predictive for chronic graft-versus-host disease (RR 2.29, p=0.02). Conclusion: Our long-term results in a large cohort of patients failed to demonstrate any survival benefit of peripheral blood stem cell over conventional bone marrow transplantation even in high-risk patients. Long-term follow-up of prospective randomized trials and QOL analyses with regard to chronic graft-versus-host disease in peripheral blood stem cell transplant recipients are highly warranted. Allogeneic hematopoietic stem cell transplantation (HSCT) has a high success rate in patients <16 years of age with a hemoglobinopathy and an HLA-matched sibling donor. Inability to identify HLA-matched related or suitably matched unrelated donors and general intolerability for any risks of transplant-related mortality have markedly limited the use of this treatment option. In an attempt to widen the pool of acceptable donors, unrelated cord blood (UCB) from HLA matched or mismatched donors have been explored as an alternative. Between 2000 and 2006, 19 patients with thalasemia major (Thal) (n=12) and high risk sickle cell disease (SCD) (n=7) underwent unrelated UCBT. All patients with Thal were transfusion-dependent; 5 patients with SCD had a history of stroke. UCB units were matched at 6 of 6 (n=4), 5 of 6 (n=6) and 4 of 6 (n=9) HLA loci with typing at antigen level for HLA A and B, and allele-level for HLA-DRB1. Median infused cell dose was 5.8x10 7 /kg. Fifteen patients received a myeloablative conditioning regimen consisting of busulfan [BU]>8mg/kg with or without ATG/ALG and other chemotherapeutic agents and 4 received a reduced intensity conditioning [RIC] regimen consisting of limited field irradiation with BU <8 mg/kg or melphalan <150mg/m² with other chemotherapeutic agents. GVHD prophylaxis consisted of cyclosporine (n=14) or tacrolimus (n=4); information was missing for one case. Thirteen of 19 patients achieved neutrophil recovery (>500mm 3 ). While none of the 4 recipients of RIC achieved sustained chimerism, 8 of 15 recipients of a myeloablative regimen did so. All 8 had complete chimerism; no patient had mixed chimerism. Five of these 8 patients are alive with a median follow up of 2 years. Four patients developed grade II (n=2) and grade III (n=2) acute graftversus-host disease (GVHD) and 1 patient had chronic GVHD. All patients with SCD are alive; 3 are disease-free and 4 have had autologous reconstitution. Seven of 12 patients with Thal are alive; 2 are disease-free and the remaining 5 survivors have had autologous reconstitution (n=3) or second transplantation after myeloablation with complete chimerism (n=2). Of the 5 deaths, 4 occurred prior to day 100 and one at 10 months after transplantation. While the numbers of subjects were small, these results suggest a peculiarly high risk of graft failure in heavily transfused patients with hemoglobinopathy, particularly after a RIC regimen. Exploration of novel approaches in prospective clinical trials is needed. (2) (1)University of Bologna (Bologna, IT); (2) University of Illinois at Chicago (Chicago, US) Background: The role of donor antigen presenting cells (APC) in human bone marrow grafts in post-transplant clinical outcome is not well established, although data from animal models suggest that they may regulate GVH responses and immune reconstitution early after transplant. Aim: In this retrospective study we tested whether marrow composition of donor APC was associated with outcome after allogeneic transplantation. Patients and methods: 81 consecutive patients undergoing allogeneic bone marrow transplantation in our institution from June 1999 through June 2006 were analyzed, provided that graft composition data were available. All but 1 patient received the marrow from HLA matched unrelated donors. Of 81 patient, 67 received a myeloablative and 14 a reduced intensity conditioning regimen. ATG was also administered in 79/81 patients. Graft numbers of CD11c+ myeloid DC (mDC), CD123+ plasmacytoid DC (pDC) and CD14+ monocytes were determined based on flow cytometry analysis. Results: After a median follow up of 346 days (interquartile 132-968), the following transplant-related events were recorded: acute GVHD II-IV in 18 and extensive chronic GVHD in 22 patients and relapse in 19 patients. Transplantrelated mortality (TRM) occurred in 22, and relapse-related mortality in 11 patients. Patients were divided based on median donor graft mDC (0.81x106/kg recipient weight, or pDC (0.67x106/kg), or monocyte (6.6x106/kg) counts. No correlation was observed between acute and chronic GVHD as well as relapse and APC counts. However, the mortality rate was significantly higher in patients who received a greater dose of monocytes (21/40 vs 12/41) (p= 0.02), with a trend for a more elevated TRM (14/41 vs 8/41) (p=0.06), as compared to patients who received a lower dose of monocytes. mDC and pDC counts did not correlate with survival after transplant. In univariate analysis, overall mortality correlated also with advanced disease (p<0.0001) and T cell (p=0.04) counts, whereas TRM correlated with advanced disease (p<0.001). However, in multivariate analysis, monocyte counts correlated with both TRM (HR 2.5, 95% CI 1.3-4.8) (p=0.06) and overall mortality (HR 2.5, 95%CI 1.2-5.3) (p=0.01). Conclusion: Donor CD14+ cell dose in bone marrow grafts independently correlates with mortality following allogeneic transplantation suggesting a possible role of donor mature accessory cells in the regulation of post-transplant immunologic events. Safety profile of plerixafor + G-CSF: results from 2 phase III studies G. Calandra* (1), J.F. DiPersio (2) , I. Micallef (3) , P. Stiff (4), E. Stadtmauer (5), R.T. Maziarz (6) , B.J. Bolwell (7), J. Angell (1) Plerixafor (AMD3100)+G-CSF has been shown in 2 phase III, multicenter, randomized, double-blind, placebo-controlled studies to be more effective than placebo+G-CSF in mobilizing hematopoietic stem cells (HSC) for autologous transplant in NHL and MM patients. The combined safety results from these studies are reported herein. Methods: Adult NHL or MM patients, in 1st or 2nd CR or PR, planned for autologous HSC transplant, were eligible. All patients received G-CSF (10mcg/kg/day SQ) on Days 1-4 and then at ~10PM on Day 4, patients received either plerixafor (240mcg/kg SQ) or placebo. Apheresis began on Day 5 (~10 hrs after study drug). Patients continued to receive daily G-CSF and plerixafor or placebo either for 4 days or until study endpoint was met. The safety profile was compared between 297 patients (150 NHL, 147 MM) treated with plerixafor + G-CSF and 296 patients (145 NHL, 151 MM) treated with placebo + G-CSF. Adverse events (AEs) and serious AEs (SAEs) were analyzed based on 3 study periods: 1) mobilization/ apheresis, 2) chemotherapy/transplant, and 3) post-transplant until 12 months. Results: Overall incidences of AEs and SAEs were 65% and 0.7% in the plerixafor group, and 43% and 0.7% in the placebo group, respectively. During Period 1, AEs occurring more frequently in the plerixafor group than in the placebo group were GI-related (diarrhea 37% vs. 17%; nausea 34% vs. 23%; vomiting 10% vs. 6%; flatulence 7% vs. 4%; abdominal pain 4% vs. 2%), injection site reactions (erythema 26% vs. 5%; pruritus 6% vs. 0.7%), dizziness (10% vs. 6%), insomnia (7% vs. 5%), night sweats (5% vs. 2%), and hypotension (4% vs. 2%). These AEs were generally mild to moderate. In this period, SAEs were limited to hypotension plus dizziness (n=1),thrombocytopenia (n=1) and non-durable graft (an SAE by per-protocol definition) (n=1; clinically stable) with plerixafor and chest pain (n=1) with placebo. One NHL patient in the plerixafor group, who had cytogenetic abnormalities prior to the study, developed MDS posttransplant. In Periods 2 and 3, and there were no other drug-related SAE and no drug-related deaths. The causes of death were similar between groups. Conclusion: Similar to previous trials involving in more than 700 patients, plerixafor was generally well tolerated in the Phase III studies, with GI and injection site reactions being the most common AEs. S. Meyer-Monard* (1), J.R. Passweg (2), C. Troeger (3), H.P. Eberhard (4), E. Roosnek (2), G. Nicoloso de Faveri (5), Y. Chalandon (2), A. Rovo (1), O. Irion (2), W. Holzgreve (3), A. Gratwohl (1), C. Müller (4), A. Tichelli (1), J.M. Tiercy (6) (1 Introduction: Allogeneic hematopoietic stem cell transplantation is a standard therapy for many hematological diseases. For patients lacking a matched sibling donor, transplantation with stem cells from umbilical cord blood (UCB) is an established therapy. A major aim of public UCB banking is to establish an inventory of UCB units with a large HLA diversity. Few studies have addressed whether UCB banks and volunteer unrelated donor (VUD) registries recruit donors with different HLA types and whether UCB banks collect units with rare alleles and rare haplotypes. Methods: This retrospective analysis compares the distribution of HLA-A, -B and DRB1 alleles and haplotype frequencies in 1441 UCB units and 3465 VUD from two centres in Switzerland with distinct migrant populations. Comparisons of allele frequencies were done for HLA-A,-B and DRB1 at 2digit level typing and at 4-digit level for DRB1. Alleles occurring with a frequency of < 0.05% in Caucasians were considered as rare. Haplotype frequencies were estimated from phenotype data as described by C. Müller et al (Hum Immunol, 2003) . The chi-squared test for proportions was used for comparisons and a cut off of 0.001 accepted as significantly different. Results: Comparison of HLA-A, -B and -DRB1 2-digit allele frequencies showed no significant difference in distribution of HLA-A, and -DRB1 among UCB donors and VUD. Significant differences in HLA-B distribution were due to minor differences in antigen frequencies. No significant differences were observed for the generic DR1-DR16, but a highly significant difference was detected comparing DRB1*11,*13, and *14 allelic frequencies. Twenty-nine different rare alleles were found in UCB donors, whereas only one was found in VUD. The frequency of 6 haplotypes was significantly different in UCB and in VUD. Four of these haplotypes occurred significantly more frequently and 2 less frequently in UCB than in VUD. Conclusions: These data should impact on UCB collection strategies. HLA allele and haplotype distribution differ between UCB and VUD. This difference is only found with intermediate/high resolution typing. Targeted recruitment of UCB units from non-Caucasian donors could further increase HLA allele diversity of available donors. It is likely that also HLA-class-I high resolution matching will turn out to impact outcome after UCB HSCT. Intermediate/high resolution DNA typing should become standard in UCB in order to identify rare alleles and avoid mismatches. We previously reported that NK cells generated after haplomismatched stem-cell transplantation are blocked at an immature state characterized by specific phenotypic features and impaired functioning having potential impact for immune responsiveness and transplantation outcome (Nguyen, Blood 2005) . In the present study we performed a prospective study of NK cells reconstitution in 29 adults (mean age 42 years), who received unrelated umbilical cord blood transplantation (UCBT) for advanced hematopoietic malignancy (52% myeloid leukemia, 41% lymphoid diseases and 7% aplastic anemia). We used a reduced-intensity conditioning regimen. The total median nucleated cell count per graft was 3.7 x 107/kg. Six of 29 patients received two UCB units. The median time to a neutrophil engraftment was 21 days (ranged 6 to 33). 31% of patients developed acute GvHD Grades II to IV. Among the 22 patients whose follow-up was more than 6 months post-graft, the survival rate was 73%. Peripheral blood was collected from patients following UCBT, and was compared with the donors and a panel of 13 healthy volunteers. NK cells generated after SCT exhibited a similar profile in all patients. The CD56bright immunoregulatory NK cells subset was significantly increased, representing a median of 50% (ranged 10 to 83%) at 2 months post-UCBT, versus a median of 8% (ranged 5 to 27) in the controls. In addition, the inhibitory receptor CD94/NKG2A was strongly over-expressed by NK cells generated after UCBT; indeed, 80% of NK cells expressed NKG2A at 2 months versus 54% in the controls. This phenotype persisted during the 6 months of the follow-up. However, a large panel of NK receptors, including NKp30, NKp44, NKp46, NKp80, NKG2D, 2B4, and LAIR-1, presented a similar profile in patients following UCBT than in controls. Interestingly, functional analysis performed either by CD107a degranulation assay and 51Chromium released assay revealed that NK cells generated from UCB rapidly acquired cytotoxic capacities, around 62% at 1-month, 51% at 3-months post-UCBT and 50% in the controls, for an effector/target ratio of 20/1. Overall, this study strongly suggested that after UCBT, NK cells presented some phenotypic features, currently observed after transplantation, but also functional characteristics, which renewed interest of alloreactive NK cells to mediate graftversus-leukemic (GvL) effect following transplantation. I. Dolgopolov*, R. Pimenov, N. Subbotina, V. Boyarshinov, I. Visochin, G. Mentkevich Haploidentical SCT is the therapeutic modality for patients lacking HLA-identical donor. RIC ensures good tolerability and safety, early stable full donor chimerism with potential GvT effect. Since 2001 we performed 42 haplotransplantations from relatives in 36 pts with poor-prognostic malignancies: 8-AML, 3-ALL, 4-CML, 5-JMML, 1-MDS, 3-NHL, 7-NB, 4-EWS and 1-melanoma. RIC regimen included Fludarabine 180 mg/m², and ATG 40 mg/kg in combination with Busulfan 8 mg/kg (n=36) or Treosulfan 30000 mg/m² (n=6). The PBSC with a median number of 7.2 (2-35)x106 CD34+cells/kg with 4.4 (2.2-12.4)x108 CD3+ cells/kg were infused after incubation in vitro with vincristine and methylprednisolone. T-depletion was not performed. GvHD prophylaxis consisted of short methotrexate and cyclosporine A. Toxicity was transient and manageable in all but two pts. The main organs involved were GI and/or liver. One pt died from regime-related toxicity on d+26. Two pts with PD at the moment of transplantation failed to engraft and died from infection. Two pts with JMML and 1 pt with AML progressed within first month after transplantation. Thirty one pts recovered with a median time to WBC> 1x109/l and PLT> 1x109/l on d+11 (9-31) and d+12 (9-40), respectively. All recovered pts experienced a full donor chimerism by d+90 or earlier. Five pts (16%) did not develop any acute GvHD, 9 pts (28%) had grade I, 12 pts (37%) had grade II, and 6 (19%) pts had grade III a GvHD at 100 days after SCT. Acute GvHD was successfully treated with steroids and ATG. Four pts were retransplanted (3-JMML, 1-CML). One pt engrafted and now is disease free for 1 year, 1 pt died due to regimen toxicity, 2 pts relapsed and died. Nine (25%) out of 31 long term survivors are alive (8 in CR) at a median follow-up 30 (6-73) mo, 16 (44%) pts relapsed, 5 (16%) pts died from sepsis and 5 (16%) from chronic GvHD and infection. Chronic GvHD was observed in 3 (local) and 3 (extensive) pts who are alive. For the whole group DFS and EFS were 34% and 18% with a median f-up of 28 and 17 mo, respectively. For pts with hematological malignancies DFS and EFS were 44% and 23% with a median f-up of 34 and 20 mo and for pts with solid tumors 20% and 8% at 10 and 8 mo, respectively. The use of GCSF-mobilised Peripheral Blood Stem Cells (PBSC) for unrelated donor (UD) transplantation has increased dramatically since 2000. The association of PBSC with more rapid engraftment and with an increase in chronic Graft versus Host Disease (GvHD), compared to bone marrow (BM) has been reported in a number of studies. More recently the use of PBSC has been associated with an increase in transplant related mortality (TRM) and decrease in survival (OS) in T-cell replete transplants. We sought to analyse the impact of PBSC compared to BM in a cohort of UD transplant recipients, where T-cell depleting agents (in-vivo campath in >90%) were included in the transplant conditioning. The study included 159 patients transplanted between January 2000 and March 2006: CML-35; acute leukaemia (AML in 61, ALL in 50); MDS-13. All had myeloablative conditioning regimens and received grafts with 9-10/10 matched HLA alleles. 96 patients received BM and 63 PBSC. There were no associations between the stem cell source and any transplant variable (including disease and stage). All evaluable patients achieved neutrophil engraftment, with a significantly faster time to engraft in recipients of PBSC compared to BM (16 vs 20 days; p=0.0001). The incidence of acute GvHD was 46% (grade I in 50%, II in 41%, III in 7%, IV in 2%). This was significantly higher in recipients of PBSC (61%) compared to BM (36%; p=0.002), however there was no increase in either II/IV (p=0.41) or III/IV (p=0.17) disease in PBSC recipients. In a logistic regression model including other predictive variables, the use of PBSC remained significantly associated with an increase in aGvHD (OR=2.5; 95% CI 1.3, 5.0;p=0.007). The TRM was 16%, 27% and 39% at 100 days, 1 and 5 years respectively. At none of these time points was the stem cell source associated with a significant difference in TRM. The 5year incidence of chronic GvHD was 56% (BM 53%, PBSC 61%; NS), extensive disease in one third, and of relapse was 56% (BM 55%, PBSC 59%; NS). The 5-years OS was 43% with a median follow-up of 4.1 years. This was 44% using PBSC and 40% using BM (NS). In conclusion, although we observed an increase in acute GVHD with PBSC this was only of grade 1 disease. We found no association between the use of PBSC and an increased risk of chronic GVHD or of a worse transplant outcome, when compared to BM, in recipients of T-cell depleted myeloablative transplants for leukaemia. Double cord blood transplantation (dCBT) has extended largely cord blood use to adults with hematological disease. We report 35 dCBT from 2004 to 2007. Twenty three patients had high risk malignant disorders (ALL=6, AML+MDS=8, secondary AL=5, CML=2, Hodgkin disease= 2) and 12 high risk of rejection (SAA=5, PNH=1 and Fanconi Anemia=6). Among all patients 9 (28%) received a dCBT as a rescue of previous non-engrafted transplants (3 AML, 3 SAA, 1 Fanconi anemia, 1 MDS and 1 CML). Analyses of T, B and NK cells phenotype were done once a month during the first 3 months and ever 2 months until 12 months. Median age was 35 years (6-55), median weight was 59kg (17-90) and median follow-up was 15 months (4-37). Conditioning regimen varied according to disease (myeloablative) or second transplant (reduced intensity) and 22 patients received ATG. GVHD prophylaxis consisted in cyclosporine+steroids in 21 patients (60%) and associated with mycophenolate in 14 (40%). Twenty four patients (71%) engrafted at a median time of 25 days (11-42). Before day 100, among 29 evaluable patients, 18 showed full donor chimerism and 6 mixed chimerism (5 autologous recovery). After day 100, 16 out of 19 evaluable patients mantained a complete donor chimerism and 3 had evidence of both CB units engraftment. Acute GVHD was observed in 18 patients (43%) (grade III-IV, n=6) and chronic GVHD in 15 out of 26 patients at risk. During the first 100 days 19 CMV reactivations were detected; 4 HSV (resistant to acyclovir); 1 HHV6-meningoencephalitis; 4 EBV reactivations; 3 adenovirus diseases, 4 VRS infections, 3 septicemias, 6 fungal infections, 2 toxoplasmosis. After day 100 we observed 5 CMV reactivations, 1 CMV disease, 1 HSV, 1 EBV-PTLD and 3 fungal infections. Delayed immune reconstitution was observed in all patients.Median numbers of lymphocytes were 352mm3 at 3 months (n=15); 460 at 6 months (n=17) and 703 at 9 months (n=13). Median numbers of CD3/CD4 at 3, 6 and 9 months were: 34, 40, and 49 mm 3 , respectively; of NK cells 223, 294 and 368mm3 and of B cells were 1, 5 and 65 mm 3 , respectively. At 12 months overall survival was 56% ± 8 and event free survival was 48% ± 9. Sixteen patients (46%) died: 2 of relapse and 7 from infections. Five out of 9 patients have been rescued of previous non-engraftment and are alive and well (5-37 months). In conclusion dCBT is an option to treat patients with high risk diseases lacking a suitable HLA matched donor despite a long lasting immune deficiency. Objective: Enrichment and expansion of mesenchymal stem cells (MSC) from different sources is a critical issue in regenerative medicine and cell therapy. Recent studies indicate that MSC can be isolated from bone marrow (BM) floating fat fraction highly enriched in CD45low D7-FIB+LNGFR+ cells (Jones et al., 2006) . The aim of this study was to compare the BM fat with BM buffy coat (BC) derived MSC. Methods: BM aspirates were obtained from 3 donors who underwent stem cell infusion for the treatment of osteopeneic disorders, following the local Ethics Committee approval. After centrifugation of BM samples by Sepax S-100 (Biosafe, Nyon, Switzerland), nucleated cells layer (BC) at interphase between the plasma and red cells pellet were collected. Cells from BM fat were obtained by collagenase digestion of the fat layer formed at the surface of waste bag (red cells and plasma). CFU-F determination, MSC in vitro expansion, flow cytometric characterization, proliferation and differentiation potential was evaluated as previously described (Urbani et al., 2006) . Results: In vivo flow cytometric analysis of freshly isolated cells from BM fat revealed a three fold increase of CD45lowLNGFR+ cells in comparison to BM BC derived cells (0,6±0,3% vs 0,2±0,2%). Gating on this cell population showed a high expression of the MSC markers CD90, CD73 and CD105. In addition, higher CFU-F/10 6 TNC (10 fold increase) was found in BM fat vs BM BC of the same donor, indicating a correlation between CFU-F counts and CD271+ cells. Proliferative and differentiation potential towards osteogeneic and adipogeneic lineages were similar to adherent MSC derived from both BM sources. In vitro expanded MSC populations were uniformly positive for the classical markers CD105, CD90, CD73, CD29 and CD166. Conclusion: These preliminary results demonstrate that BM fat is rich of CD45lowLNGFR+ stromal progenitor cells and TNC contains 10 fold higher CFU-F than BM BC. These findings show that BM fat, usually discarded following BM processing, results in a better MSC precursors recovery and is relevant in terms of both experimental and clinical use. (2), V. Ghazarossian (3) ( Introduction: Traditional percutaneous large bore needle aspiration methods for harvesting bone marrow (BM) for bone marrow transplantation (BMT) are crude, tedious and expensive and usually require general anesthesia. A novel device, the MarrowMiner (MM) was developed for the minimally invasive harvest of BM which may enable the rapid, convenient outpatient harvest of large quantities of BM derived cells under local anesthesia for use in BMT and an increasing array of developing BM based regenerative therapies. The MarrowMiner utilizes a single marrow entry site into the ileac bone, through which the flexible, powered, MM catheter is able to access the majority of the accessible marrow space and aspirate rich marrow. The device has been tested extensively in human cadavers & in large animal trials. Methods: In 5 juvenile pigs, standard 1 and 6-port harvest needles were used to aspirate marrow at three locations along one iliac crest. On the opposite ileac, the MM was used to collect BM either along the crest or longitudinally down the plane of the iliac wing. Harvests were collected in: small (2-5ml), medium (5-20ml), & large (>20ml) volumes. BM viability, cell counts & CFU-F assays were performed. Results: The MM was observed to be safe, with the catheter remaining within the marrow cavity in up to 20cm long passes. >60 ml of marrow could be aspirated in a single pass. BM aspirated along the iliac crest by the MM contained a significantly higher (20 fold) concentration of nucleated cells and CFU/ml than by standard needles. As expected, the CFU concentration decreased with increasing volume aspirated by standard needles while no CFU dilution occurred with the MM. The MM yielded significantly higher CFU/ml than the standard needles with medium and large volume aspirates, where the MM harvested 9 fold more CFU-F/ml than the 6-port needle (figure). Conclusions: The study demonstrated that the MM can successfully & safely harvest large volumes of BM, with significantly higher stem cell content than traditional needle aspiration in a porcine model. The flexible shaft allowed for aspirating bone marrow of the highest cellularity along the iliac S148 crest, a path unfeasible for rigid needles. This study suggests that this novel device may enable significantly improved clinical BM harvest in a more rapid, reproducible and less invasive manner, particularly if directed to harvest along the iliac crest. Following FDA and CE Mark approval human trials are now underway. Immunological reconstitution after allogeneic stem cell transplant (ASCT) is a critical issue since a prolonged immune deficiency leads to an increased susceptibility to infections. Stem cell source has a strong impact on immune reconstitution since bone marrow (BM) and peripheral blood stem cells (PBSC) differ with respect to their content of progenitors and mature cells. The aim of this study was to determine in a single institution series the impact of stem cell source on: immune reconstitution, incidence of infections, graft versus host disease (GVHD) and relapses. Between 1993 and 2007, 135 patients with hematologic malignancies underwent ASCT after myeloablative conditioning: 127 were included in an immunological study performed on days +30, +90, +120, +180, +270, +365, +547, +730, +1092, +1461 with monoclonal antibodies (anti-CD3, -CD4, -CD8, -CD19, -CD16, -CD56). Stem cell source was BM in 91 cases and PBSC in 36: graft characteristic are in table 1. Median age of BM patients was 39 years (11-59), of PBSC 38 (16-59). Unrelated donors were 28% and 25%. Median time to neutrophils recovery was 21.5 days in BM and 12.5 in PBSC; median time to platelets recovery was 23 and 13 days respectively. The infections rate and the incidence of aGVHD and cGVHD at different times from transplant are in table 2. OS and DFS were 45% and 33% in BM and 39% and 21% in PBSC (p=ns). CD3+ lymphocytes reached the lower normality range on day +120 in BM and +30 in PBSC: the mean value/mmc on +30 was 235 vs 578 (p = 0.002): the difference was significant again on +730 (1359 vs 1834, p = 0.0475); +1092 (1296 vs 1752, p = 0.0499); +1461 (1240 vs 2136, p = 0.0011). CD3+/CD8+ reached the lower range on +90 in BM and +30 in PBSC, with difference on +30 (139 vs 315.4, p = 0.0041). CD3+/CD4+ reached the lower range on +547 and +365 respectively with a significance on +30 (79.97 vs 191.3, p = 0.0005); +90 (133.8 vs 216.5, p = 0.0068); +730 (442.1 vs 690, p = 0.0018); +1092 (485.4 vs 744.5, p = 0.0038); +1461 (498.4 vs 922.3, p = 0.0005). CD19+ reached the lower range on +120 in the two groups with a significance on +30 (2.678 vs 14.67, p=0.0054), CD3-/CD16+/CD56+ on +90 and +30 respectively, with a significance on +30 (178.1 vs 358.9, p = 0.0001). Stem cell source had a significant impact mostly on the early immune reconstitution since only CD3+/CD4+ showed a persistent difference. No significant differences were observed in survival, infections and GVHD. Cryopreservation after transportation may impair haematopoietic stem cell function and engraftment for allogeneic peripheral blood stem cells M. Lioznov*, C. Dellbrügger, A. Sputtek, B. Fehse, N. Kröger, A. R. Zander University Medical Centre Eppendorf (Hamburg, DE) Recent data suggest that the practice of using frozen allogeneic grafts is becoming increasingly common among transplant centres. Therefore we retrospectively analysed 31 frozen allogeneic peripheral blood stem cells (PBSC) grafts by flow cytometry with regard to their CD34+ content, membrane integrity (7-AAD) and stem cell-specific enzyme activity (aldehyde dehydrogenease, ALDH) in relation to individual transplantation results. Membrane integrity of CD34+ cells was significantly (p<0.01; Student's t-test) impaired in cryopreserved PBSC compared to unfrozen allografts: 69.6% ± 9.4% and 96.4% ± 5.6%respectively. For further analysis, the 22 PBSC samples with normal SSCloALDHbr numbers (0.54% ± 0.23%) were united into group I, whereas those 9 of the 31 frozen probes with particularly low relative SSCloALDHbr numbers (0.15% ± 0.03%) comprise group II. Interestingly, mean CD34+ percentages and membrane integrity in the two groups did not differ significantly from each other. We next compared the numbers of CD34+ vs. ALDHbr cells actually transplanted per kg in this two groups ( Fig. 1 ): no significant (p=0.82) difference in CD34+ numbers was observed after thawing, even when dead cells were excluded using 7-AAD staining. In contrast, we found ( Fig. 1 ) significantly lower numbers of SSCloALDHbr cells transplanted per kg in group II as compared to group I (group I: 5.2 ± 2.6 x10 6 /kg vs. group II: 2.0 ± 0.8 x10 6 /kg; p<0.01). Importantly, thosed lower SSCloALDHbr cell numbers in the 9 transplants from group II correlated well (r 2 =0.42; p<0.05, Pearson correlation) with the observed loss of CFU-GM potential in that group (Fig. 2) . It is noteworthy that 7 of 9 patients who received decreased SSCloALDHbr cell numbers (group II) had no engraftment; 2 engrafted patients never reached full chimaerism and suffered an early relapse. In contrast, all other 22 patients (group I) showed well timed leukocyte engraftment at day 15.9±3.4 and 19 of them showed full donor chimaerism. Since all 9 grafts from group II were collected from unrelated donor, it cannot be excluded that transportation from the donation centre followed by cryopreservation involved too much "stress" for HSC and progenitors. We therefore conclude that allogeneic PBSC grafts become much more sensitive to cryopreservation after transport and/or storage. The engraftment potential of frozen HSC grafts may reliably predicted by measuring ALDH activity. J.A.E. Somers* (1), E. Meijer (1), E. Braakman (1), K. Sintnicolaas (2), J. Lie (3), M. Oudshoorn (3), L.F. Verdonck (4), E.J. Petersen (4), A. Brand (5), J.J. Cornelissen (1) (1 Background: Umbilical cord blood (UCB) is considered an important alternative source of hematopoietic stem cells. Due to the limited number of nucleated cells in a single cord blood unit, which is associated with graft failure, adult patients often disqualify for a single umbilical cord blood transplantation (UCBT). UCBT using two (partially) HLA-matched units may be associated with improved engraftment. Objective: To explore the feasibility of double UCBT in adult patients lacking a suitable matched unrelated donor. Methods. Units were matched for HLA-A and B loci on serology split level and HLA-DRB1 on high resolution. Conditioning regimens were myeloablative using ATG/cyclophosphamide/TBI 2x6 Gy in 2 patients and nonmyeloablative using ATG/fludarabine/TBI 2Gy in 12 patients. The non-myeloablative regimen was supplemented with cyclophosphamide in 6 patients. Three patients did not receive ATG. Graft-versus-host-disease (GVHD) prophylaxis consisted of cyclosporine A and mycophenolate mofetil. Results: 14 double UCBTs were performed. A high-risk hematological malignancy was diagnosed in 13 patients and aplastic anemia in 1 patient. Median age of recipients was 48 years (17-61). 11 UCBs were mismatched at 1 antigen and 16 UCBs were mismatched at 2 antigens. The median number of total nucleated cells infused was 5.3 x107/kg (3.7-7.0).To date, the median follow up of survivors is 10 months (4-18 months). 9 out of 14 patients are alive and well, 1 patient has died due to relapse and 4 patients (29%) have died due to TRM. Primary and secondary graft failure were observed in 2 and 1 patients, respectively. Cumulative incidence of neutrophil recovery in 12 evaluable patients is 75% with a median time to neutrophil recovery of 26 days. After hematopoietic recovery, hematopoiesis was derived from a single unit (5 patients) or from both units (5 patients). Acute GVHD gr II-IV developed in 3 patients, grade 2 in one patient and grade 3 in two patients. Among 9 patients who survived beyond 90 days after UCBT, chronic GVHD developed in 2 patients. Other major complications consisted of EBV-related lymphoproliferative disease (1), EBV reactivation (1), CMVreactivations (3) and invasive fungal infections (3). Conclusion: These results suggest that double UCBT is feasible and may lead to sustained engraftment in the majority of patients transplanted for high-risk hematological malignancy. Background: Angiogenesis plays an important role in haematological malignancies. The aim of our study was to investigate bone marrow (BM) microvessel density (MVD) before and after stem cell mobilization with granulocyte colony-stimulating factor (G-CSF) alone and in combination with chemotherapy. Methods: 26 patients (pts) with haematological malignancies were included in the study -21 male and 15 female 17-60 years old (median, 34,5). In 7 pts MM was diagnosed, in 11 -HD, in 6 -NHL, in 12 cases -acute leukemia (AL): in 7 -AML and in 5 -ALL. In 12 cases mobilization was performed with G-CSF alone subcutaneously 10 microg/kg per day for 5-7 days. In other 24 cases G-CSF 5 microg/kg was used in combination with chemotherapy (in 12 cases with cyclophosphamide (CY) 6 g/m² and in 12 cases with DexaBEAM). The bone marrow biopsies for histological and immunohistochemical analysis were performed before the G-CSF administration and on the last day of PBSC collection. Control group (10 donors of BM, 7 male and 3 female, aged 17-59 years, median, 29) was also included in the study, BM biopsy was performed during BM harvesting. Blood vessels were highlighted by immunostaining endothelial cells with a monoclonal antibody to CD34 (Novocastra Lab Ltd). The MVD was calculated in 10 HPF and expressed as vessels/0,491 mm². Results: Before mobilization MVD was 46,7±-3,6 in AL pts, 71,2±5,5 in MM pts, 47,7±3,5 in HD and NHL pts. In the control group MVD was 45,5±3,0. So, there were no significant differences in BM MVD between pts with AL in remission, HD and NHL without BM involvement and the control group. MVD was significant higher (p<0.0001) in pts with MM in partial or complete then in control group. The role of G-CSF for the induction of BM angiogenesis was seen when the mobilization was performed with G-CSF alone. In this group there were 9 pts with AL and 3 pts with NHL. In those cases MVD increased from 44,8±3,3 to 67,2±4,6. In cases when mobilization was performed with G-CSF in combination with CY statistically significant increase of MVD from 66,7±4,2 to 83,7±4,0 (p<0,0001) was also seen. After the mobilization with G-CSF and DexaBEAM in pts with HD and NHL increase of MVD was maximal (73,2±4,9, that 66,7% higher then the results before mobilization). Conclusion: Stem cell mobilization with G-CSF alone and in combination with chemotherapy is accompanied by statistically significant increase of BM MVD. DMSO and stem cell infusion toxicity remains a concern within the transplant community and for regulatory authorities. In 2004 the stem cell product transfusion protocol was changed at our institution from rapid injection via 50ml syringe at 4°C to a 5-minute infusion at 15-20°C. In a retrospective analysis we aimed to determine the impact of this protocol change on early adverse events (AE). Commonly discussed parameters supposed to affect AE rates like amount of DMSO infused and preparation method (8% DMSO in autologous plasma + saline vs. 10% DMSO in autologous plasma) were also analysed. Leukocyte content was ≤1x10 6 /ml in all preparations. Methods: Stem cell product transfusion forms of 203 consecutive autologous peripheral blood stem cell transplantations from 01/01/2003 to 30/11/2007 have been reviewed, and AE occurring within 2 hours of transfusion were graded according to Common Toxicity Criteria version 3. Typical odour and mild coughing were excluded from the analysis as they occured in virtually every patient. p-values were calculated using Fisher's exact test. Results: No grade 4/5 adverse events were observed in this series. Grade 3 AE occurred in 2 of 42 (5%) injections at 4°C and 3 of 161 (2%) transfusions at 20°C. These included newly induced tachyarrhythmia absoluta, severe symptomatic hypertension (3x) and syncope. AE of any grade were documented in 18 of 42 (43%) cases in 2003 and 32 of 161 (20%) cases with the new protocol (p=0,0042). Most frequent observations were hypertension, nausea and bradycardia. Intravenous medication was applicated in 4 patients (atropin 3, verapamil 1). Time to engraftment remained unchanged. 109 patients had received 1 single bag of 100ml (8ml DMSO) with 22 AE (1 grade 3 event), whereas 94 patients with 2 or more bags experienced 30 AE (4 grade 3 events), p=0,0757. No difference at all was seen between the preparation methods / DMSO concentrations. Conclusions: Transfusion of stem cell transplants cryopreserved with DMSO is generally a safe procedure. Overall toxicity is significantly lower when the product is transfused over 5 minutes at 20°C compared to injection at 4°C while engraftment is not affected. Higher amount of DMSO infused is associated with higher AE rates and more grade 3 AE, although not statistically significant due to the low number of events. High-dose lenograstim allows collection of autologous haematopoietic stem cells in elderly patients with acute myeloid leukaemia or myelodysplastic syndrome M. Bernardi, M. Tassara, A. Crotta, C. Messina, J. Peccatori, A. Assanelli, D. Clerici, S. Mastaglio, E. Guggiari, S. Gattillo, L. Malabarba, F. Ciceri San Raffaele Scientific Institute (Milan, IT) Background: allogeneic (allo) stem cell transplantation (SCT) is the only potentially curative strategy for acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Patients (pts) older than 60 can rarely benefit from alloSCT because of the high risk of procedure-related toxicities; alternatively, in elderly pts consolidation of complete remission (CR) with intensified-dose chemotherapy and autologous (auto) SCT can prolong DFS. Unfortunately, mobilization and apheresis of autologous peripheral stem cells (PSC) in these pts often fails because of decreased marrow reserve. At our center we usually administer high dose lenograstim (10 microgr/kg/die) after consolidation chemotherapy to mobilize and collect autologous PSC. Data on feasibility of this procedure in our elderly pts are here reported. Aim: to retrospectively evaluate data on mobilization and collection of PBSC in elderly AML and MDS pts. Methods: period 2/1999 to 9/2007, 72 pts, median age 66.4 (60-76.3). Diagnosis (WHO): AML 23, AML MD 33, MDS 15, undifferentiated AL 2. All pts received a conventional induction treatment. Mobilization of PSC was attempted in 38 cases, 2 with lenograstim alone, 36 with consolidation/mobilization chemotherapy + high dose lenograstim (10 microgr/kg/die). Consolidation/mobilization regimens (total 36): FLAG-IDA 2, FLAG 2, FLAG-daunoxome 1, dauno-cytarabine (3+7) 6, mitoxantrone-cytarabine 1, A8 (total cytarabine 8 gr/sqm for 4 days) 22, HD cytarabine (total > 8 gr/sqm) 2. Leukapheresis were started when PSC (CD34+ MNC) reached the absolute count of 15/microl; the COBE spectra blood separator was utilized. Results: CR rate was 68% (49/72) after 1 to 3 induction cycles, 55.5% (40/72) after the first cycle. Median number of chemotherapy cycles before leukapheresis: 2 (range 0-3). In 33/38 pts (86.8%) mobilization and collection of PSC was successful: mean 11.4 CD34+MNCx10e6/kg (0.95-42). Subsequent CR consolidation with intensified-dose chemotherapy and autoSCT was performed in 24/33 (72.7%) pts. Conclusions: mobilization and collection of PSC is feasible in most elderly pts with AML or MDS, if adequate marrow stimulation is performed. In our hands cytarabine at a total dosage of 8 gr/sqm followed by high dose lenograstim proved to be the most appropriate strategy, also for its low toxicity profile. One third of initial pts finally received an intensified consolidation and autoSCT. Petersen, B. Fehse, M. Lioznov, A. Muth, H. Lellek, M. Hartwig, U. Bacher, H. Kabisch, R. Erttmann, N. Kröger , A.R. Zander University Medical Center (Hamburg, DE) Objectives: Compared to bone marrow (BM), the use of peripheral blood stem cells (PBSC) for allogeneic stem cell transplantation has been associated with quicker engraftment and higher incidence of chronic graft-versus-host disease (cGvHD). Further data suggest a beneficial role for patients with high risk disease, however data on long term endpoints are less available. We performed a retrospective analysis of S151 259 consecutive patients who underwent unrelated donor (UD) allogeneic stem cell transplantation between December 1993 and October 2005 in our center and analyzed the impact of stem cell source on outcome. Patients and methods: 110 patients received PBSC and 149 patients BM. 194 patients recieved HLA-matched und 65 patients HLA-mismatched unrelated grafts. Recipients of PBCS had more CD34 cells/kg BW while more patients in this group were positive for CMV. Other relevant patient and donor characteristics were matched between the two groups. Conditioning consisted of a TBI or busulfan based myeloablative regimen including anti-thymocyte globulin (ATG-Fresenius) for prevention of serious GvHD. Results: Leukocyte and platelet engraftment were faster in the PBSC vs. BM group (15 vs. 17 days, p< 0.001 and 17 vs 23 days, p< 0.001). For recipients of PBSC and BM respectively outcome was as follows: grade II-IV aGvHD (51 vs 39%, p= 0.9), III-IV aGvHD (26 vs 19%, p= 0.1); cGvHD (61 vs 43%, p= 0.1); TRM at 2 yrs (29% in both groups); overall survival (OS) at 5 yrs (51 vs 54% p = 0.8); disease free survival (DFS) at 5 yrs (51 vs. 41% p= 0.7); relapse rate at 5 yrs (26 vs 42% p= 0.3). In the multivariate analysis of all patients the following factors were of prognostic significance: age negatively influenced TRM; bad risk disease negatively influenced OS, DFS and relapse; patient's CMV seropositivity negatively influenced OS, DFS and TRM. CD34+ cells/kgBW positively influenced TRM; conditioning with TBI negatively influenced relapse rate. TRM and OS for both good and bad risk disease were independent of stem cell source. There was a trend to better DFS (66 vs 52%, p= 0.3) and lower relapse rate (16 vs. 29%, p= 0.3) for good risk patients who received PBSC compared to BM. DFS and relapse rates for bad risk patients were independent of stem cell source. Conclusion: Our results indicate that PBSC are at least as good as BM for unrelated donor allogeneic stem cell transplantation for good as well as bad risk disease stages when in vivo T cell depletion with ATG is included. It is known that HSP is influenced by temperature. Here we searched the effects of temperatures on preservation of hematopoietic stem cells (HSC) and the changes of HSP according to the temperatures and cryopreserving agents. The G-CSF-mobilized peripheral blood HSCs were collected from a healthy donor. DMSO, EG and GLY were used for cryopreserving agents. HSP were measured by real-time quantitative PCR, and cell viability were measured by trypan blue stain and MTS assay, respectively. Testing temperatures were 4°C, room temperature (RT, 20°C) and 37°C. Samples were HSCs stored according to testing temperatures without cryopreserving agents (cycle 0), HSCs thawed after first cryopreservation with various cryopreserving agents (cycle 1). The measure was done daily from D0 to D5 at cycle 0, and in 30 minutes interval (0, 30, 60, 90, 120 min) at cycle 1. Whereas more than 98% of cells were viable till 5 days after the harvest of cycle 0 at 4°C, RT and 37°C showed rapid decrease of viable HSCs. By day 3, less than 10% cells were viable at 37°C. At cycle 1, cell viability was rapidly decreased in both DMSO and GLY after 120 min stay at all temperatures. In case of EG, the same patterns were found at RT and 37°C. However, cell viability remained relatively well at 4°C. The expression of HSP 27 were over-expressed by day 2 and suppressed thereafter at all temperatures of cycle 0. At 4°C of cycle 0, the over-expression of HSP 27 was greater than those at RT and 37°C. The expression of HSP 70 was mostly suppressed along times. The suppression at 37°C was greater than those of other temperatures. In case of HSP 90, the overall expression was suppressed except for day 2 at 4°C and RT. At all cases of cycle 0, the expressions at 37°C were lower than those at 4°C and RT. At 4°C of cycle 1, DMSO showed decreased expression of HSP27 and HSP70. In contrast to DMSO, the expression of HSP27 and HSP70 was increased in case of EG and GLY at 4°C of cycle 1. The overexpression of HSP90 at 4°C of cycle 1 in EG and GLY was very low compared with those in DMSO. Overall expressions of HSPs in GLY were lower than in DMSO or EG at RT of cycle 1 except for HSP27. The expression of HSP27, HSP70, HSP90 were all decreased at 37°C of cycle 1 except for HSP70 in EG and HSP90 in DMSO. In conclusion, the rapid apoptosis at 37°C could be caused by changed expression of anti-apoptotic HSPs. The expression of HSPs influenced by temperatures and cryopreserving agents may affect viability of HSCs. Mesenchymal stem cells (MSCs) derived from several sources can differentiate into multiple mesenchymal and non mesenchymal lineages, representing a promising tool for tissue repair. The aim of this study was to compare MSCs derived from cord blood (CB) and Wharton's Jelly of the human umbilical cord (UC) regarding morphology, the success rate of isolating MSCs, colony frequency, expansion potential, multiple differentiation capacity and immune phenotype. To isolate MSCs the CB MNC fraction was cultured in a-MEM culture medium, supplemented with Lglutamine and 10% foetal bovine serum (FBS). In order to isolate MSCs from the Wharton's Jelly, blood vessels were removed and Wharton's jelly had been digested using collagenase, hyaluronidase and trypsin. In both cases cultures were maintained at 37ºC in a 5% CO2 atmosphere and nonadherent cells were removed after 24 hours. To assess the presence of primitive cells in both sources colony-forming unit fibroblast (CFU-F) assays had been performed and the immunophenotype of MSCs was verified with 3-colour flow cytometry. Gene expression was examined with reversetranscriptase polymerase chain reaction (RT-PCR). To induce osteogenic, chondrogenic and adipogenic differentiation cells were cultured into the appropriate media. Differentiation outcome was evaluated using histocytochemistry and RT-PCR. In UC primocultures the mean expression of MSC markers was 91% for CD105, 90.5% for CD90 and only 1.0% were CD45, whilst in CB primocultures the MSCs markers expression was 18%, 0.9% and 95% respectively. Long-term cultures of pure MSC populations were the outcome of 95.2% of UC and 3.6% of CB primocultures. MSCs obtained from both sources shared the same immunophenotype and expressed stem cell genes, such as oct-4, Nanog, FGF2, LIF. This phenotype was maintained throughout culture period. The frequency of CFU-F in UC and CB was found to be 1/841 and 1/2.12 x 106 cells respectively. Finally, while properly induced UC-MSCs differentiated into osteocytes, chondrocytes and adipocytes, CB-MSCs failed to differentiate into other cell type than osteoblasts. This data indicates that UC, compared to CB, is a better source of MSCs. Taking into account all the advantages and disadvantages of the UC and CB, depending on the therapeutic indication, the clinical implication may be based on differentiation capacity, but more likely on the abundance, frequency, and expansion potential of the cells. C. Regidor*, I. Krsnik, R. Forés, R. Cabrera, G. Bautista, E. Ruiz, I. Sanjuan, S. Gil, J.A. Garcia-Marco, B. Navarro, E. Ojeda, P. Palomo, A. De Laiglesia, J. Cartier, M.N. Fernandez Hospital Universitario Puerta de Hierro (Madrid, ES) Engraftment of UCB transplants is dependant on their content on hematopoietic progenitors (HP) assayed as CD34+ cells. TNC is at best a surrogate of that. None of these evaluate proliferative capacity of HP that is evaluated by functional assays like CFU. We report our experience related to data received from CBBs, on which supposedly the best units were selected for 65 adult patients. Between Feb/96 and Aug/07, 74 units were received in our laboratory. Because of inadequate shipment 2 units were discarded; 7 were infused directly after thawing, 65 diluted using the first step of the Rubinstein procedure and 9 (unfractionated or ABO incompatible) washed; 9 patients received a single unit transplant and 56 received co-infusion of mobilized CD34+ cells from a third party donor (TPD) which exerted the effect of a "bridge transplant" thus favouring final CBU engraftment (Haematologica, 2006; 9:640) . Because of failure or early rejection 3 received a 2nd unit that engrafted; 1 received 2 other units that also failed and 1 a 2nd transplant after late relapse. Data provided by the CBBs included CD34+ cells added to TNC for 61 units, CFU also given for 48 (31 post-thaw controls). The final transplanted product showed TNC recovery (median, range) of 93% (50-121). Data for CFU and CD34+ were 81% (0-403) and 200% (41-633). These results reflect to a certain extent inter-lab variation, what imply limited adequacy of performing these controls only at the transplant centre. Similar results were obtained for diluted and washed units. For patients receiving TPD HP cells we have obtained CB engraftment and full chimerism cumulative indexes of 96% and 91%, with primary CB graft failure in 3 cases: 1 an endstage LMC in whom 2 subsequently units also failed. Units initially given to the other 2 patients showed TNC recoveries of 50.6 and 60.5%, implying TNC/Kg 1.57 and 2.1x10 7 /Kg (within range of the other patients), being the only ones without CFU growth. These 2 patients and the one with early rejection maintained normal ACN from the TPD until engraftment of the 2nd unit. Conclusion: It would be convenient that CBBs perform and report post-thaw reliable control assays on recovery of both CD34+ and CFU cells, to avoid transplantation of CBU of inadequate engraftment potential. Our procedure of coinfusion of TPD HP has proved a safeguard for this possibility of graft failure. Acknowledgment: Work supported by grants from EU (Allostem) and Research Agencies of Spain and CAM P594 Successful engraftment with double umbilical cord blood transplantation in adult patients with high-risk acute leukaemia I. Baltadakis*, D. Karakasis, C-G. Balotis, M. Tsirogianni, V. Kyriazi, A. Manaka, Z. Poulopoulou, I. Bika, M. Garofalaki, M-E. Karatza, I. Apostolidis, N. Harhalakis, E. Nikiforakis Evangelismos Hospital (Athens, GR) The major barrier of umbilical cord blood transplantation (UCBT) is the limited graft cell dose, especially for adult patients. Among the proposed methods to increase cell dose, the combined transplantation of 2 partially HLA-matched UCB units has proven to be feasible and can extend the applicability of UCBT to virtually all adult patients, who do not have a timely available HLA-matched donor. From 8/2006 to 10/2007, 10 patients (female:male= 6:4) with high-risk acute myeloid (AML, n= 8) or lymphoblastic (ALL, n= 2) leukemia received double UCBT at our center. The patients had a median age of 33.5 years (range: 16-57), and a median weight of 60 kg (range: 53-94). The median time from diagnosis to UCBT was 17.5 months (range: 3-96). At the time of transplant, 3 patients were in 1st complete remission (CR), 4 in 2nd CR, 1 in 3rd CR and 2 in resistant relapse. The conditioning regimen was myeloablative in 8, and nonmyeloablative in 2 patients. Cyclosporine plus mycophenolate mofetil was used as acute graft-versus-host disease (aGVHD) prophylaxis. The majority of UCB units (19/20) were 4/6 antigen matched to recipient for HLA-A, -B, and -DRB1, and 5-7/10 matched at the allele level for HLA-A, -B, -C, -DRB1 and -DQB1. UCB units were washed prior to infusion according to the New York Blood Center protocol. The median doses of nucleated and CD34+ cells at infusion were 4.4 x 10 7 /kg (range: 2.9-5.4 x 10 7 /kg) and 1.27 x 10 5 /kg (range: 0.6-2.0 x 10 5 /kg), respectively. All patients achieved sustained neutrophil recovery at a median of 18.5 days (range: 6-41, Figure 1 ). Engraftment of both units was observed in 5 of 10 patients on day 28. In all cases, one unit finally predominated. The cumulative incidence of platelet recovery (ƒ®50,000/uL) was 70% by day 150, and occurred at a median of 96 days (range: 33-117). All patients developed aGVHD (grade II: 9, grade III: 1) responsive to treatment with steroids. Two of 5 evaluable patients, with follow-up greater than 100 days, have developed chronic GVHD. Three patients died due to treatment related mortality (TRM). One patient had relapse of AML 8 months post transplant, but achieved complete remission after withdrawal of immunosuppression. Seven patients are alive and in CR, 3-17 months after UCBT. In conclusion, double UCBT resulted in stable donor engraftment with acceptable TRM in this high-risk adult patient group. Further improvement is anticipated with the application of the method at earlier disease phases. The Austrian Bone Marrow Donor Registry is the central search coordinating unit for international MUD donor searches in Austria. We have analyzed 98 international donor searches -searches that have been started in the years 2005, 2006 and 2007 which have been terminated in 2007. 26 (26%) of the searches were closed without success: Without a suitable donor for transplant. 72 (74%) of the searches were successfully terminated: 1 donor with 2/10 allele mismatches, 8 donors with 1/10 mismatches and 63 donors with 10/10 matches were accepted as suitable donors by the transplant physicians. All patient donor pairs were typed at an allele level including the HLA-loci HLA-A,-B,-C,-DRB1 and DQB1. In order to predict the success of the international bone marrow donor search, we have used an already published algorithm (Tiercy et al.: The probability of identifying a 10/10 HLA-allele matched unrelated donor is highly predictable. Bone Marrow Transplantation 40, 515-522 (2007) for prediction of the search result. According to the already published algorithm, three categories of patients were established at the beginning of the search: Patients with low prognosis, patients with intermediate prognosis and patients with high prognosis. We were able to identify a compatible donor in 14/34 (41%) cases in the low prognosis group of the patients, in 25/33 (75%) cases in the intermediate prognosis group -and in 27/31 (87%) cases in the high prognosis group. Thus, the already published algorithm for the prediction of the probability of identifying a matching donor turned out to be useful also in Austrian patients. Prediction is correct in 75% of the cases and will be used for pre-informing colleagues from the transplantation units. A bone of contention: best approach for harvesting bone marrow to maximise TNC and CD34+ cell counts V. Antonenas*, F. Garvin, K. Mickelthwaite, I. Kerridge, K. Background: Bone marrow (BM) has been utilized as a source of stem cells for transplantation for many years. Although the use of BM has decreased with the advent of mobilized stem cells, utilization is increasing once again due to the lower rate of chronic GVHD associated with BM as a stem cell source. There is no generally accepted technique for harvesting BM. Protocols vary both in relation to the volume of each aspirate and the number of aspirates performed at each puncture site. Aim: To investigate whether differences in the volume and number of aspirates taken at the time of BM harvest affect the number of mononuclear cells (particularly CD34+ and CD3+) collected. Method: Consented adult donors were positioned in the prone position for collection of bone marrow from the posterior iliac crests. Triplicate samples of 5, 10 and 20ml aspirate volumes were taken at the start. Subsequent harvesting was performed using 10ml aspirate volumes. Triplicate samples were taken from the 10ml aspirates when harvest volumes of 250, 500, 750 and 1000mls were attained. Samples were analysed for total nucleated cell (TNC) count, CD34+ and CD3+ cell counts. Results: The following Table shows results from 4 BM harvests. Total cell counts (± SEM) taken from 5, 10 and 20 ml aspirate volumes at the beginning of harvest are shown in the third column. Cell counts from 10ml aspirates after 250, 500, 750 & 1000ml of BM have been taken are shown in the last 4 columns: Conclusion: The aspiration of increasing volumes of BM does not produce a proportional increase in total numbers of NC, CD34+ or CD3+ cells collected. The highest ratio of CD34+ to CD3+ cells is observed with 10ml aspirates. The data suggest that for any given volume of marrow, multiple aspirations of smaller volumes will result in collection of greater numbers of all cell types. This conclusion needs to be verified by appropriate study. The data also indicate that there is a sudden drop in yield of both CD34+ and CD3+ cells once 750ml of BM has been aspirated in 10 ml aliquots. Further harvesting beyond this point is unlikely to be of benefit and if insufficient cells have been obtained, a second harvest may be a better option than continued aspiration on the same occasion. Structure and functional characteristics of human umbilical cord blood monolayer culture Objectives: It's known that during cultivation, adherent cells of umbilical cord blood (UCB) are forming monolayer reminiscent, in its composition, of stromal monolayer of bone marrow (BM) culture. But the presence of mesenchymal stem cells (MSC) still remains controversial. This study has been performed to investigate the structure and some functional characteristics of MSC-like cells presented in the cord blood monolayer culture. Materials and methods: 43 samples of UCB were used. All UCB samples were obtained through full-term delivery. To perform a monolayer culture, UCB mononuclear fraction cells, was cultivated in a culture medium containing DMEM, 20% FCS, 1% Pen/Strep. The phenotypic structure of UCB culture was characterized by the following monoclonal antibodies: CD34; CD117; CD45; CD14; CD3; CD19; CD31; CD90; HLA DR; HLA AB. To determine functional characteristics of MSC's presented in UCB culture we analyzed their differentiation ability and hemostimulation activity. Results: In most cases the cell culture which had been attached to the plastics was heterogeneous. Spindle-shaped cells and polygonal cells were observed. In some samples we could see clonal growth, however their number did not increase 100 cells. Only 3 out of 43 samples of UCB under study showed great colonies. As evidenced by immune phenotypic characteristics of the studied monolayer UCB cultures are polymorphous and dissimilar in each sample. Most of cells present in the culture were macrophages (CD45+) but in different concentrations we found out the cells of mesenchymal origin (CD90+CD31-) including endothelial cells (CD34+CD31+) (Tabl). It was shown that the cells of the UCB monolayer culture have capacity to differentiate into adipocytes and osteoblasts which fact was proved by their specific staining. However in some cultures the induction of differentiation initiated the detachment of the greater parts of cells. Hemostimulation ability of UCB monolayer culture depended from the phenotype of culture. Cells of CD45+ and CD14+ phenotype are, evidently, stimulant for formation of GM-colonies but also non-hematopoietic UCB culture populations shows direct correlation between level of expression in CD90+CD31-phenotype population culture and the number of CFU-GM colonies. Conclusion: UCB contains fraction of non-hematopoietic cells which functional characteristics were confirmed. But low number of these cells call in a question use of UCB as a source for therapeutics strategies. A. Pontari* (1), G. Pucci (1), E. Spiniello (1), D. Marcuccio (1), R. Monteleone (1), G. Irrera (1), T. Moscato (1), G. Gallo (1), D. Princi (1), A. Musella (1), G. Strati (1), M. Antonucci (2), R. Idotta (3), E. De Pino (4), P. Baccillieri (5), C. Ermio (6), F. Chiaravalloti (7), M. Talarico (8), N. Lisanti (9), S. Marino (10), P. Iacopino (1) (1)AO Bianchi Melacrino Morelli (Reggio Calabria, IT); (2 The Calabria Cord Blood Bank (CCBB) has been created in January 2006 at the Regional Bone Marrow Transplant and Cell Therapy Center "Alberto Neri", Azienda Ospedaliera "Bianchi-Melacrino-Morelli" in Reggio Calabria (RC). Since this date, CCBB received n°1333 cord blood units collected in 23 Birth Centres and validated by 12 Transfusion Medicine Units. Umbilical Cord Blood (UBC) is an acceptable source of hematopoietic cells for transplantation for patients without family matched donors. In fact, cord blood is easily available thanks to the many mothers who decide to donate cord blood at the time of the birth of their child, increasing the likelihood to find a donor for the patients awaiting transplant. In our experience we evaluated the correlation between high UBC volume (≥100 ml) and cell count. UCB samples were screened, processed and evaluated for the cell count. Our evaluation of an adequate collection is according to the cell count criteria as N° TNC >1,1 x 10 9 . We examined 811 UBC units received at CCBB; of these 354 were obtained from caesarean birth and 457 after natural delivery. The mean volume obtained for UBC units from caesarean section is 94,95 ml (ES+2,7), for UBC units of natural delivery is 84,9 ml (ES+1,4), and it is statistically significant (P<0,0001). Nucleated Cell Count (NCC)/ml for UBC units from natural delivery is higher than UBC units of caesarean section. The difference between the rate is statistically significant (5,9 ES+0,17 vs 4,48 ES+0,21; P<0,0001). Ultimately, it seems that natural childbirth can get a TNC value higher than caesarean birth even if the volume is lower. This is probably due to the increased blood volume because of the saline infusion during the anaesthetic drug in pregnant women undergoing caesarean section, therefore the sample is hemodiluted. Thanks this comparison, it is feasible that for the caesarean birth is necessary to collect higher volume than natural delivery in order to obtain a TNC adequate to our banking standard. High-dose therapy followed by autologous haematopoietic stem cell transplantation (auto-HSCT) is an essential part of treatment of many oncological and oncohematological diseases. Haematopoietic stem cells (HSC) for transplantation are most often collected from peripheral blood (PB) after previous mobilization with chemotherapy and/or granulocyte colony stimulating factor (G-CSF). In up to 20% of patients mobilization is ineffective and does not allow for collection of at least 2x10 6 CD34-positive cells/kg necessary for save transplantation. Aim of the study: Evaluation of factors influencing effectiveness of mobilization of HSCs. Materials and methods: The analysis included 66 mobilization courses performed for 62 patients (31 males and 31 females in age of 20 to 248 months). The indications for HDT were: neuroblastoma (31), Ewing's sarcoma (12), lymphoma/leukemia (10), brain tumor (6), other (3). G-CSF was administered subcutaneously in dose of 10, 17 or 24 mcg/kg/day either alone in steady state of PB leukocyte count (50) or in combination with myelosuppressive chemotherapy (16). Apheresis was triggered when the number of CD34+ cells in PB had reached 10 cells/mcl and were repeated until collection of at least 3x10 6 CD34+ cells/kg. Effectiveness of mobilization was scored according to total number of collected CD34+ cells/kg: 0 for <2x10 6 , 1 for 2-3x10 6 , 2 for >3x10 6 , 3 for >3x10 6 collected in one apheresis. Multinomial logistic regression was used to asses significance of factors supposed to influence the effectiveness of mobilization. Results: The effectiveness of mobilization was scored as 0 in 13 (19.7%), 1 in 13 (19.7%), 2 in 20 (30.3%) and 3 in (30.3%) cases. At least 2x10 6 CD34+ cells/kg were collected in 55 (88.7%) patients, in two cases after two courses of mobilization. Longer time from diagnosis to mobilization was associated with significantly lower probability of effective mobilization (p=0.02, fig.1 ). Mobilization tended to be most effective in patients with lymphoma/leukemia and less effective in patients with brain tumors, however the difference was not significant (p=0.07, fig.1 ). Interestingly, neither the dose of G-CSF nor the type of mobilization significantly influenced the effectiveness of mobilization. Radiotherapy performed prior to mobilization did not appear important, either. Conclusion: Time from diagnosis to mobilization is the most important factor facilitating collection of number of PB HSC sufficient for auto-HSCT P600 Positron emission tomography identifies a differential pattern of bone marrow FdG uptake in "poor" and "good" peripheral stem cell mobilisers G. Marcacci*, C. Caracò, F. Frigeri, L. Aloj, G. Corazzelli, F. Russo, G. Capobianco, C. Becchimanzi, M. Arcamone, S. Lastoria, A. Pinto National Cancer Institute, Fond. Pascale (Naples, IT) Objectives: G-CSF use is associated to the increase of bone marrow (BM) fluorodeoxyglucose (FdG) uptake as detected by Positron Emission Tomography (PET). In contrast, no data is available as to changes in BM FdG-uptake during peripheral blood stem cell (PBSC) mobilization. This study was aimed at investigating patterns of BM FdG-uptake during mobilization as quantified by Standardized Uptake Value (SUV)determinations.We also evaluated whether PET scanning may turn of value for identifying good and poor mobilizers.To our knowledge, this is the first PET-based study in this setting. Methods: Seventeen patients(pts)(M/F=10/7), median age 51 yrs (r28-65), with relapsed lymphoma (NHL/HD=13/4) without BM involvement, were accrued after informed consent. Baseline PET was obtained at relapse, before salvage therapy and any CSF administration.After salvage regimes, pts were mobilized by VRL/CTX or idARA-C; G-CSF(10ug/kg/day)was given from day 6 through apheresis. PET scans were obtained on day 9 or 10 (after nadir with a WBC>1000/ul). SUVmax and average(avg) were measured (whole lumbar spine and bilateral iliac regions) and compared to SUV of the same BM regions at baseline PET. The aim was to calculate a BM specific Delta-SUV (mobilizing vs steady-state Delta-SUV) for each single patient. Results: Twelve pts mobilized PBSC (median CD34 peak 39.99/ul, r 23.2-280.5/ul; median CD34 in the harvest 3.3x10 6 /Kg,r 2.1-12.5) while 5 pts were poor mobilizers(median CD34 peak 10.9/ul, r 7.5-14.1/ul). In the group of good mobilizers, apheresis was performed at CD34 peak (day 11-14), with a median of 1 apheresis/pt(r 1-2).Unexpectedly, effective mobilization was associated with a low BM uptake of FdG: median BM Delta-SUVmax and Delta-SUVavg of 2.0 (r 1.0-3.8) and 2.3(r 0.9-3.9), respectively. In contrast, poor mobilizers displayed a median Delta-SUVmax and Delta-SUVavg of 4.7 (r 2.4-12.8) and 5.9 (r 4.1-14.2), respectively. Conclusions: While FdG-BM uptake usually increases upon CSF administration, our results suggest that PBSC mobilization may be associated with a more complex metabolic pattern of BM as detected by PET. We documented that, 48 to 72 hrs before CD34 peak, poor mobilizers display a higher FdG-BM uptake (Delta-SUV>3)as compared to good mobilizers (Delta-SUV<3). These preliminary results indicate that BM PET may represent a new tool for early identification of poor mobilizers allowing a timely modification of the mobilization strategy to possibly rescue the procedure. (1) (1)Cell and Tissue Bank (Pisa, IT); (2)University of Pisa (Pisa, IT) Aim of the study: The small percentage of cord blood units (UCBs) stored that are actually used for transplantation contributes to raise the already elevated costs of their processing and cryopreservation. The identification of gestational predictors allowing to identify and select only optimal UCBs would contribute to decrease these costs . Methods: In 350 banked UCBs, we evaluated the correlation between neonatal/gestational parameters and laboratory data used to assess the quality of UCBs. Results : Weigh at birth and placental weight showed a positive correlation with total nucleated cells count, volume, CD34+ cell counts , total CFU and BFU. Biometric parameters (biparietal diameter in both genders, femoral length and abdominal circumference) evaluated by ultrasound at the end of the gestational period also correlated with the above-mentioned laboratory parameters. Among all gestational parameters examinated, the number of CD34+ cells showed a statistically significant correlation with abdominal circumference, but not with biparietal diameter, cranic circumference and femoral length; WBC showed a positive correlation with cranic circumference and biparietal diameter but not with abdominal circumference and femoral length. Conclusion: Abdominal and cranic circumference and biparietal diameter might represent useful parameters for selecting UCB donors. Evaluation of CD 34 cell subsets composition in leukapheresis products and their impact on the haematopoietic recovery after autologous transplantation A. Dmoszynska*, T. Gromek, M. Cioch, M. Wach, A. Walter-Croneck, D. Jawniak, J. Manko, W. Legiec Medical University of Lublin (Lublin, PL) Introduction: Rapid engraftment is one of the most important factors for safety of haematopoietic stem cell transplantation (HSCT). It is known that recovery of neutrophil number after PBSCT is related to graft CD34+ counts. Predicting the speed of PLT recovery is more complicated. Peripheral blood stem cells can be mobilised using different type of chemotherapy. There is a substantial variability in the yield of HSC between patients. The effect of different mobilisation regimen on the composition of leukapheresis product is not clear. Aim: To analyse CD34+ cell subsets in leukapheresis products and to evaluate influence of HSC subsets on the recovery of WBC and PLT. Material: Thirty patients (18 women and 12 men) who underwent autologous PBSCT were included into this study (20 pts with MM, 7 with NHL, 2 with HL and 1 with AML). PBSC were mobilised using different mobilising regimens: high dose (HD) cyclophosphamide, HD etoposide, HD cytarabine or FLAG. G-CSF was used at a dose 10 ug/kg/day. Leukapheresis procedures were performed with blood cell separator (Baxter Healthcare Corp.). Patients received a median graft dose of CD34+ cells 3.4 x 10 6 /kg. Methods: CD34+ cell subsets were identified by multi parametric flow cytometry (Calibur, Becton Dickinson). The following panels of monoclonal antibodies were assayed: anti CD34/CD33/CD90/CD38, anti CD34/CD133/CD7/CD38, CD34/CD123/CD90/CD117, CD34/HLADR/CD41a/CD38. Results: Distribution of CD34+ cell subsets in leukapheresis product is shown in table below. We found statically significant correlation between time of granulopoiesis recovery and number of infused CD34+/CD123+/CD90+ subset. We also observed that the use of different mobilisation protocols influence the CD34+ cell subsets contents. Conclusions: The most effective mobilising regimen is HD cyclophosphamide providing the highest number of CD34+/CD123+/CD90+ subset. Higher contents of this subset correlates with rapid and durable recovery of granulocytes and platelets. Cobe spectra haematopoietic progenitor cell apheresis outcome improved using Gambro BCT automated version 6.1 software S. Loaiza*, P. Elsey, E. Bray, J. Goddard, K. Patel, E. Olavarria, J. Davis Hammersmith Hospital (London, UK) A number of factors impact on apheresis processing. High WBC apheresis (>400x10 9 /L) requiring dilution increases consumable use, volume stored and DMSO/fluid load given to patients. Immunoselection validation limits stipulate maximum WBC levels for processing with one or two reagent kits. Of relevance in the allogeneic setting where engineered low Tcell doses may be required, residual T-cell doses are higher in selections carried out on high WBC products. Automated version 6.1 (v6.1) apheresis collection software offers the potential to improve harvest quality and address some of these issues. We compared apheresis collected using Spectra v4.1 software with v6.1 introduced into our centre in 2006. Data analysis (Table 1 ) was carried out on apheresis collected on Multiple Myeloma patients (MM) and normal donors (ND) (V4.1 MM=43, ND=34) (V6.1 MM=37, ND=32). Within the subject groups there were no differences in mobilisation efficiency and all were successful in achieving target dose. Apheresis using v6.1 for both MM and ND subjects demonstrated reduced collection volumes and WBC counts. For v4.1 5/43 MM and 23/34 ND required dilution, in contrast no MM or ND apheresis requiring dilution using v6.1. With v6.1 MM and ND volumes stored were significantly reduced (MM median v4.1=342 Vs v6.1=200 p≤0.0001; ND median v4.1=439.5 Vs v6.1=251 p≤0.0001). Should ND apheresis require immunoselection, only 1/32 harvests using v6.1 exceeded the 6x10 10 threshold and would require two reagent kits. In contrast 26/34 ND using v4.1 would require the use of two reagent kits. Apheresis CD34% and as fold increase over PB CD34% data reflect mononuclear cell enrichment. In MM and ND the CD34% were higher for apheresis using v6.1 (p=0.0032 and 0.0109 respectively). Although CD34 fold increase was higher in ND with v6.1 (p=0.0055) there was no significant difference in MM (p=0.6527). In conclusion our data demonstrate that v6.1 offers a number of operational, financial and clinical benefits. Apheresis do not require dilution and immunoselections rarely require more than one reagent kit. The final stored volumes are considerably reduced as is the fluid/DMSO load administered to patients. Single umbilical cord blood (CB) is an accepted alternative among patients with no available related HLA-identical donor. The co-infusion of CD34+ cells from a non-HLA-identical donor (dual SCT) has also shown to be useful. We describe retrospectively the main clinical results of both strategies in our institution. S157 Methods: Six patients underwent single unit umbilical CB transplantation between September-06 and June-07. On the other hand, ten dual SCT were performed in 9 patients from March-04 to January-07. All patients had high-risk hematologic malignancies. There were no significant differences between age, sex, HLA disparities, median of transplanted cells, number of therapy lines before transplantation, and pre-SCT status. TABLE 1. Results: Table 2 . Engraftment of ANC>500/µL was reached at +23 (15-29) for CB pts and +16 (9-28) days for dual pts (p=0.05); platelets> 20,000 at +48 (44-168) and +29 (9-41) days respectively (p=ns). One dual SCT pt showed primary graft failure and received a second dual SCT. The time to achieve complete CB chimerism in peripheral blood and bone marrow was not different. Both groups showed similar rates of infectious complications, without serious bacterial infections. There was a visceral tuberculosis case among dual transplanted patients. No GvHD ≥ grade III were present in any patient. Relapse rate was 0% for the CB group, probably due to short survival and short follow-up, and 44% for the dual group (3 patients died and 1 underwent a second non related SCT successfully). With a median follow-up of 7.2 months in the CB group the mortality at 100 and 180 days was 50% (3/6). Causes of death were: 2 multiorgan toxicity and 1 post-SCT lymphoma. One additional pt died due to pulmonary CMV at +270 d. In the dual SCT group the median follow-up was 35 months with 22% mortality at 100 days and 30% at 180 days; causes of death were: 2 relapses and 1 multiorgan toxicity. One additional pt died due to relapse at +270 d. Conclusions: In our experience, CB SCT with co-infusion of CD34+cells from a 2nd donor showed faster neutrophil engraftment. Remarkably, mortality rate was similar in both groups, however causes of death were different: CB SCT showed mortality due to toxicity while mortality associated to dual SCT was mainly due to relapse. More cases and a longer follow-up are needed to assess this matter. Comparison between lenograstim and filgrastim plus cyclophosphamide for peripheral blood progenitor cell mobilisation R. Ria*, M.A. Barletta, G. Iodice, A. Vacca, F. Dammacco University of Bari Medical School (Bari, IT) Purpose: Recombinant human (rHu) granulocyte-colony stimulating factor (G-CSF) has been widely used to treat neutropenia as well as mobilize bone marrow blood progenitor cells (BMPCs) for autologous and allogeneic BMPCs transplantation. G-CSF shortens the duration of neutropenia, and is thus used for lowering frequency of neutropenic fever. Beside the effects on proliferation and differentiation of myeloid precursor cells, G-CSF also alters functional activity of mature neutrophils, thus influencing chemotaxis, respiratory burst, and antigen expression. Patient response to hematopoietic progenitor-cell mobilizing regimens seems to vary considerably, hence preventing comparison between regimens. Here we compare the number of progenitors mobilized into blood after rHu nonglycosylated (filgrastim) and glycosylated (lenograstim) human G-CSF on days 1 to 12. Patients and methods: Eighty-six patients with multiple myeloma, non-Hodgkin and Hodgkin lymphoma were consecutively enrolled to receive mobilization regimen with cyclophosfamide 3 to 7 gr/mq plus lenograstim (55 pts) or filgrastim (31 pts) on days 1 to 12, and underwent leukaphereses. The hematopoietic progenitor-cell content of each collection was determined. In addition, toxicity, day of leukaphereses needed to reach the minimum target of progenitor cells, and day to WBC and platelet recovery were evaluated. Results: Lenograstim gave mobilization of more CD34(+) cells (15.34±3.1x106 CD34/kg) vs. filgrastim (11.04±2.4x106 CD34/kg; paired t test, P<.01). The ability to achieve a target collection of ³3x106 CD34/kg using two leukaphereses was 75% for lenograstim and 48% for filgrastim. No significant difference between the two regimens for toxic events, day to WBC and platelet recovery were seen. Moreover, no differences were seen in the multiple myeloma, non-Hodgkin and Hodgkin lymphoma subgroup. Conclusions: Our data show that patients treated with highdose cyclophosphamide plus lenograstim mobilize more progenitors, with no differences as toxicity or day to WBC and platelet recover. Moreover, they achieve adequate mobilization and the target collection of progenitor cells in less time, and need less number of leukaphereses. Stem cell grafts quality estimation E. Babenko*, A. Golovatcheva, A. Alyansky, A. Pugachev, N. Mikhaylova, B. Afanasyev Pavlov State Medical University (Saint Petersburg, RU) Objectives: The outcome of the hematopoietic stem cell transplantation depends on different factors, including number of transfused mononuclear cell (MNC), CD34+ cells and colony forming unit-granulocyte-macrophage (CFU-GM) assay. Recent studies demonstrate high enzymatic activity of Aldehyde dehydrogenase (ALDH) in hematopoietic stem cells. The aim of this study is to compare ALDH activity level with expression of CD34 marker and colony-forming ability (CFU-GM assay) in fresh and frozen samples of bone marrow (BM) and peripheral blood stem cells (PBSC). Material and methods: 70 samples of BM and PBSC were characterized by measuring CD34+, ALDHbr+ and CFU-GM assay. For CD34+cells enumeration FACSCalibur flow cytometer was used with triple staining protocol, ALDHbr+ cells were identified using ALDEFLUOR reagent. CFU-GM assay was performed using Methylcellulose-Based Colony Assay Media. Results: Average number of CD34+, ALDHbr+ and CFU-GM is summarized in Table. Fresh allogenic samples (BM and PBSC) exhibited a good correlation between the percentage of CD34+ cells and ALDH activity and the number of CFU-GM (p=0,0001).In fresh autologous samples (BM+PBSC) the number of CD34+cells was almost identical ALDHbr+ cell(p=0,0001). Nevertheless, there was no correlation between CFU-GM and CD34+ and ALDHbr+ (p=0,96, p=0,456 respectively).In thawed autologous samples number of CFU-GM was in good correlation with ALDHbr+cells (p=0,03), but not with CD34+ counts. According to this study cryopreservation of PBSC resulted in loss of CD34+cells and ALDH br+ cells: 46% and 16% respectively. BM stem cells were more stable: there was no loss in ALDH br+ cells and the number of CD34+ cell decrease to 85% to compare with initial number. Conclusion: Measuring of ALDH activity can be used as an additional stem cell marker, associated with their repopulating potential. Background: Mobilized peripheral blood stem cells (PBSC) have become the main source for autologous or allogeneic transplantation following myeloablative therapy in patients with haematological malignancies or solid tumours. Classical strategies for PBSC mobilization include administration of granulocyte colony-stimulating factor (G-CSF) alone or in combination with other cytokines or myelosuppressive chemotherapy. PBSC mobilization and collection have been optimized in numerous clinical trials, but a significant proportion of patients failed mobilization. The aim of the study was to establish the influence of multiple clinicl and laboratory factors on the outcome of peripheral blood stem cell mobilization. Patients and methods: 216 patients with haemathological malignancies and solid tumors were included in the study (Hodgkin's lymphoma (n=49), non-Hodgkin lymphomas (n=52), multiple myeloma (n=44), acute leukaemias (n=27), solid tumors (n=30)). There were a few patients wth CML (n=8), MDS (n=6), but they were excluded from the study because of their minority. 156 patients (72%) were mobilized with G-CSF, a combination of chemotherapy and G-CSF has been used in 60 patients (28%). 92 patients (42%) received more than six courses of chemotherapy and 124 (58%) less than six respectively. Bone marrow involvement was diagnosed in 76 (35%) patients. We also analyzed the level of leukocytes, thrombocytes, CD34+ in the peripheral blood. We used the following criteria for the mobilization outcomesuccessul mobilization when ND34+/kg>2x106. Results: There were no influence of disease type, age and sex on stem cell mobilization. BM involvement does not seem to be an poor prognostic factor of PBSC mobilization (p= .78). In patients received less than six courses of chemotherapy stem cell yield was significantly higher (10.0±2.2x106CD34+/kg against 5.5±1.7x106 CD34+/kg(p= .006)). Better outcome was seen in patients mobilized with chemotherapy plus G-CSF than G-CSF alone (8.12±1.12x106 CD34+/kg against 6.9±0.9x106CD34+/kg.(p= .008)). Leukocytosis and the number of PB CD34+ were independent risk factors. Conclusions: Diagnosis, age, sex, bone marrow involvement does not influence outcome of stem cell mobilization. Better stem cell yield was seen in patients received less than six courses of chemotherapy and in patients mobilized with cytokines combined with chemotherapy than cytokines alone.Leukocytosis and the number of PB CD34+ were obtained as independent risk factors. Background: Umbilical cord blood (UCB) is increasingly used as an alternative source in adults who need an allogeneic transplantation but lack a suitable donor. Double UCB grafts help to overcome the cell dose limitation. Methods: Between December 2001 and May 2007 thirteen patients (male: 7, female: 6) with a median age of 34.5 years (range: 21-54) and a median weight of 64 kg (range: 47-82) underwent 16 UCB transplantations. All recipients had relapsed or resistant disease (8 AML, 2 ALL, 1 AUL, 1 CML with blast crisis, 1 Hodgkin's disease). Myeloablative conditioning was performed in seven and reduced intensity conditioning in six patients. GvHD prophylaxis consisted of ATG (5mg/kg from days -4 to -1), cyclosporine A (starting on day -1 with 3mg/kg/d iv.) and methotrexate (10mg/m² on days +1 and +3). A second UCB transplantation was performed in two patients because of graft failure (only ATG was given before the 2nd graft) and in a third patient because of chemosensitive relapse. Due to low cell count a double UCB graft was used in one patient. Grafts (total number = 17) were matched 6/6 (1), 5/6 (5), 4/6 (9) and 3/6 (2). The median nucleated cell dose was 3.8 x107/kg (range: 2.5-6.3). Results: Neutrophil recovery without G-CSF administration occurred at a median of 35 days (range: 25-51). A selfsustained platelet count above 20x10 9 /L was achieved at a median of 42 days (range: 27-61). Graft failure was observed in 2 cases (HLA match 3/6 and 4/6). Six patients died because of infections and 4 because of progression or relapse of disease. Five patients developed acute GvHD (grade I, n=1; grade II, n=4). No chronic GvHD has been observed. In December 2007, three of 13 patients (23%) are disease-free with a follow-up of 72, 17 and 16 months. All of them have a Karnofsky index of 100% and are back at work. Conclusion: The outcome of our patients with advanced stage of disease treated with UCB transplantations is comparable with published data using a suitable HLA-matched donor. Therefore UCB transplantation should be considered if no suitable donor is available in time. An important graft-versus-leukemia (GVL) response caused by Natural Killer (NK) cells after haploidentical stem cell transplantation, without causing significant graft-versus-host disease, is mainly referred to a KIR-ligand (Killer Immunglobulin-like Receptor) mismatch between donor and recipient cells. Adoptive transfer of NK cells could be an attractive tool for cancer immunotherapy. Difficulties regarding the isolation of sufficient numbers of tumor-reactive NK cells lead to limited data about their safety and clinical effects. We have developed a novel culture system without the use of feeder cells for the ex vivo generation of NK cells from CD34+ hematopoietic progenitor cells. This two-step NK cell generation system uses mainly cytokines such as SCF, Flt3-L, IL-2 and IL-15 and glycosaminoglycans to direct first expansion phase and second differentiation phase. The developmental phase and the final NK cell product is controlled and characterized by immunophenotyping using multi-colour flow cytometry and CFSE-based cytotoxicity assays against various tumor cells. The described system generates a homogeneous cell product of CD56+/CD3-cells with a purity >95% of total cells. A total cell expansion of 10.000 fold allows to generate 10 10 NK cells from 10 6 CD34+ stem and progenitor cells within 5 weeks of culture. During the expansion phase of two weeks we expand CD34+ cells more than 100 fold. The phases of development show a decrease of stem cell-specific antigens (i.e. CD34, CD117) during the first three weeks, whereas antigens specific for NK cells and NK cell progenitors (i.e. CD56, CD94, CD161) are up-regulated after initiating differentiation at day 14 ( Figure 1 ). The effective differentiation of the expanded progenitor cells into NK cells is characterised by the expression of NK cell-specific antigens including CD56, CD94, KIR, NKG2A, NKG2D and NKp receptors as well as homing receptors such as CD62L, CXCR4 and CCR7. The NK cell product shows high expression levels of inhibitory and activating receptors such as KIR indicating that the final product is highly activated. Cytotoxicity assays demonstrated robust lysis of more than 90% against AML as well as melanoma tumor cell lines ( Figure 2 ). This system, with its huge expansion potential to generate highly activated NK cells with homing capability, is the basis for a first clinical trial in 2008, to infuse haploidentical NK cells generated from CD34+ cells in poor-risk AML patients. Human CD34+ myeloid leukaemic progenitor cells are susceptible to lysis by minor histocompatibility antigen LRH-1-specific cytotoxic T lymphocytes I. Overes (1) CD8+ T cells recognizing minor histocompatibility antigens (MiHA) on leukemic stem and progenitor cells play a pivotal role in effective graft-versus-leukemia (GVL) reactivity after allogeneic stem cell transplantation. Previously, we have identified a novel hematopoietic cell-restricted MiHA, designated LRH-1, which is presented by HLA-B7 and encoded by the P2X5 purinergic receptor gene. Interestingly, tetramer analysis showed a direct association between in vivo expansion of LRH-1-specific CD8+ T cells and the disappearance of Bcr-Abl positive tumor cells in the accelerated phase CML patient from whom LRH-1-specific CTL was originally isolated. Furthermore, we demonstrated that P2X5 mRNA is significantly expressed in CD34+ leukemic subpopulations from chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. These findings indicate a role for LRH-1 in inducing GVL immunity against myeloid leukemic stem and progenitor cells. Here, we investigated susceptibility of CD34+ leukemic progenitor cells to LRH-1-specific CTL lysis and the applicability of LRH-1 as a specific target for immunotherapy of myeloid leukemia. We demonstrated that LRH-1-reactive CTL specifically recognize and efficiently kill CD34+ myeloid leukemic progenitor cells obtained from chronic phase CML patients, while cytokinematurated CML cells are only marginally lysed due to >10-fold down-regulation of P2X5 mRNA expression. Furthermore, targeting of IFNg-stimulated CD34+ blasts from the accelerated phase CML patient by LRH-1-specific CTL resulted in complete growth inhibition within 3 days. Finally, tetramer analysis revealed that LRH-1-specific CD8+ T cells could be detected in two other myeloid leukemia patients following DLI treatment. Remarkably, 6.9% LRH-1-specific CD8+ T cells were present in an AML patient treated with preemptive DLI, who was in clinical remission without GVHD. This finding correlates with the hematopoiesis-restricted expression of the P2X5 gene. In conclusion, our data provide a rationale for use of P2X5-encoded MiHA as immunotherapeutic targets to treat residual or persisting myeloid malignancies after allogeneic stem cell transplantation. The effector function of natural killer (NK) cells is regulated by activating and inhibitory receptors (KIRs). In haploidentical Tcell depleted transplantation the donor/recipient KIR mismatch significantly impacts on clinical response, particularly in acute myeloid leukemia (AML). Therefore, haploidentical KIRmismatched NK cells may be used as adoptive immunotherapy for high risk AML patients in nontransplantation settings. Thirty-one AML patients entered a phase I-II study of NK-cell based immunotherapy and were screened for the availability of one haploidentical KIR ligand mismatched donor. Ten of them resulted as having one suitable donor. NK cells were enriched from steady-state leukaphereses by using an immunomagnetic separation system (Miltenyi Biotec,Germany). CD56+ NK cells were enriched from 8.28±3.7% to 85.9±3.2% (recovery 67.9±15%, viability >92%). So far, 9 patients (1 partial remission, 4 progressions and 4 complete remissions) received NK cell infusion which was preceeded by administration of fludarabine 125 mg/mµ and cyclophosphamide 4g/mµ. AML patients received 6 subcutaneous injections of interleukin 2 (IL-2) after infusion. The median number of reinfused NK cells was 3.6x10 6 /Kg and contaminating CD3+ T cells were always less than 1x10 5 /Kg. NK cells were capable to kill in vitro NKsensitive K562 cells. The procedure was well-tolerated and no significant toxicity, including graft versus host disease (GVHD, was observed. The patient in partial remission obtained a complete remission, which lasted for 6 months; among the 4 patients with progressive disease 3 patients showed the persistence of disease and one patient died during the aplastic phase. The 4 patients in complete remission are stable after 5,3,4 and 1 months of follow up. By using a RT-PCR-based chimerism assay, we demonstrated the presence of adoptively transferred NK cells in 2/4 patients with a peak in circulating NK cell number at day 12 after infusion. Functional studies demonstrated the presence of alloreactive NK cell clones in all donors (at the frequencies of 11±4%) and in 3/4 patients (at the frequences of 16±5%) only at day 3 after infusion of NK cells. In summary, enrichment of CD56+ NK cells allows the collection of a suitable number of target cells to be used as adoptive immunotherapy in AML patients. Infusion of cryopreserved NK cells is feasible and safe and adoptively transferred NK cells can be detected in the peripheral blood after infusion. Cytomegalovirus (CMV) disease is a significant cause of morbidity and mortality after allogeneic stem cell transplantation, especially in CMV seropositive (CMV+) patients transplanted with a CMV-donor. Although we and others have demonstrated the feasibility and possible clinical effectiveness of in-vitro selection and adoptive transfer of CMV-specific CD8+ memory T cells from CMV+ donors, no suitable method was available for the induction of primary immune responses against CMV for the treatment of persistent CMV reactivation in the high risk group of patients transplanted with a CMV-donor. In the current study we investigated the possibility to induce and isolate CMV-specific T cells from CMV-healthy donors by in-vitro priming and selection. We used as responder cells CD45RO-PBMC from HLA-A1, A2, A3, B7, or B8 positive CMV-donors (n=16). By CD45RO depletion we removed the majority of regulatory T cells capable of inhibiting the initiation of the response. Naïve donor T cells were cocultured in the presence of IL-7 and IL-15 with mature monocyte-derived dendritic cells loaded with a cocktail containing 1µg of each relevant CMV pp65, pp50, or IE1 derived 9-mer peptide. At day 10, the responses were restimulated with peptide loaded autologous PBMC. At day 20 CMV-specific CD8+ T cells were detected by specific tetramer or pentamer staining, and isolated by flowcytometric cell sorting or magnetic bead isolation, or further enriched by another restimulation, followed by isolation of CD137 or IFNg expressing T cells at day 21. In 16/16 CMV-donors CMV specific T cells could be detected at day 20 of the immune response in frequencies ranging from 0.01-0.4%. T cells against all 3 major immunogenic CMV proteins pp65, pp50, and IE1 were detected and isolated with different dominant responses detected in different donors. To further illustrate the primary nature of these immune responses, responses were successfully generated against known minor histocompatibility antigens (3/3) . The isolated T cells were capable of vigorous in-vitro expansion, expressed high affinity T cell receptors, and were highly cytotoxic against both peptide loaded target cells and targets presenting endogenously processed antigen. In conclusion, we have developed a method for the in-vitro induction and isolation of functional CMV-specific CD8+ T cells from CMV-donors. This may allow the treatment of serious CMV-related complications in CMV+ patients transplanted with a CMV-donor. Donor lymphocyte infusions (DLI) are often used preemptively for a loss of donor T cell chimerism, but carry a substantial risk of inducing graft-versus-host disease (GVHD). We and others have shown that CD8-depleted DLI can be administered with a reduced risk of GVHD. In this study, we investigated the impact of prophylactic CD8-depleted DLI on T cell chimerism after allogeneic stem cell transplantation in patients with hematologic malignancies who were conditioned with fludarabine, melphalan, and alemtuzumab. Eighteen patients received between 1 and 4 times CD8-depleted DLI in increasing doses starting with 1x10 6 /kg bodyweight after the immunosuppressive medication had been tapered. The median time of the first DLI was day +119 (range, 60 -194). Four patients developed acute GVHD of grade III or IV and 3 patients had extensive chronic GVHD during a median observation period of 15 months after the first DLI (range, 2-36). We also analyzed 16 patients following the same protocol who did not qualify for DLI due to acute GVHD (n=14) or unavailable donors (n=2). The patients' characteristics in both groups were comparable with a median age of 55 (range, 35 -64) years in the DLI group and of 56.5 (range, 29 -67) years in the non-DLI group. The donor types were matched sibling (DLI: n=5; non-DLI: n=2), matched unrelated (DLI: n=7; non-DLI: n=7), and mismatched unrelated (DLI: n=6; non-DLI: n=7), respectively. For chimerism analysis, donor T cells were isolated by immunomagnetic cell sorting and chimerism was determined by standard STR-based assay. Ten of the 18 patients in the DLI group had a secondary loss of donor T cell chimerism and one patient's T cells showed prolonged mixed chimerism. In the non-DLI group, 4 out of 16 patients had a secondary loss of complete donor chimerism and only in one patient of these, the mixed T cell chimerism reconverted spontaneously. In contrast, all 10 patients with the secondary loss of donor as well as the single patient with ongoing mixed T cell chimerism converted to durable full donor T cell chimeras after CD8-depleted DLI. Our results demonstrate that prophylactic CD8-depleted DLI are able to reverse a decrease in donor T cell chimerism. A randomized trial in patients with a secondary loss of donor T cell chimerism should now investigate whether CD8-depleted DLI compared with undepleted DLI lead to an improved clinical outcome with regard to GVHD and disease relapse. E. Chernykh* (1), V. Stupak (2), O. Leplina (1), E. Shevela (1), I. Pendurin (2), S. Nikonov (1), A. Ostanin (1) (1 Objectives: Despite resent advances in radio-, chemotherapy and surgical treatment, the prognosis for patients with malignant glioma is still very poor. Therefore, the development of new treatments, such as immunotherapy is very important. Dendritic cells (DCs) are antigen presenting cells that play a central role in the initiation and modulation of immune response. In this study we investigated the safety and immunologic and clinical response of tumor lysate-pulsed DC therapy for patients with malignant glioma. Methods and Materials: Thirty nine patients with anaplstic astrocytoma (AA, n=24) and glioblastoma (GB, n=15) were enrolled in this study. Controls represented the population of 80 patients (AA=47 and GB=33) who received surgical resection and radiation therapy. The patient's peripheral blood DC were generated with granulocyte macrophage colonystimulating factor plus interferon-alpha, and pulsed with an autologous tumor lysate. DCs were used for the generation of CTL (that were inoculated in the cavity of rejected tumor) and the course of 4-6 biweekly subcutaneous vaccinations after radiation therapy. Results: The protocol was well tolerated and was not associated with an evidence of toxicity or serious adverse effects. Sixty five percent of patients developed systemic immune response according to proliferation and positive delayed-type hypersensitivity skin test to autologous tumor lysate. The level of 1-2-and 3-year survival in this group was significantly higher in compare with controls (74 vs 52,5%, 61 vs 27,5 and 50 vs 19%, respectively). The most effect was observed in GB patients. A median survival in this group was 14 months vs 8 months in controls (p=0,003). Conclusions: Together, our results showed the safety and clinical response of autologous tumor lysate-pulsed dendritic cell therapy for malignant glioma patients. Prevention of relapse by donor lymphocyte infusion for mixed chimerism 7 months after T-cell depleted allogeneic stem cell transplantation W. Marijt*, I. Baas, P. von dem Borne, R. Barge, P. Deutz, F. Beaumont, I. Starrenburg, W. Fibbe, R. Willemze, J. Falkenburg Leiden University Medical Center (Leiden, NL) T cell depleted allogeneic stem cell transplantation (TCD alloSCT) is associated with both an increased relapse rate (RR) and mixed chimerism (MC) compared to non-TCD alloSCT. Since donor lymphocyte infusion (DLI) for hematological relapse of acute leukemia cures only 20-30% of patients (pts), pre-emptive DLI might restore complete chimerism (CC) and decrease the RR. We evaluated whether low dose DLI administered 6-9 months (mo) after transplantation (Tx) for acute leukemia in pts with MC not suffering from GVHD could decrease the RR without inducing severe GVHD. The effect on RR in this group of patients was compared with that of a historical control group. In group 1 (DLI group) 42 pts (AML 25, ALL 12, high risk MDS 5) and in group 2 45 pts (AML 29, ALL 13, HR-MDS 3) were transplanted in CR after conditioning with TBI and cyclophosfamide. Pts in group 1 received stem cells from HLA-identical sibling donors (27), HLA-matched unrelated donors (MUD)(13), or 1/2 antigen mismatched family donors (2) . Pts in group 2 were transplanted with an HLA-sib (41) or a MUD (4). Grafts were T cell depleted with 20 mg Campath. Only MUD Tx pts received ciclosporin for 6-12 weeks. 6 Mo after Tx 9/42 pts in group 1 had relapsed, and 6 pts had died due to infections. 7/26 MC pts had GVHD, which resolved in 3 pts. 3 MC pts did not receive DLI due to logistical problems. In total, 20 MC pts received a 1st DLI (3x10 6 CD3+ T cells/kg body weight) at a median of 7 (range 5-22) mo after Tx. When no CC and no GVHD was present next DLI were administered in escalating doses of 1x10 7 (4x), 3x10 7 (3x), and 1x10 8 T cells/kg (1x). The median follow-up after the 1st DLI was 16 (range 2-41) mo. In 14 pts the percentage patient cells decreased to <1% after DLI (p<0.05). In 6 pts MC did not change. In 6 pts GVHD grade I-III developed after DLI of whom 1 pat died. 4 Pts in group 1 not receiving DLI relapsed within 9 mo after Tx. None of the 20 MC pts treated with DLI developed a relapse from 9 mo post Tx onward (total RR in group 1 13/42). In group 2 13 pts relapsed in the first 9 mo post Tx. Thereafter, 6 additional patients developed a relapse. This difference was statistically significant (p<0.05). Nonrelapse mortality in both groups was identical (19% group 1, 22% group 2). In conclusion, our results indicate that low dose DLI projected to be administered at a median of 7 mo after Tx decreases the degree of MC in the majority of pts and significantly reduces RR without severe GVHD. Polyclonal memory CD8 T-cell responses to PRAMEspecific peptides occur in patients with acute leukaemias and chronic myeloid leukaemia K. Rezvani*, A.M. Yong, B. Jafarpour, R. Eniafe, S. Mielke, J. Barrett National Institutes of Health (Bethesda, US) PRAME (Preferentially expressed antigen of melanoma), highly expressed in various solid tumor cells and normal testis, is also aberrantly expressed in hematological malignancies. PRAME may therefore be a useful antigenic target for immunotherapy in leukemia. To determine whether PRAME is naturally immunogenic in leukemia, we studied CD8+ T-cell responses to four previously identified HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201 positive patients with acute lymphoblastic leukemia (ALL), acute myeloid S162 leukemia (AML), chronic myeloid leukemia (CML) and healthy donors. Using HLA-A2 tetramer analysis and intracellular (IC) interferon-gamma production by CD8+ T-cells pulsed with PRAME peptide, responses were detected in 4/10 patients with ALL, 6/10 with AML, 3/10 with CML and 3/10 healthy donors. The frequencies of PRAME-specific CD8+ T cells as measured by IC interferon-gamma production to peptide stimulation was greater in patients with myeloid leukemias and ALL compared to healthy controls. All four peptides were immunogenic in patients with AML, CML and ALL. PRA100, PRA142 and PRA300 were highly antigenic, each eliciting responses in 3/20, 4/20 and 7/20 of patients with myeloid leukemia (CML and AML) and 1/10, 3/10 and 3/10 patients with ALL respectively. In healthy donors however, PRA300 was the only immunogenic peptide, eliciting responses in 3/10 donors. PRAME-tetramer specific CD8+ T cells displayed both central memory (CD45RO+CD27+) and effector memory phenotypes (CD45RO-CD27-). We determined PRAME gene expression on patient peripheral blood (PB) by quantitative reverse-transcription PCR (qRT-PCR). Expression levels of >0.001 PRAME /ABL were significantly more likely to to be associated with CD8+ T-cell responses to 2 or more PRAME epitopes compared to those with expression levels <0.001 (4/6 vs. 0/24, P = 0.01), suggesting a PRAME-driven CD8+ Tcell response. The diversity of the peptide epitope amino acid sequences indicated a polyclonal T cell response to PRAME. The presence of memory T cell PRAME responses which are polyclonal in patients should facilitate the generation of robust immune responses using multiple peptide vaccines to treat leukemia in or outside the context of allogeneic stem cell transplantation. The beneficial effect of allogeneic SCT in MM is based on graft-versus-myeloma (GVM) reactivity. The proof of principle is the observation of clinical responses after donor lymphocyte infusions (DLI). In this study feasibility of T cell-depleted RICallogeneic SCT followed by DLI is studied, and the application of recipient dendritic cells (DC) as adjuvant immunotherapy to boost GVM reactivity. 17 MM patients received a T cell-depleted allogeneic SCT after conditioning with fludarabine 30 mg/m 2 /d and cyclophosphamide 1200 mg/m 2 /d on days -6 to -3. After discontinuation of CsA, patients without GVHD and minimal residual disease (MRD) will receive low dose DLI. Patients with persistent residual disease will be vaccinated with ex vivo generated, monocyte-derived DC from recipient origin. Prior to SCT, PBMC from all these patients were collected by leukapheresis and cryopreserved (2-4 bags of 6-12 x 109 cells/patient). Validation experiments showed that thawed monocytes can be used to generate mature monocyte-derived DC with a good yield. Functional assays demonstrated that the generated products have a mature phenotype (>80% CD80+CD83+), good migration capacity towards lymph-node chemokines and ability to stimulate T cells in a MLR reaction. All 17 patients engrafted. Eight patients developed mild GVHD, 7 patients grade II and 1 patient grade I. Seven patients with residual disease were treated with low-dose DLI (1.0 x 106 T cells/kg), and 5 patients received a second DLI (5 x 106 T cells/kg). None of these patients developed GVHD after DLI. Five patients with MRD have received 3 vaccinations with recipient DC. The number of DC varied from total 4 x 10 6 to 48 x 10 6 per vaccination. One-third of the DC vaccine was given i.d. and two-third i.v. Recipient DC vaccinations were given with limited toxicity. Three patients developed fever and local induration at the injection site. Remarkably, none of these patients developed GVHD after recipient DC vaccination. In 1 patient, we observed a very good PR with an ongoing decline of light chains. Another patient became in CR after DCvaccination. These responses will be objectivated by IgH-PCR. Further immunomonitoring is currently performed. Combination of T cell-depletion and RIC is feasible in MMpatients following autologous SCT. Thawing-and culture procedures were optimized to use monocytes collected before SCT for culturing mature DC. Five patients have received DCvaccinations without significant toxicity or GVHD. CD8 Leukemia-associated-antigens including proteinase 3 (PR3) and elastase (ELA2) are self-antigens which induce low frequency autoreactive T-cells in normal individuals. PR1, an HLA-A*0201 epitope shared by PR3 and ELA2, is expressed in normal neutrophils and overexpressed in myeloid (but not lymphoid) leukemias. T-cells against PR1 have been linked to graft versus leukemia (GVL) in myeloid leukemia. We looked for PR1-specific CD8+ T-cells in 28 patients (13 chronic myeloid leukemia (CML), 10 acute lymphoblastic leukemia (ALL), 5 solid tumors) in the first 30-120 days following T-cell depleted SCT, using PR1/HLA-A2 tetramers and intracellular interferon-gamma staining, and correlated these with ELA2 and PR3 expression (using qRT-PCR) and GVL effects. Ten CML, 6 ALL and 3 solid tumor patients had detectable PR1 responses post-SCT. PR3 and ELA2 expression was strongly associated with emergence of PR1-specific T-cells. Disappearance of PR3 and ELA-2 expression coincided with disappearance of PR1-specific T-cells (P<0.001). The in-vivo anti-leukemia effect of the PR1 response was assessed in CML patients by BCR-ABL qRT-PCR. Nine of ten patients with early PR1 responses were BCR-ABL negative at day 90 post-SCT compared to 0/3 without (P<0.001).This GVL association was restricted to CML: in ALL, using WT1 qRT-PCR to measure minimal residual disease (MRD), 2/5 patients with PR1 responses and 3/5 patients without were MRD+ on day 90 (P=0.36). Since PR1 responses were not CML restricted and all patients had 100% donor myeloid chimerism by day 30, the recovering donor marrow was the likely antigenic source of PR3 and ELA2 driving the PR1 response. We found a significant correlation between ELA2 and PR3 gene expression levels, neutrophil-monocyte count and the emergence of PR1 CD8+ T-cell responses in patients with ALL and solid tumors. Our findings suggest that the post-SCT milieu is favorable for exaggerating weak autoimmune responses to self-antigens such as PR1. GVL may follow if the self-antigen is also expressed on the leukemia as in CML. These results suggest that vaccination together with induction of T-cell homeostatic proliferation is likely to enhance the antileukemia response of transplantation. The in vitro generation of mature T cells from human hematopoietic stem and progenitor cells (HSPC) could fulfill two existing needs. First, it could enhance T cell immune reconstitution after stem cell transplantation. Second, the generation of tumour antigen specific T cells could provide an efficient therapy for numerous malignancies and could enhance GVT effect in the context of allogeneic SCT, without aggravating GVHD. A few years ago, it was shown that T cells could be generated from human HSPC by culturing these cells on the murine stromal cell line OP9, that was transduced with the Notch ligand Delta-like-1 (OP9-DL1). HSPC from human umbilical cord blood (UCB) (CD34+38-) and postnatal thymus (PNT) (CD34+) were introduced into our in vitro coculture system with OP9-DL1, in the presence of the cytokines Flt-3L (5 ng/ml), SCF (2.5 ng/ml) and IL-7 (5 ng/ml). Every 3-5 days cells were harvested and transferred to fresh OP9-DL1 cells. Each time an aliquot was analyzed phenotypically (flowcytometer). We found that under the described conditions, HSPC from both UCB and PNT mature into CD4+CD8b+ double positive (DP) T cell precursors. Some cells acquire TCRab, others TCRgd, with a more efficient generation of gd T cells in control conditions. Also the final maturation stages, characterised by the acquisition of CD69, then CD27, then the loss of CD1 and finally the loss of CD69, are reached more efficiently and rapidly in TCRgd cells compared to TCRab cells. These phenotypically mature cells also appear to be functional as illustrated by their proliferation in response to PHA in the presence of irradiated mononuclear cells. In contrast, when a g-secretase inhibitor (GSI), that inhibits the proteolytic intracellular processing of notch and thefore inhibits downstream signalling, is added to cultures containing CD4+CD8b+ T cell precursors, the development of TCRab is favoured relative to TCRgd development, and the maturation of both cell types is enhanced, suggesting that the final maturation of T cells is inhibited by Notch signalling, which has not been shown before. We can conclude from this study that it is feasible to generate phenotypically and functionally mature T cells from human HPSC by coculturing them with the murine stromal cell line OP9-DL1. We have also demonstrated that a decreased notch signal from the DP stage on, leads to a growth advantage for TCRab cells compared to TCRgd cells, and to an enhanced maturation of these populations. (1) Donor lymphocyte infusion is an effective form of adoptive immunotherapy for hematological malignancies after allogeneic stem cell transplantation. Graft-versus-Leukemia reactivity, however, is often accompanied with Graft-versus-Host Disease (GvHD) due to recognition of ubiquitouslyexpressed minor histocompatibility antigens (mHags) on non-hematopoietic tissues. Transfer of T cell receptors (TCRs) recognizing hematopoiesis-restricted mHags to virus specific T cells may be a powerful anti-tumor therapy with a low risk for GvHD. The purpose of this study is to develop an optimal TCR-encoding multi-cistronic retroviral vector and an efficient method to generate TCR-engineered virus specific T cells for adoptive immunotherapy. Retroviral vectors encoding the TCR for hematopoiesis-restricted mHag HA-2 with and without selection marker gene were compared for TCR surface expression and HA-2 specific lysis. In addition, two different methods, i.e. peptide stimulation of CD8+ cells and pentamerbased isolation of antigen specific T cells, were investigated for their efficiency to generate TCR-transduced virus specific T cells. Bi-cistronic retroviral vectors without selection marker gene most efficiently mediated TCR surface expression and HA-2 specific lysis. Furthermore, both methods were useful for generating gene-modified virus specific T cells, but the purity of virus specific T cells after pentamer isolation was higher as compared to peptide stimulation of CD8+ cells. Finally, the capacity of gene-modified cells to express the transgenic TCR at the cell surface markedly differed between virus specific T cells and correlated with lysis of relevant target cells. In conclusion, our data support the use of bi-cistronic retroviral vectors and pentamers for generating TCR-engineered virus specific T cells and illustrate the relevance of selection of gene-modified T cells with appropriate surface expression of the transgenic TCR for clinical gene therapy. M. Casucci* (1), S. Perna (1), A. Bondanza (1), Z. Magnani (1), M. Bernardi (1), A. Crotta (1), A. Cignetti (2), F. Caligaris- Cappio (1), F. Ciceri (1), C. Bordignon (1), C. Bonini (1)(1 Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the first clinical success of cancer immunotherapy. Donor lymphocyte infusion (DLI) after allo-HSCT allows to cure chronic myeloid leukemia but is less effective against acute myeloid leukemia (AML), suggesting the need for more potent therapeutic approaches. Myeloid blasts have the unique ability to differentiate into dendritic cells (DCs), offering a model in which the most potent antigen presenting cell and the tumor cell coincides. In principle, leukemic dendritic cells (LDCs) could be used to improve the anti-tumor effect of DLI. Current protocols for differentiation of AML blasts into LDCs are based on cytokines cocktails. Unfortunately, these protocols allow the differentiation of less than 30% of primary AMLs. The aim of this study was to evaluate the ability of primary and secondary AML to differentiate into functionally competent LDC by the use of the calcium ionophore A23187, able to overcome cytokine receptors, in combination with IL-4. In a cohort of 11 AML patients, we observed that a short (48 hrs) exposure to A23187+IL4 allows the LDC differentiation of a large proportion of AML blasts (9/11 AMLs) and shows an optimal toxicity profile (mean of cell yield: 30%). LDCs significantly up-regulated the expression of CD86, CD80, HLA-DR, CD54 and CD58, markers involved in the immunological synapse. Such up-regulation correlated with an increase in T-cells allo-stimulatory capacity that proved similar to that of healthy DCs. In addition, LDCs were able to produce immunostimolatory (IL-12) but not tollerogenic (IL-10) cytokines. Most importantly, LDCs differentiation upon exposure to A23187+IL4 allowed preserving the expression of disease surface markers (CD34 and CD117) and possibly leukemic antigens, ultimately leading to the priming and expansion of leukemia-reactive allogeneic lymphocytes. This protocol has been able to directly induce the acquisition of a mature phenotype in 44% of LDCs, with the highest frequency observed among AML secondary to myelodisplasia. The differential ability of primary AML and AML secondary to myelodisplastic syndromes to rapidly differentiate into functionally mature LDC suggests the existence of molecular factors committing blasts to DC lineage. The efficacy of A23187+IL4 in differentiating both primary and secondary leukemic blasts into LDCs provides the rational for novel adoptive immunotherapeutic approaches to treat high risk AML. The aim of the study was to evaluate feasibility and preliminary efficacy of dendritic cell (DC) based vaccination intended as an adjuvant therapy. Twelve patients with indolent B cell lymphomas in remission following a standard chemotherapy (CHT) received a median of 6 (range 2-10) injections of irradiated lymphoma/DC hybrids or DCs pulsed with tumor lysate. Lysate-pulsed DCs were used for booster injections, whenever the amount of collected viable lymphoma cells was not sufficient to complete vaccination with the use of cell hybrids. Autologous lymphoma cells electrofused with either autologous or allogeneic DCs were administered to uninvolved lymph nodes under USG guidance. Monocytederived DCs were generated from the peripheral blood (PB) adherent cells stimulated with GM-CSF and IL-4. If only possible, autologous DCs were generated also from the bone marrow DC progenitors. Four patients with less than complete remission after CHT discontinued vaccination after 2-6 injections due to disease progression. Three of these patients died. One patient with multiple myeloma remains in partial remission 20 months (mos) after the start of vaccination but developed a myelodysplastic syndrome. Eight patients were vaccinated during remission following CHT. One patient with mantle cell lymphoma (MCL) relapsed 15 mos after the start of vaccination performed following the second-line CHT, and died 10 mos later; 9 years since MCL diagnosis. One patient with MCL presenting with IgM paraproteinemia relapsed 32 mos after starting immunization ( Fig. 1 Current methods for the detection and isolation of antigenspecific T cells require the availability of peptide/MHC multimers or are restricted to cells that produce cytokines after antigen contact. We herein demonstrate that de novo cell surface expression of the TNF receptor family member CD137 identifies currently activated, but not resting, human virusreactive CD4 and CD8 T cells. Antigen-triggered CD137 expression was first detectable upon 6h of stimulation, and reached peak intensity at 24h, allowing the determination of a clear-cut population of CD137+ T cells at this time point. The median frequencies of cytomegalovirus (CMV) pp65-reactive CD137+ cells measured ex vivo in 12 different CMV+ healthy donors after 24h of stimulation were 4.5% (range, 0.5-8.6) in CD8 T cells and 2.7% (range, 0.4-6.4) in CD4 T cells, respectively. PBMC derived from the same donors and left unstimulated, as well as PBMC from CMV-seronegative donors stimulated with CMV peptides contained less than 0.4% CD137+ cells per total CD4 and CD8 T cells, confirming the specificity of antigen-induced CD137 expression. We next established an in vitro approach allowing the activation and subsequent CD137-based magnetic cell sorting of virusreactive CD4 and CD8 T cells at the same time. We demonstrated the suitability of this assay to isolate CMVpp65reactive CD4 and CD8 T cells from PBMC of 10 CMV+ healthy individuals. Enriched fractions had a median purity of CD137+ cells of 63.8% (range, 12.7-94.1) among CD4 T cells and 76.5% (range, 28.5-93.4) among CD8 T cells, respectively. The CD137+ populations could be expanded in vitro and showed CMVpp65-specific cytokine production by CD4 and CD8 T cells as well as a strong enrichment of CMVpp65/HLA tetramer-binding CD8+ T cells. We finally compared the efficiency of the CD137 assay with the IFN-g secretion assay to isolate CMVpp65-specific CD4 and CD8 T cells from PBMC. Although both methods were performed at optimal conditions, the numbers of CD137+ T cells measured before and after enrichment were approximately 2-fold higher than those of IFN-g+ cells (n=5), suggesting that CD137 detects a broader repertoire of virus-reactive T cells. In conclusion, activation-induced CD137 expression provides a means for the rapid identification and sorting of viable virusreactive CD4 and CD8 T cells. The CD137 assay is most attractive for the simultaneous targeting of both T-cell subsets in monitoring studies and adoptive immunotherapy trials. Ex vivo generation of antigen specific T-cells for adoptive T-cell therapy: antigen-dependent prestimulation followed by Dynabeads CD3:CD28-based expansion G. Borelli* (1), T. Aarvak (2) , G. Kvalheim (1) Adoptive T cell therapy has been used for treatment of infectious diseases and cancer. To achieve clinically required T cell numbers and break tolerance induced by the tumor ex vivo expansion might be a required step. We have developed a T cell expansion method where we have optimized the culture conditions to retain the antigen specific T cells during expansion. T cells are expanded using Dynabeads coupled with anti-CD3/CD28 (Dynabeads ClinExVivo CD3/CD28). These beads mimic the engagement of the TcR/CD3 complex and the CD28 molecule during antigen presentation. If the patient samples contain a low frequency of antigen specific T cells a prestimulation step using antigen loaded-dendritic cells (DC) or peripheral blood mononuclear cells (PBMC) prior to Dynabeads expansion increases the total number of antigen specific T cells. Therefore, a prestimulation step might be included in a clinical protocol where reinfusion of virus-specific or tumor specific T cells will be performed. As a model system for optimizing the T cell expansion protocol we have measured the number of virus specific CD8+ T cells before and after expansion using pentamers. Since regulatory T cells (Treg) have been shown to reduce the immune response we have depleted Treg cells with CD25 Dynabeads prior to 9 to 10 days expansion with Dynabeads CD3/CD28 in presence of IL-2. Our data show that by including a prestimulation step we obtained up to 47% CMV or EBV specific CD8+ T cells, even in donors who had no detectable levels of these cells in PBMC. When stimulated with Dynabeads CD3/CD28, the virus specific CD8+ T cells showed more than 300-fold expansion. By manipulating the ratio of anti-CD3/CD28 on the Dynabeads the final product was enriched for CD4+ (Dynabeads antiCD3/antiCD28high) or CD8+ cells (Dynabeads antiCD3/antiCD28low). A high Dynabeads: T cell ratio (3:1) deleted the virus specific T cells, which could be retained by reducing this ratio. By depleting Treg cells with CD25 Dynabeads the expansion of all T cell subsets (including virus specific T cells) was increased. After expansion the CD8+ T cells showed an early-intermediate effector phenotype with increased expression of CCR7 and CD62L, and the cells were cytotoxic. This protocol has been scaled up and a clinical T cell platform employing Dynabeads CD3:CD28 and Wave Bioreactor will be used for both virus and tumor specific T cell production. R. Zappasodi* (1), M. Di Nicola (1), C. Carlo-Stella (1), R. Mortarini (1), A. Molla (1), C. Vegetti (1), S. Albani (2), A. Anichini (1), A.M. Gianni (1)(1 Objectives: Effective adoptive T cell therapy requires the exvivo generation of functional T lymphocytes with a long lifespan in-vivo. For this reason, we evaluated the advantages in T cell expansion conferred by a new artificial antigen presenting cell (aAPC)-based system able to reproduce the natural APC membrane reorganization during the immune synapse formation. Methods: Our aAPCs were generated by clustering activating (anti-CD3), co-stimulating (anti-CD28) and adhesion (anti-LFA-1) biotinylated monoclonal antibodies (mAbs) in microdomains (MDs) held on a liposome scaffold by neutravidin rafts. The co-localization of T cell ligands in MDs and the targeting of an adhesion protein, increasing the efficiency of immunological synapse formations, represent the novelties of our system. The activity of our aAPCs was compared with that of anti-CD3/-CD28 coated immunomagnetic microbeads and immobilized anti-CD3 mAb (OKT3 clone), the only two commercially available systems suitable for clinical usage. A single artificial stimulation was provided to CD3+ T cells isolated from healthy donors and to anti-MART-1 CD8+ T cells, enriched after being cultured with the cognate peptide for 15 days. Two weeks after the artificial stimulation, expanded T cells were counted and apoptosis was assayed in order to compare the fold increases in viable cell number obtained with the different systems. Moreover, the maturation and the activation levels of expanded T cells were evaluated and functional assays were performed to verify their cytotoxic activity. Finally, to allow the feasibility of the procedure for large-scale production of T cells in clinical setting, several experiments were performed by reducing the dose of aAPC or increasing the concentration of T cells in cultures. Results: Our aAPCs exhibited high efficiency (1) in expanding functional polyclonal CD3+ T cells and tumor-specific CD8+ T cells, (2) preserving the highest cell viability in culture compared to the other commercial artificial systems, (3) enriching the fraction of T cells expressing a central memorynaïve phenotype, (4) without increasing the frequency of CD25high FoxP3+ regulatory T cells; (5) moreover they proved to be suitable for large scale application in clinical setting. Conclusion: our aAPCs might represent an efficient tool to rapidly obtain a sufficient number of functional T cells for adoptive immunotherapy in cancer patients. We previously showed the possibility of expanding large-scale amounts of anti-tumor cytotoxic T-cell (CTL) lines derived from peripheral blood lymphocytes (PBL) in compliance with GMP standards. We are currently evaluating the feasibility and safety of adoptive immunotherapy consisting of lymphoablative chemotherapy, to favor T cell proliferation in vivo, followed by CTL infusions for patients with advanced solid tumors. Four patients with (metastatic) renal sarcoma, extraosseous PNET, endometrial stromal sarcoma, and renal cell carcinoma, with large tumor masses progressing after failing conventional therapies, have been treated so far. In all subjects we were able to generate CTL lines displaying high levels of cytotoxic activity against patient tumor cells but not against non-malignant cells. Patients received lymphoablation with fludarabine/cyclophosphamide followed by CTL infusions on day +14 and +28, and every 2 months thereafter. Patient #1 and #2 received 6 CTL infusions. The median number of CTLs administered/infusion was 206x106 (150-280) and 313x106 (120-600), respectively and disease progressed 6 and 5 months after the last CTL infusion. Patient #3 so far received 12 infusions of higher doses of CTL (median 676x106, range 100-1726) and remains in stable disease at >2 years after starting CTL therapy. Patient #4 received 15 infusions of high doses of CTL (median 1.600x106, range 830-4.000) and remained in stable disease for 2.5 years, then progressed. No adverse events related to CTL infusions occurred. Immunological monitoring, performed in three patients, showed an increased frequency of tumor-reactive cells in PBL, persisting for 3-4 weeks after infusions. We have shown that autologous ex vivo generated anti-tumor CTL lines, can be infused safely, and are able to increase tumorspecific responses measured in vitro. In the two patients in whom large quantities of antitumor CTLs were infused, we observed long-lasting disease stabilization. Optimization of this immunotherapy strategy (frequency of lymphoablation and CTL infusion schedule) is currently under investigation. Introduction: rituximab has made a significant change on the biology and natural course of non-Hodgkin's lymphoma (NHL) since its introduction. Yet, little is known on the effect of rituximab in patients after autologous stem cell transplantation (SCT). Patients and methods: Thirty seven patients (24 males and 13 females) suffering from B cell NHL were treated with rituximab following autologous SCT (36 conditioned with TECAM, 1 with BEAM). The median was age 49.6 years (range 19-71). Basic disease was diffuse large B cell NHL (16), follicular NHL (9), mixed (3), T cell rich B cell (3), primary mediastinal lymphoma (3), mantle (2), and SLL (1). Disease status upon SCT was CR (12), PR (15) and relapsed/persistent disease (11). Rituximab dose used was IV 375 mg/m 2 in all patients and intrathecal 25 mg (2 patients). The treatment was prophylactic or therapeutic in 20 and 18 patients respectively and the median time from SCT to treatment was 158.5 days (range 41-4230). A range of 1-3 cycles given and a median of 4 doses per cycle (range [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] with an interval ranging from every week to every 3 months. Rituximab treatment was accompanied by cytokine treatment (interferon alpha and/or interleukin 2) in 21 patients. Results: With a median follow up of 18.25 months (1-90 months) , 25 patients are alive and most are disease free (figure 1). Cause of death was disease progression in most patients. Therapy related complications included neutropenia (1) and recurrent infections (1) . Conclusion: we conclude that the addition of rituximab to post autologous SCT prophylactic or therapeutic regimens seems promising although this should be further verified in controlled clinical trials. Grafting T cells by tumor-antigen specific T cell receptors (TCR) could trigger the initiation of effector function and redirect T cell cytotoxicity towards tumors. We utilized various HLA-A2.1 transgenic mice to bypass human MDM2-and p53specific self-tolerance. The efficiency of double chain (dc) TCR modified T cells could be affected by the incorrect TCR a/b chain pairing between endogenous and transgenic TCR constructs to form hybrid TCR potentially leading to autoimmunity. To address this concern, chimeric A2.1restricted peptide-specific murine single chain (sc) TCRs were constructed (Va-Li-VbCb) and retrovirally transduced into human T cells. Despite detectable surface expression, these chimeric receptors were not able to convey any MDM2-or p53-specific cytolytic activity. Therefore we developed a truncated TCR-alpha domain (Ca) comprising solely the TCRa signal peptide, the ecto-domain, the transmembrane region as well as the cytoplasmic tail and cotransduced these construct with the scTCRs. We anticipated that Ca would stabilize scTCR expression by interacting with the single chain beta chain. Indeed, this approach not only led to increased expression levels of the chimeric scTCRs, but also induced specific lysis of A2.1 positive MDM2 or p53 peptide-pulsed target cells as well as solid tumor cell lines. Recognition of malignant targets by p53 specific scTCR transduced CD4 and CD8-positive T cells was equivalent to that observed with double-chain p53 TCR gene modified effector cells. To test whether this concept is applicable to human TCRs as well, we constructed a human gp100-specific scTCR and a human Ca domain. In contrast to the gp100-specific double chain TCR, only a marginal expression pattern was observed for the human scTCR / Ca constructs. Introduction of an additional disulfide bond within the constant domains in order to stabilize TCR surface expression showed no effect. Since murine TCR are expressed on human T cells to a much higher extent, the human constant beta-domain of the scTCR was replaced by murine Cb. Comparable to the murine scTCR concept, the chimerized scTCR coexpressed with murine Ca demonstrated high cell surface expression and triggered cytotoxicity of malignant A2.1/gp100-positive targets. In summary, our results lay a commonly applicable conceptual basis for the construction of therapeutic scTCR to prevent recombination of natural and transgenic dcTCR alpha and beta chains. Imatinib and its role in allogeneic stem cell transplantation D. Bund* (1), R. Buhmann (2) , H. Kolb (2) (1)Helmholtz Center Munich (Munich, DE); (2)LMU Großhadern (Munich, DE) It was shown that Imatinib, a potent and selective inhibitor of the BCR-ABL tyrosine kinase, can induce durable haematological and major cytogenetic responses in a high percentage of CML patients. However, the disease recurs in most patients when Imatinib is discontinued. In contrast, allogeneic stem cell transplantation (ASCT) is considered to be the only curative treatment and it does cure by the immune effect of donor T-cells against CML progenitor cells. The role of Imatinib is contentious. On the one hand it may improve the results of ASCT by reducing the tumour load, on the other hand it may also reduce the effect of donor lymphocyte transfusions (DLT) by impairing the function of T-cells and the capacity of myeloid cells to present antigen. Patient derived CML-cells were investigated to stimulate allogeneic HLA-matched and mismatched T-cells without and with Imatinib (1, 2 or 5 micro M). The proliferative response of the T-cells was evaluated after a 5 day culture in presence of various Imatinib concentrations and various responder-tostimulator ratios. Thus, proliferation was detected via CFDA. The activation profile (CD25) and the expression of the TCRalpha/beta on the T-cells was determined by FACS. The cytotoxicity of CD8+ T-cells was assessed in [51Cr]-release assays after a 7 day culture of CML cells with HLAmismatched T-cells. Additionally, we characterized the antigen-presenting profile (HLA-DR, CD40, CD80, CD86, CD54 and CD58) of the CML cells over a 5 day culture in the presence and absence of Imatinib. The proliferative capacity of allogeneic T-cells was inhibited in the presence of Imatinib in a dose-dependent manner. Moreover, the T-cell activation marker CD25 and the expression of TCRáâ was reduced in the presence of the different Imatinib concentrations. Pretreatment of CML cells with Imatinib for 48 hours had only slight impact on the proliferation and activation of T-cells. Besides, Imatinib impaired the cytotoxic function of HLA-mismatched T-cells also in a dose-dependent manner. Finally, the antigenpresenting profile of the myeloid leukemia cells, mostly costimulatory molecules (CD86, HLA-DR), was downregulated by increasing concentrations of Imatinib. In summary, Imatinib may impair both, the T cellular immune response and the antigen presenting profile of the CML cells in vitro. These results might have an impact on new strategies of treatment of chronic myeloid leukaemia with immunotherapy. (1) The occurrence of allo-reactive NK cells after KIR-ligand mismatch HSCT may be correlated with a favorable influence on relapse rates, engraftment and incidence of GvH disease. To benefit from NK cell effects after HSCT it is crucial to 1. Understand the mechanisms that regulate NK cells cytotoxicity and 2. Predict the resulting NK cell repertoire and the functional consequences after HSCT. We investigated the influence of different HLA class I on cytotoxic responsiveness of NK cells which express only one defined HLA class Ispecific inhibitory receptor (mono-KIR NK cells). Further we analyzed cytotoxic reactivity of hyporesponsive mono-KIR and KIR-NKG2A-NK cells. Immuno-magnetically enriched mono-KIR and KIR-NKG2A-NK cells from donors of different HLA class I (=KIR-ligand) background were stimulated with HLA class I negative (control) and single HLA class I transfected target cells. Cytotoxic reactivity was analyzed by flow-cytometry based CD107A degranulation assay. KIR2DL2/3-and KIR2DL1-mono-KIR NK cells from HLA-C1 homozygous donor confronted with different target cells showed the following stimulation indices ( Cw0505 and inadequate ligand Cw0303, respectively. KIR-NKG2A-NK cells of same donor, but either co-cultured with target cells alone or together with cytotoxic active mono-KIR2DL1 NK cells resulted in the following SI: K562 10.02 and 48.08; K562-Cw0505 3.60 and 18.17; K562-Cw0303 4.19 and 21.00, respectively. Thus, we observed inhibition of NK cells cytotoxicity by HLA class I, which is not ligand to the HLA class I specific inhibitory receptor of the NK cells. State of hyporesponsiveness of NK cells, finding in their donors no appropriate ligand to HLA class I-specific inhibitory receptor or lacking expression of them, is variable. Cytotoxic active NK cells may recruit hyporesponsive NK cells to augment anti-neoplastic efficacy. Comparison of the cytotoxic effects of 4 commercially available preparations of anti-T-cell globulins in various haematological malignancies F. Ayuk, N. Maywald, U. Larsen, S. Hannemann, A.R. Zander, N. Kröger University Medical Center (Hamburg, DE) Objective: Polyclonal Anti-T-cell globulines (antithymocyte globulines) are widely used in allogeneic stem cell transplantation mainly due to their anti-T cell and immunomodulatory activity. We have previously reported potent anti-myeloma activity of ATG-Fresenius®. Similar data were later reported for Thymoglobulin® Various ATG preparation have been shown to induce apoptosis in malignant B cell lines. We thus sought to compare the activity of the 4 commercially available ATG preparations in myeloma, B-NHL, myeloid leukemia as well as healthy T cells. Materials and methods: The cell lines RPMI-8226, EJM, KMS-12-BM, OPM-2, U266 (myeloma), HL60, BV173, K562 (myeloid leukemia) and Jurkat (T-lymphoblastic leukemia) were used. Primary T cells were obtained from healthy donors by negative selection using Rosettsep for T cells. Primary CLL samples were obtained from patients and crypreserved after Ficoll gradient centrifugation until use. Cells were incubated at 1 x 10-6 ml-1 with 10-500ug ml-1 of ATGs. For complementdependent cytotoxicity, 50% active human serum (pooled from 5 healthy donors and cryopreserved until use) was added as source of complement and cells incubated for 45 minutes prior to staining with 7AAD and flow cytometry. For complementindependent cell cytotoxicity, cells were incubated with ATG for 20 hours prior to staining and flow cytotometry. Results: 1. All ATG preparations showed potent complementdependent cytotoxicity against primary T cells. 2. The cytotoxic effect of Atgam® was weaker than that observed for the other ATG preparations 3. Atgam® also showed a weaker anti-myeloma effect compared to the other ATG preparations. 4. ATG-Fresenius®, Thymoglobulin® and Lymphoglobulin® had similar anti-myeloma effects 5. The ATG preparations showed variable complementdependent cytotoxicity against myeloid leukemia cell lines, but 6. Little or no complement-independent cytotoxicity could be observed against myeloid leukemia cell lines Conclusion: Polyclonal ATG preparations all possess potent activity against hematological malignancies in particular those of lymphatic origin. We suggest that this potential be taken into consideration when designing future conditioning regimens. Our findings may also be helpful in choosing the optimal dose for each ATG preparation, when targeting hematological malignancies. Limited efficacy of donor lymphocyte infusions despite high donor chimerism and low lymphocyte counts in relapsing non-CML diseases H. Guillaume*, P. Chevallier, J. Delaunay, S. Ayari, P. Moreau, F. Mechinaud, M. Mohty, J.L. Harousseau CHU Nantes (Nantes, FR) Donor lymphocyte infusions (DLI) are generally performed either to treat residual or relapsing disease or to convert mixed chimerism following allogeneic transplantation. The efficacy of DLI to treat relapsed malignancies is still debated. Chimerism and low lymphocyte count prior to DLI have been reported to have a favorable impact on disease response and survival. This report assessed the outcome of 54 pts receiving DLI for various indications (disease relapse or progression, n=46; mixed chimerism (<90% donor) without relapse, n=5; EBV lymphoproliferation, n=3). Diagnoses included MDS or AML, n=18, myeloma, n=14, NHL, n=7, Hodgkin's disease, n=3, CML, n=6, CLL, n=3, and myelofibrosis n=1. Median age was 47.5 (range, 1.5-62) y. 89% of donors were HLA-matched siblings, while 11% were MUD. Reduced intensity conditioning was used in 27 patients (50%; fludarabine, busulfan and ATG), and 27 patients received a standard myeloablative TBI/cyclophosphamide-based regimen. GHVD prophylaxis was CsA alone in RIC patients, and CsA+Mtx in the myeloablative group. The graft was T-cell depleted in 8 cases. Median time to DLI post grafting was 247 (range, 19-1573) days. In addition to DLI, 22 pts (47%) received prior (n=9, 19%) or subsequent chemotherapy to enhance disease control. The median pre-DLI lymphocyte count was 1.05x109/L (range, 0.05-4.38). Prior to DLI, 60% of the pts were in full (> 95%) donor chimerism. Thirteen (28%) among those who have received DLI for relapse are still alive in CR with a median follow-up of 1165 (range, 230-3527) days. Overall, acute and/or chronic GVHD developed in 18 of 52 (35%) evaluable pts. However, GVHD was not predictive of CR and was not associated with a better survival in this group of pts. Three patients died from acute GVHD following DLI. Two of the 3 pts who received DLI for EBV lymphoprolif. responded, in three out the 5 mixed chimerism were corrected. Four out 6 CML pts (66%) responded to DLI, whereas only 9 pts (19%) had tumor response in the non-CML group. The overall probability of survival following DLI was 43.2% (95% CI, 35.1-62.9) at 4 years for the whole group. For those receiving DLI in the non-CML group, the overall survival probability from DLI was 36.2 % at 4 years. Full donor chimerism prior to DLI or lower lymphocyte counts (below the median value of 1.05 x 109/L) were not predictive of survival (full chimerism, 45.3% vs. 50.5%). We conclude that DLI has limited efficacy as salvage therapy in relapsing or progressive non-CML diseases. Background: Human class I leukocyte antigens are ligands for killer cell immunoglobulin receptors (KIRs). These receptors are expressed by NK and T-cells and thus modulate innate and adaptive immunity. KIR mismatches have been suggested to modify the outcome after stem cell transplantation. The clinical impact of killer cell receptors may vary between disease entities and pre-and post transplant therapeutic regiments. We have therefore performed a retrospective study with patients transplanted in our centre between 1996 and 2007. Patients and methods: Out of a consecutive cohort of 147 allografted AML-patients, samples from 54 donor/patients pairs were evaluable for retrospective KIR-ligand matching. The median patient age was 47 (25-66) years. Patients were transplanted with G-CSF-stimulated PBSC (n=51) or bone marrow (n=3) from HLA matched unrelated (n=31) donors or family related donors (n=23). KIR typing was performed as previously described. Patients were categorised according to their HLA inhibitory KIR ligand group C1, C2, Bw4, A3/A11 and presence or absence of KIR. Results: The overall survival (OAS) of all 54 evaluable AMLpatients was 65%. A total of 15 patients died due to relapse (n=10) or non-relapse mortality (NRM, n=5). Patients with KIR-mm had a lower NRM (1/31 vs. 4/23, p=0.06) and a lower mortality due to relapse (3/31 vs 7/23, p=0.17). OAS was superior in patients with KIR-ligand mismatches (KIR-mm) compared to the group of patients without mismatches (85% versus 42%, log rank p= 0.004). Patients with 2 KIR mismatches (C1 and/or C2) had even better survival compared to patients with single KIR mismatches. Conclusions: Our results demonstrate that patients with KIR mismatch have a reduced mortality due to both relapse and as well as NRM, resulting in superior overall survival. Patients with AML benefit from KIR-ligand mismatched allografts. KIR typing would be a useful tool to be included to define optimal histocompatibility of donor patient pairs. (29) or matched unrelated donors (12) using a reduced intensity conditioning (RIC) regimen consisting of fludarabine, busulphan and ATG combined with alemtuzumab added to the graft. In 38 patients mixed chimerism was present after transplantation for which donor lymphocytes were given with a median dose of 5 x 10E6/kg CD3+ cells (range 1-5) at a median time point of 7 months after SCT (range 5.1-15). After DLI, 35 patients (92%) developed an immune response as defined as a major decrease in the patient chimerism. In 26 patients full donor chimerism was achieved, in 9 patients a low patient signal remained present (1-2%) . Patient chimerism decreased from median 12% (range 1 to 86%) to 0% (range 0-2%). In 31 of 38 patients measurable disease was present before DLI. In 22 of these patients conversion from mixed to full donor chimerism coincided with clinical GVT responses. Seven patients with active myeloid disease all achieved complete remission (4 AML/MDS patients with 8-65% blasts, 2 patients with progressive CML and 1 with active CMMOL). All these patients are still in CR after a median time of 22 months. In 8 myeloma patients 5 complete and 1 partial response were observed. Most myeloma patients subsequently developed bone or extramedullary relapses without bone marrow disease. In 13 patients with lymphoid disease 9 responses were observed. One patient with an immunocytoma and one with T-PLL achieved CR. In 7 CLL patients 3 CR and 1 PR were observed, notably two CR occurred in patients with massive bone marrow involvement. Two of four patients with aggressive NHL showed a CR to DLI in combination with rituximab and one patient a PR to DLI. Although no tumor regression was observed in three patients with renal cell carcinoma after DLI, disease progression was halted for several years. Grade 3-4 acute GVHD developed in 22% of patients, grade 1-2 in 39%. Limited or extensive chronic GVHD was observed in 30 and 23% of patients, respectively. GVHD responded to therapy in most patients, only in 9% of patients chronic GVHD did not resolve. Mortality due to acute and chronic GVHD was 10%. In conclusion, after T cell depleted RIC SCT a state of mixed chimerism is induced in most patients, which can be converted into full donor chimerism with DLI in more than 90% of patients. This conversion is accompanied with clinical GVT responses in a high percentage of patients with acceptable GVHD. The contribution of NK cells to graft-versus-malignancy mechanisms following HLA-identical peripheral blood stem cell transplantation (PBSCT) remains unclear. To determine the role of the grafted NK cell dose and the donor's repertoire of activating and inhibitory killer cell Ig-like receptors (KIRs) for relapse-free and overall survival following T-cell-replete PBSCT from HLA-identical siblings, 83 consecutive transplants for haematological malignancies were retrospectively evaluated. A score based on the presence of the inhibitory KIR3DL2 missing its ligand, HLA-A3/A11, the activating KIRs 2DS2, 2DS4, 2DS5, and a high NK cell dose was associated with reduced relapse incidence (23% versus 61%; p=0.0013), superior relapse free survival (56% versus 23%; p=0.0011) and overall survival (61% versus 26%; p=0.068 for all transplants; 55% versus 9%; p=0.027 for reduced intensity conditioning transplants). These results support the importance of NK cells for graft-versusmalignancy alloreactivity in HLA-identical PBSCT. While HLA-C incompatibility likely dominates NK alloreactivity in HLAmismatched PBSCT, KIR3DL2 missing HLA-A3/11 and specified activating KIRs may contribute to NK reactivity following HLA-identical PBSCT. Donor regulatory T-cells characterised by the CD4+CD127low/neg phenotype correlate with graftversus-tumour effect after donor lymphocyte infusion Y. Hicheri (1) Background: Regulatory T-cells (Treg) play a pivotal role in the control of graft-versus-host disease (GVHD) and might thus also influence the graft-versus-tumor (GVT) effect after allogeneic hematopoietic stem cell transplantation (HSCT). Recent studies have demonstrated that the majority of Foxp3+ Treg cells have a weak expression of CD127 (interleukin-7 receptor alpha chain) corresponding to a CD127low/neg phenotype. We aimed to assess the role of Treg on the clinical outcome after donor lymphocyte infusions (DLI) by analyzing the Treg frequencies in DLI products, using the CD25 and Foxp3 classical Treg markers but also the membranous CD127 expression. Patients and Methods: Treg frequencies in DLI products were quantified by flow cytometry in 25 patients treated for relapse of various hematological malignancies (14 acute leukemia, 5 chronic myeloid leukemia, 6 others) after HSCT from a sibling (n=21) or matched unrelated donor (n=4). We compared (i) patients who developed significant GVHD (grade II to IV, n=5) vs. those who did not (grade 0 or I, n=20), and (ii) those with durable complete (n=6) vs. no/partial remission (n=18) of their malignancy after DLI (n=1 not evaluable). The mean follow-up after DLI was 20 (2-61) months. Four of the 6 patients in complete response after DLI developed GVHD while only 1 of 5 patients developing GVHD did not reach complete remission (p=0.006). Results: Our study reveals that patients in durable complete remission had received a lower, relative or absolute, number of FoxP3+CD25+ or FoxP3+CD127lo/-Treg cells compared to the group of patients with no or partial remission after DLI (p=0.04 for both). The same difference was observed when analyzing the DLI content in CD4+CD127lo/-T cells (p=0.04, Figure 1 ). Patients developing GVHD after DLI also received lower numbers of Treg but this difference did not reach statistical significance. In multivariate analysis adjusted for diagnosis (CML vs. others), sex mismatch, and the CD3+ Tcell dose infused, the CD4+CD127lo/-Treg content of DLI products was the only factor significantly correlated with the hematological response (p=0.05). Conclusion: CD4+CD127lo/-Treg cells could exert an inhibitory effect on the GVT effect after DLI. This phenotype characterization of Treg could be relevant to anticipate DLI efficiency or to manipulate this cell-population in order to increase the GVT effect of DLI in relapsing patients. Rejection of murine high grade B-cell-lymphoma by a foreign antigen requires interferon gamma signalling A. Gerbitz* (1), M. Sukumar (2) , A. Wilke (2) , J. Mautner (3), H. To date mechanisms of T-cell mediated immunity and immune-escape in B-cell-lymphomas are poorly understood. In fact even when lymphomas express foreign antigens such as viral antigens derived from Epstein-Barr-Virus, they are neither recognized nor rejected. Using a transgenic mouse Bcell-lymphoma model we show here that lymphoma specific expression of chicken ovalbumin (OVA) after retroviral transduction (OVA-IRES-GFP) mediates the rejection upon transfer of lymphoma cells into wild type host animals. 50% of the recipient mice rejected OVA containing lymphomas whereas we observed a 100% lymphoma growth of IRES-GFP control transduced lymphomas in GFP transgenic recipients that are tolerant for GFP. Developing OVA expressing lymphomas displayed a loss of GFP expression indicating a selection for non transduced cells. In spleens from mice successfully rejecting OVA-containing lymphomas we found up to 1.5% (±0.12%) SIINFEKL specific T-cells. After transfer we observed an induction of MHC surface expression on transferred lymphoma cells expressing OVA of approximately 10 fold for class I and up to 50fold for class II compared to cells before injection. This induction could be mimicked in vitro by stimulation with interferone gamma and was lost in STAT1-/-lymphoma cells. To further gain mechanistic insights of lymphoma rejection, we transferred OVA transduced lymphoma cells into Stat1-/-and IFNg-/recipients. Lack of STAT1-/-on the recipient side or inability to secrete interferon gamma was associated with faster lymphoma progression and was not different when compared to IRES-GFP transduced cell lines injected into GFP transgenic hosts. Although we found 1.6% (±0.52%) SIINFEKL T cells in spleens of lymphoma bearing STAT1-/animals, interferon gamma production was significantly decreased. Surprisingly transfer of primed OTI T-cells in STAT1-/-recipients of OVA containing lymphoma did not result in the rejection of the lymphoma. These results suggest that IFNgamma signalling is crucial for rejection and that interferon signalling is required on the host side, i.e. in the stroma. In addition these results point towards the ability of B-cell lymphomas to induce tolerance or anergy towards specific antigens. Identification of phosphatidylinositol 4-kinase type II beta as HLA class II associated minor histocompatibility antigen involved in graft-versus-leukaemia reactivity M. Griffioen*, E.D. van der Meijden, M.W. Honders, C. Rutten, S.A.P. van Luxemburg-Heijs, P.A. von dem Borne, R. Willemze, J.H.F. Falkenburg Leiden University Medical Center (Leiden, NL) Patients with hematological malignancies can be successfully treated with HLA-matched T cell-depleted allogeneic stem cell transplantation (alloSCT) and subsequent donor lymphocyte infusions (DLI). The efficacy of DLI is mediated by donor T cells recognizing minor histocompatibility antigens (mHags) on malignant recipient cells. Since HLA class II molecules are predominantly expressed on hematopoietic cells, mHag specific CD4+ T cells may selectively mediate Graft-versus-Leukemia (GvL) reactivity without Graft-versus-Host Disease (GvHD). The aim of this study was to identify the HLA class II associated mHag that is recognized by CD4+ T cells induced in a patient with relapsed chronic myeloid leukemia (CML) after HLA-matched alloSCT who developed strong GvL reactivity with mild GvHD of the skin after treatment with DLI. We previously developed recombinant bacteria cDNA expression libraries based on delivery of exogenous antigens and used this method for identification of the first autosomal HLA class II (HLA-DQB1*0603) associated mHag LB-PI4K2B-1S. LB-PI4K2B-1S has a population frequency of 40-50% and is encoded by the broadly-expressed phosphatidylinositol 4kinase type II beta gene. In the patient with CML, a polyclonal CD4+ T cell response against LB-PI4K2B-1S and simultaneous mHag specific CD8+ T cells were demonstrated. LB-PI4K2B-1S specific CD4+ T cells were shown to recognize and lyse the CD34+ CML cells of the patient and other leukemic cells, as well as high HLA-DQ-expressing normal hematopoietic cells. HLA-DQ expression on normal cells of non-hematopoietic origin was moderately upregulated by IFNg and not sufficient for recognition by LB-PI4K2B-1S specific CD4+ T cells. In conclusion, the data suggest that (1) LB-PI4K2B-1S specific CD4+ T cells mediated tumor rejection by directly eliminating the malignant cells of the patient as effector cells and stimulating the induction and maintenance of CD8+ T cell immunity as helper cells, and (2) HLA-DQ associated mHags may be appropriate targets for T cell therapies with the aim to selectively stimulate GvL after HLAmatched alloSCT with a low risk for GvHD. High response rate and improved graft-versus-host disease following bortezomib as salvage therapy after reduced-intensity conditioning allogeneic stem cell transplantation for multiple myeloma Despite progress in terms of TRM, a significant proportion of multiple myeloma (MM) patients may still progress after reduced-intensity conditioning allogeneic stem cell transplantation (RIC allo-SCT). This report describes the results of 37 MM patients who received bortezomib (1.3 or 1.0 mg/m² intravenously, days 1, 4, 8, and 11) as a salvage therapy after RIC-allo-SCT. Overall, 32 patients (86%) received bortezomib salvage in progressive disease, while 5 patients (14%) were in PR. Median time between allo-SCT and bortezomib initiation was 20 (range, 1-65) months. At time of bortezomib initiation, the majority of patients (n=26; 70%) did not have any symptoms of chronic GVHD, while 8 patients S171 (22%) had some form of limited chronic GVHD, and 3 patients (8%) had extensive signs. The median number of bortezomib cycles administered was 6 (range, 1-15). Peripheral neuropathy was frequently observed after bortezomib (n=13; 35%; 4 grade 2 and 9 grade 1). Mild thrombocytopenia not requiring platelets transfusions was observed in 9 cases (24%). Fatigue was also observed in 7 patients (19%) . None of the patient had to discontinue the treatment because of a life-threatening adverse event, and no treatment-related toxic deaths were observed. In terms of GVHD, patients did not experience reactivation or worsening of GVHD symptoms. Interestingly, two patients among the three patients with extensive GVHD signs at the beginning of bortezomib experienced a significant improvement and were staged as limited chronic GVHD at last follow-up. In all, 27 patients (73%; 95%CI, 59%-87%) achieved an objective disease response (7 CR, 7 VGPR, and 13 PR). Prior use of thalidomide and/or DLI did not influence response to bortezomib. There was also no difference in response when using bortezomib with or without dexamethasone. With a median follow-up of 9 (range, 3-42) months from bortezomib initiation, 25 patients (68%; 95%CI, 53%-83%) still had a sustained objective disease response (5 CR, 5 VGPR, and 15 PR). Ten patients (27%) died and 27 are still alive with a median overall follow-up after allo-SCT of 80 (range, 18-153) months. The majority of deaths were directly attributed to disease progression (n=8; 80% of all deaths). Most importantly, patients achieving an objective disease response (CR, VGPR or PR) after introduction of bortezomib enjoyed a significantly higher overall survival as compared to nonresponding patients (P=0.002). Collectively, these data demonstrate that bortezomib is a safe and potentially efficient option for MM patients failing RIC allo-SCT. Donor treatment with recombinant human interleukin-11 and recombinant human granulocyte colony-stimulating factor preserves the graft-versus-leukaemia effects after allogeneic bone marrow transplantation X.J. Huang* University of Beijing (Beijing, CN) Background and objective: Donor treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and recombinant human interleukin-11 (rhIL-11) in combination significantly reduced the lethal graft-versus-host disease (GVHD) of recipients after bone marrow transplantation (BMT). The impact of rhG-CSF and rhIL-11 in combination on graft-versus-leukemia (GVL) effects is currently still unclear. We, therefore, investigated whether the protection from GVHD and the maintain of GVL effects could be afforded by the combination of rhIL-11 and rhG-CSF simultaneously. Design and methods: We used a leukemic murine model to determine the GVL effects after transplantation of bone marrow grafts from donor treated with rh-G-CSF and rhIL-11 either alone or incombination. T cell subgroups, long-term engraftment were analyzed using flow cytometry. Lymphocyte proliferation ability/ mixed lymphocyte culture (MLC) was determined by monotetrazolium (MTT) assay. Cytotoxicity of T cells was examined using lactate dehydrogenase LDH-release cytotoxicity assay. Results: The grafts from donor treated with rhIL-11 and rhG-CSF incombination had significantly lower lymphoctes and CD3+, CD4+ and CD8+ T cells than those with rhIL-11 (2.01±0.75) or rhG-CSF (2.04±0.54) alone or control (1.82±0.33) (P<0.01). The ratios of CD4+ cells/CD8+ cells were also significantly lower in the grafts from donor treated with rhIL-11 and rhG-CSF in combination (1.52±0.47) (P<0.05). rhG-CSF and rhIL-11 either alone or in combination treatment to donors significantly decreased the lymphocyte proliferation in recipients after BMT to host alloantigens when compared with control (P<0.05). The killing potentials of T cells from donor treated with rhIL-11 and rhG-CSF either alone or in combination were significantly higher than those of control by day 12 after transplantation (p<0.05). Meanwhile, the allografts from donors treated with the rhIL-11 plus rhG-CSF was better in maitaining GVL effect and preventing GVHD after BMT than that treated with rhIL-11 or rhG-CSF alone. Interpretation and conclusions: Donor treated with the combination of rhIL-11 and rhG-CSF efficiently maintains the GVL potential of allografs. Key words: recombinant human interleukin-11; recombinant human granulocyte colony-stimulating factor; graft-versusleukemia effects; combination; T cell P641 Long-term outcome after sibling allogeneic haematopoietic stem cell transplantation plus modified prophylactic donor lymphocyte infusion for advanced leukaemia X.J. Huang* University of Beijing (Beijing, CN) Objective: To evaluate the long-term feasibility and efficacy of a modified prophylactic donor lymphocyte infusion (DLI) approach with advanced leukemia. Patients and Methods: Thirty-five patients with advanced leukemia received modified prophylactic donor lymphocyte infusion (The core content consists of two elements: granulocyte colony stimulating factor-mobilized peripheral blood progenitor cells (GPBPC) instead of steady donor lymphocyte infusion and the use of short-term CsA or MTX for prophylaxis of infusion-related GVHD) after HLA identical sibling allogeneic hematopoietic stem cell transplantation (allo-HSCT). Thirteen patients had AML, five were transplanted with primary resistant disease, eight were relapsed patients; twelve had ALL, eight were transplanted with primary resistant disease including four Ph+ ALL, four were relapsed patients; five had CML in AP and BP; four had myelodysplastic syndrome-refractory anemia with excess of blasts in transformation (MDS-RAEBT); one patient had myeloproliferative disease with 8p11 in refractory relapse. Results: Forty-two infusions were performed in 35 patients, of which 30 were before +90 days post-SCT. The median mononuclear cells (MNC) and CD3+ cells infused for GPBSCI before +90 days was 1.3x108/kg and 0.54 x108/kg, respectively. In addition, those after +90 days were 2.27x108/kg and 1.32x108/kg, respectively. Eight patients experienced II-IV grade acute GVHD. Twenty-two patients developed chronic GVHD, of which 15 cases were extensive. Twelve patients did not suffer transfusion-related GVHD. No GVHD related death or transfusion-related pancytopenia was observed. Fifteen patients were in disease-free survival after 46.7 (range, 14-58) months of follow-up. With an 18-month median follow-up, overall survival (OS) at 1 and 1.5 years was 70.9% and 48.8%, respectively; the respective leukemia-free survival (LFS) was 68.6% and 45.1%. One-year cumulative incidence of leukemic death and non¨Crelapse-mortality was 16.4% and 15.2%, respectively. Conclusion: The chosen sequential strategy of allo-SCT followed by modified prophylactic donor lymphocyte infusion might represent a step forward in the treatment of advanced leukemia. [Key words] advanced leukemia, donor lymphocytes infusion, granulocyte colony stimulating factor, hematopoietic stem cell transplantation (1) The presentation of leukemic antigens can be improved by conversion of leukemic cells to leukemia-derived DC (DCleu), thereby forming a platform for the generation of leukemiaspecific cytotoxic lymphocytes. We could already show:1) the generation of DC/ DCleu is possible from every AML/MDScase, independent of karyotype and FAB-type with at least one of 3 different DC-generating methods 2) DC/DCleu can be quantified by combination of suitable blast and DC-antigens and 3)The antileukemic reactivity of DC-trained T-cells can be specified in a fluorolysis assay (Schmetzer 2007). Aim: We want to enlight the role of the composition and quality of DC/ DCleu and (DC-trained) T-cells to mediate leukemiacytotoxic reactions ex vivo or to predict or correlate the clinical response to a DC/DLI-based immunotherapy in vivo. Methods: DC were generated from 27 AML-cases in blast-rich phases with the best of 3 DC-generating methods and used to train T-cells (autologous patients'-(n=10), allogeneic donor-(n=11) or T-cells at relapse after allogeneic SCT (n=6)) in a 'Mixed lymphocyte culture' (MLC). The leukemia-lytic activity of DC-(or blast-or untrained) T-cells against naïve blasts was quantified after 3/24 hours. Results: 1) DC can be generated in all 27 AML-cases. 2) In Ø 44% (n=12) of the cases T-cells gained a CTL-activity after DC-training. 3) The leukemia-cytotoxic T-cell training efficacy with DC was superior to a blast-training (Ø 42% vs 34% lysed blasts). 4) A comparison of cases with or without a gain of lytic T-cell activity showed 68 vs 60% DCleu, 52 vs 30 % mature and 27 vs 17% migratory DC and 50 vs 40% proliferating T-cells, 53 vs 46% memory T-cells, 68 vs 56% CD4+ T-cells. 5) In cases with a training with > 65% DCleu/ >40% mature DC/>65% CD4+ T-cells 70%/80%/80% of trained T-cells gained a lytic activity . 6) In AML-patients with(n=7)/ without (n=9) successful response to a GM-CSF-DLI-based therapy of relapse after SCT we could demonstrate 43 vs 29% DC; 78 vs 56% mature DC; 61 vs 52% blasts convertible to DCleu. Moreover we found, that cases with more vs less than 60% blasts convertible to DCleu the overall survival from transplantation to endpoint was 612 vs 168 days. Summary: That means, that the generability and composition of DC/ DCleu and of DC/MNC-trained T-cells could contribute to predict the clinical course of the disease after SCT in individual cases and could help to create specific antileukemic T-cells for immunotherapy of AML. Short tandem repeat allele matching between donor and recipient is associated with disease relapse after HLAidentical allogeneic stem cell transplantation C. Manzano*, D. Barroso, P. Balsalobre, D. Serrano, A. Gómez-Pineda, J.L. Díez-Martín, I. Buño Hosp. G.U. Gregorio Marañon (Madrid, ES) Background: Short tandem repeats (STR) are routinely used to distinguish donor (D) and recipient (R) DNA and therefore analyse chimerism after allogeneic stem cell transplantation (SCT). However, the influence of particular STR alleles in D and R on the outcome of SCT has been scarcely studied. Objective: To evaluate the association between D/R STR matching and the dynamics of chimerism and the development of complications post-SCT (rejection, relapse, graft versus host disease -GVHD-and exitus). Patients and methods: 32 patients that received a SCT from an HLA-identical related donor were analysed. Multiplex PCR was performed to amplify 11 STR (SGM AMPflSTR Plus, Applied Biosystems) loci from D and R genomic DNA (QIAamp Blood Minikit, Qiagen) obtained from peripheral blood samples before SCT. PCR products were revealed by capillary electrophoresis and analysed using the Genotyper software (Applied Biosystems). Overall D/R STR matching was analysed, being "0" the result for a D/R pair fully mismatched for each of the 11 STR markers, and "22" that of a D/R pair in which all 11 STR loci have both alleles identical. Results were analyzed using Fisher's exact test due to the reduced sample size. Results: Results varied from 9 to 18 (matched alleles out of the 22 possible variants) in the present series. Patients transplanted from highly matched donors (16-18 STR alleles matched) showed a statistically significant greater incidence of disease relapse than those transplanted from more mismatched (9-15 STR alleles matched) donors (4/7; 57.1% vs 4/25; 16%; p=0,046). Discussion: These data suggest that a greater "genomic identity", as measured through overall STR matching, between donor and recipient would be associated with a lower graft versus leukemia effect and therefore with an increased risk of relapse. Analysis of a larger sample is warranted in order to confirm these results and to establish D/R STR matching as a means to predict the outcome of HLA-identical related stem cell transplantation. HLA-DP as specific target for graft versus leukaemia reactivity in HLA-class II expressing haematological malignancies C.E. Rutten*, S.A.P. van Luxemburg-Heijs, M. Griffioen, E.W.A. Marijt, I. Jedema, M.H.M. Heemskerk, E.F.M. Posthuma, R. Willemze, J.H.F. Falkenburg Leiden University Medical Center (Leiden, NL) Mismatching for HLA-DPB1 in unrelated donor hematopoietic stem cell transplantation (URD-SCT) has been associated with a significant decreased risk of disease relapse, indicating that HLA-DP may represent a target for a graft versus leukemia (GVL) effect in HLA-class II expressing hematological malignancies. To investigate whether HLA-DP specific T cells could mediate GVL reactivity following HLA-DPB1 mismatched URD-SCT, we analyzed the immune response in a patient with refractory immunocytoma responding to DLI. Patient and donor were fully matched for HLA-A, -B, -C, -DR and -DQ but differed for both HLA-DP alleles (donor HLA-DPB1* 0402,0501; patient HLA-DPB1* 0201,0301). Following a non-myeloablative conditioning regimen the patient received a T cell depleted URD-SCT resulting in mixed chimerism (75% donor) without graft versus host disease (GVHD). After SCT persistent disease was observed and the number of malignant cells gradually increased to 58% in BM. Therefore, a single dose DLI was administered 7 months after SCT. DLI caused a profound antileukemic effect resulting in complete remission and conversion to full donor chimerism in the absence of GVHD. During the clinical response, the emergence of leukemia reactive CD4+ T cells was demonstrated using IFN-g ELISPOT analysis. These leukemia reactive CD4+ T cells were clonally isolated from peripheral blood and bone marrow and characterized. All CD4+ T cell clones (n=21) produced INF-g in response to patient leukemic cells but not to donor cells. To determine whether these CD4+ T cell clones were capable of lysing patient leukemic cells, a CFSE based cytotoxicity assay was performed. Eleven CD4+ T cell clones showed 20-84% specific lysis of the leukemic cell population. Using blocking studies, panel studies and retroviral transduction experiments of both mismatched HLA-DPB1 alleles, we identified HLA-DPB1*0201 and HLA-DPB1*0301 as the targets of this immune response. Furthermore, these HLA-DPB1 specific CD4+ T cell clones were capable of recognizing and lysing several HLA-DP expressing myeloid and lymphoid hematological malignant cells. Since the expression of HLA-DP is mainly restricted to hematopoietic cells, HLA-DP may be used as a specific target for immunotherapy following T cell depleted URD-SCT. Therefore, in patients with HLA-class II expressing hematological malignancies HLA-DP mismatched SCT may be preferable over a fully matched SCT allowing DLI to induce a potent GVL effect. So far, the Graft-versus-Leukemia (GVL) effect was a strictly clinically described phenomenon following allogeneic hematopoetic stem cell transplantation (HSCT). GVL effect is one of the most prominent examples showing the potential of the immune system to eliminate malignant diseases. However, in the last years T-cell responses against tumorassociated antigens (TAA) could partly be set in correlation with clinical benefit. Previously, TAA such as WT1 and proteinase-3 have been proposed as the targets for T-cells to establish a GVL effect. Now, we examined in addition other TAA (MUC1 and HM1.24) as possible T-cell targets of GVL related immune responses. We have defined new peptide epitopes from the MUC1 and HM1.24 antigens by the reverse immunology approach to increase the number of patients who can be screened and to expand the repertoire of immunologic monitoring as well as therapeutic approaches. A total of 25 patients after allogeneic stem cell transplantation have been screened and we are able to detect T-cell responses to both the MUC1 and HM1.24 antigens on top of the WT1 and the proteinase-3 antigen. Interestingly, we could detect a significant relationship between relapse and the absence of a T-cell response to TAA: Only 1/10 patients (10%) with TAA-specific CTL relapsed in contrast to 8/15 patients (53.3%) without TAA-specific CTL responses (p < 0.05). Furthermore, we demonstrated MUC1 peptides presented by HLA A*6801, B*0702 and B*4402 to be specifically recognized by CD3+/CD8+ T-cells. In conclusion, CD8+ T-cell responses directed to TAA might contribute to the GVL effect and are not limited to WT1 and proteinase-3. These observations clearly highlight both the importance and the potential of immunotherapeutic approaches in allogeneic stem cell recipients. Advanced tumor-stage mycosis fungoides (MF) and Sézary syndrome (SS) are characterized by very poor prognosis, mainly due to chemoresistance and rapid disease progression. Because skin is a frequent target of graft versus host disease (GvHD), allogeneic hematopoietic stem cell transplantation (HSCT) may represent a potential strategy of cure in patients with cutaneous T-cell lymphomas (CTCL), mainly by its immunomediated action. In our Institution, between 09/2000 and 12/2007, seventeen patients with stage IIIB-IVB refractory MF/SS underwent allogeneic HSCT either from HLA-identical sibling, or from HLA-matched unrelated donor (MUD). A reduce intensity conditioning (RIC) was used in 15 pts, whereas 2 pts underwent myeloablative conditioning (MAC) due to bulky tumor disease. All had previously received at least two conventional lines of treatment. RIC regimens included fludarabine/cyclophosphamide/TBI200 (3 pts) or pentostatin/TBI200 for related donor-HSCT (12 pts) and melphalan/CP1H/fludarabine/TBI200 for MUD-HSCT (1 pt) or pentostatin/TBI200 for UCB-HSCT (1 pt); MAC regimen comprised thiotepa (15 mg/kg) and CTX (150 mg/kg). All patients (10 males and 7 females; median age 49 years, range 38-66) had a clinical diagnosis of MF (13 pts) or SS (4 pts) confirmed by histopathology, immunohistochemistry and molecular biology. Median time from diagnosis to transplant was 48 months (range 13 to 252). GVHD prophylaxis included: Cy-A and MMF in RIC-HSCT; standard Cy-A and MTX in MAC regimen. Source of stem cells was PB for related donors, BM or UCB for unrelated donors. Following RIC-HSCT, a full donor chimerism was achieved in 33% of pts at day +30 and in 80% at 6 months. A complete CR was obtained in 11 pts, 2 of whom subsequently died (1 for grade IV hepatic GVHD and 1 for sepsis). Among other pts, 2 died before engraftment, 1 from septic complications and 1 from disease progression. Six patients experienced grade I-III aGvHD in the skin while chronic GvHD (extensive in 2 cases) occurred in 6 patients. After a median follow-up of 32 months (range 2-84), nine patients are alive, 8 of whom in CR. Both patients who underwent allogeneic MAC-HSCT are alive in VGPR after a follow up of 9 and 10 months, respectively; after immunosuppression withdrawal, 1 pt experienced evident regression of residual tumor lesions. We conclude that immunomediated GVL contributes significantly to the effectiveness of allogeneic HSCT in pts with refractory MF/SS. The antileukemia effect of donor lymphocyte infusions (DLI) in patients with leukemia relapse after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is generally accepted now. Better outcome was observed in patients with CR at time of DLI, but the most effective schedule of DLI is still not well defined. The goal of our study was to investigate the effect of different DLI schedules: 1) at time of relapse, 2) after CR achievement, 3) during myelosupression after reinduction chemotherapy. Methods.: 13 adult patients (9 -AML, 4 -ALL) with acute leukemia relapses (hematological -12, cytogenetic-1) after HLA-matched allo-HSCT received treatment including DLI. Due to repeated relapses DLI were applied twice to 3 patients. Thus we analyzed the results of 16 DLI procedures. Donor lymphocytes were infused once a week, 4 times, total lymphocytes dose varied from 3,5*108 to 8,2*108 cell/kg. DLI were applied in 6 pts at relapse without preceding chemotherapy, in 4 pts -after CR achievement, and in 6 ptson day 5-7 after completing reinduction chemotherapy (in cytopenia). IL-2 (2-9 MUE) was used in 11 cases. Chimerism was detected and monitored by PCR analysis (VNTR and STR), and by FISH-analysis for centromers of X and Y chromosomes. Results: Mixed chimerism was detected in all pts before DLI. When DLI were applied to pts in relapse without preceding chemotherapy only 2 (33%) AML-pts attained second CR: in 1 pt disease free survival was 26 month followed by 2nd relapse, and in the other pt with cytogenetic relapsecomplete molecular remission and complete donor chimerism was registered at 2nd month after DLI+ IL-2 and is preserved now for 6 months. None of other 4 pts from this group (2-AML, 2-ALL) responded to DLI. When DLI were applied after CR achievement, CR sustained for 2, 3, 5 and 34 month, but all pts died: 3 from relapse and 1 -from B-cell lymphoma originated from "host" hematopoiesis. Among 6 pts treated with DLI at cytopenic phase after reinduction chemotherapy CR with complete donor chimerism was achieved in 5 (83%) pts (4-AML,1-ALL): 3 of them are alive for 6, 10 and 56 months after DLI; 2 patients died at 4 and 5 months after DLI due to GVHD-related infections. Only 1 ALL-pt did not respond to this DLI schedule. Conclusion: Our results suggest, that a new approach to infuse donor lymphocytes in aplasia after reinduction chemotherapy is effective in relapsed after allo-HSCT acute leukemia pts and needs further investigation. Donor lymphocyte infusion as treatment for molecular and morphological relapse after allogeneic SCT: 15-year single-centre experience D. Sairafi*, M. Remberger, O. Ringdén, J. Mattsson Karolinska Institutet (Stockholm, SE) Infusion of donor derived CD3+ T-cells have been used in more than two decades as a way to augment the graft versus leukaemia effect after allogeneic stem cell transplantation (aSCT). In the present study we performed a retrospective analysis of 118 patients with haematological malignancies who had received donor lymphocyte infusion (DLI) due to morphological relapse (n=44), molecular relapse, i.e. detectable levels of BCR/ABL in PCR analysis and/or mixed chimerism, (n=40), or cytogenetic relapse of CML (n=12). Twenty two patients were treated due to other reasons, which included extramedullary relapse, rejection, graft failure or EBV-lymphoma. The main objective was to look for difference in outcome when DLI was given at different stages of relapse. Also, we sought to assess the safety of the treatment, mainly by evaluating the incidence of acute graft versus host disease (GVHD). The relapse-free survival was significantly higher for those who had received DLI as a treatment for molecular relapse. The treatment was not as effective if the disease was allowed to progress to full haematological relapse. In the group with molecular relapse 1 and 3 year survival was 85 % and 68% respectively, whereas the group with morphological relapse had a survival rate of 51% and 36% respectively (p<0.001). In this material DLI administrated as a bulk dose regimen (BDR) was not associated with a higher incidence of acute GVHD grade III-IV compared to escalating dose regimens (EDR); 9% and 8% respectively. We could however see that the patients who developed GVHD grade III-IV after BDR had received a significantly higher cell dosage. Median cell dosage leading to acute GVHD grade III-IV was 180*10 8 /kg body weight (range 1,3 *10 8 to 360*10 8 ) in this group, compared to 2,0*10 8 (range 0,51*10 8 to 2,4*10 8 ) for grade II, and 1,2*10 8 (range 0,4*10 6 to 6,4*10 8 ) for grade I. In contrast there was no association between severity of acute GVHD and cell dosage in cases with EDR. In conclusion, a positive impact on outcome is seen in cases where DLI has been administrated as treatment for molecular relapse after aSCT. This positive effect seems to be of even greater importance in patients with acute leukaemias, which makes frequent analysis of chimerism status early after aSCT crucial in these cases. The incidence of acute GVHD grade III-IV seems to be on a fairly acceptable level, just below 10%. /dose x 5 days, melphalan 140 mg/m²/dose for one dose and rabbit antithymocyte globulin (ATG) 5mg/kg for 2 doses pre-SCT and 2 doses post-SCT. CSA was given for GVHD prophylaxis at standard doses. The median CD34 dose was 7.8 x 10 6 /kg (range, 4-11 x 10 6 /kg). Results: the regimen was well tolerated. nine patients (75%) have sustained engraftment and adequate immune reconstitution with a median follow up of 19 months (range, 11-30 months). Chimerism (assessed by short tondom repeats) ranged between 9-100% (median 47%) for myeloid cells and 19-100% (median 64%) for lymphoid cells. The median times to an absolute neutrophil count (ANC) of ≥ 0.5 x 10 9 /L and platelet count of ≥ 20 x 10 9 /L were 12 days and 28 days respectively. One patient died with sepsis and 2 pts rejected their grafts. Conclusion: non-myeloablative HSCT for MHC II deficiency may offer adequate immune reconstitution with a reduction in the short and long-term toxicity associated with conventional conditioning. Long term follow up is needed to assess long term graft stability. (1), M. Uzunel (2) (1)Center for allogeneic stem cell transplantation (Stockholm, SE); (2) Clinical Immunology (Stockholm, SE) Background: In this retrospective study we evaluated the chimerism status and outcome in 58 patients (64 transplants) with non-malignant disorders treated with allogeneic stem cell transplantation (ASCT) between 1997 and 2007. Patients and Methods: The patient material included 19 different diseases. The largest patient groups were Fanconi anemia (9) and Hemophagocytic lymphohistiocytosis (11). The donors were related in 20 cases and unrelated in 44 cases. Reduced intensity conditioning (RIC) was given in half of the transplants. Donor chimerism (DC) was defined as <5% and mixed chimerism (MC) between 5 and 99% recipient cells. Two consecutive samples showing >30% recipient cells was also defined as high chimerism (high MC). Patients with high MC and the management of these patients was analysed in more detail. Results: The overall survival in 58 patients was 87 % with a median follow up of 4,3 years (range 0.4-10.6). 23 transplants (36%) were DC and 41 transplants (64%) showed some degree of MC of which 26 were high MC. The incidence of MC was 78% and 50% after RIC and myeloablative conditioning, respectively (p=0.04). Acute GVHD II-III was more common in patients with DC (39%) than in patients with MC (8%) (p=0.002). In multivariate analysis, DC was the only significant risk factor for developing severe acute GVHD (OR 6.1, 95% CI 1.7-22.6, p=007). All 9 rejections occurred in patients with high MC. Donor lymphocyte infusions (DLI) were given in 17 cases. The level of MC was decreased in 7 cases, unchanged in 4 cased, increased in one case and 5 patients rejected their grafts. A second ASCT was performed in 6 cases with rejections (4 are in complete remission (CR) with DC, 1 is in CR with high MC and 1 patient also rejected the second graft). Out of the 58 patients, 49 are in CR, 2 in partial remission, 1 patient has an ongoing rejection and 6 patients have died. Conclusion: We conclude that patients with non-malignant disorders that develop MC post ASCT has less risk of GVHD but high MC, however, increases the risk of rejection. Despite the high incidence of MC, the overall survival is promising with high remission rates. P. Svenberg* (1), J. Mattsson (1), O. Ringdén A patient with congenital disorder of glycosylation type ia and bone marrow failure: transplantation with favourable outcome K.-W. Sykora, M. Sauer, U. Baumann, A. Das, A. Broll, K. Welte Hannover Medical University (Hannover, DE) Background: congenital disorder of glycosylation type ia (CDG-Ia) (McKusick 212065) is the most frequent form of CDG, with more than 500 patients reported worldwide. It is caused by a deficiency of phosphomannomutase (PMM2), a cytosolic enzyme that converts mannose 6-P to mannose 1-P, leading to truncation of mannose-containing carbohydrate chains on proteins (Marquardt, Eur J Pediat 2003) . Clinically, it is characterized by hypotonia, psychomotor retardation, peripheral neuropathy with inability to walk, internal strabismus, inverted nipples, feeding problems and failure to thrive. Thrombopenia is a common feature, but trilineage hypoplasia is definitively not. Patient: During the first 3 years of life, the now 8 year old boy developed all the clinical features of his disease listed above. In addition, a slowly progressive pancytopenia became apparent at the age of 3 years which finally resulted in persistent transfusion dependence for thrombocytes and erythrocytes 3 years later. G-CSF was necessary to maintain the ANC above 500/ul. The course was complicated by multiple -including gastrointestinal and pulmonary-bleeding episodes, and multiple episodes of bacterial septicaemia. Bone marrow aspirates were initially uncharacteristic, but at last showed the morphological picture of refractory cytopenia. No chromosome abnormalities were present. The patient was conditioned with Fludarabine (3 x 30 mg/m²), Thiotepa (3 x 5 mg/kg) and ATG Fresenius (3 x 20 mg/kg) and received a BM graft from 10-10 allele identical unrelated donor, GVHD-prophylaxis was CSA and MTX. Results: Regeneration was delayed, and GM-CSF was given from day +18. Granulocyte engraftment (500/ul on day 28) was accompanied by a febrile engraftment syndrome with pulmonary involvement, requiring high-dose steroids. Maximum GVHD was grade III skin, responding to a prolonged course of CSA and prednisone. At 8 months followup the patient is fully engrafted, with 100% donor chimerism and good immune reconstitution. Discussion: To our knowledge, this is the first patient transplanted with CDG. It is possible that an unusual PMM2 gene mutation in our patient caused not only thrombopenia but also pancytopenia making CGD a model for bone marrow failure. CDG alters many potentially immunogenic proteins in the recipient suggesting the possibility of unsual immunopathology after BMT. We did not observe this in our patient, illustrating the feasibility of BMT in CGD. Whether other features of CGD are improved by BMT will be seen in further follow-up. We describe a 6-year old patient with X-linked anhidrotic ectodermal dyplasia, osteopetrosis and combined immunodeficiency due to a mutation of the NEMO (NFkB essential modulator) gene. From early infancy, he suffered from severe opportunistic infections (CMV, PCP) and osteopetrosis without lymphedema. Despite being under PCPprophylaxis and regular IVIG, he developed a Staphylococcus aureus osteomyelitis and a Streptococcus pneumoniae pneumonia. Since April 2005, he suffered from an intestinal Mycobacterium avium intracellulare (MAI) infection. He was treated with 4x-tuberculostatic therapy and gamma-Interferon for more than 1 year. At 6 years of age (Feb 2006), a combined cord blood and bone marrow HSCT from his 1-year old HLA-identical sister was performed. The conditioning consisted of 14 mg/kg Busilvex, 160 mg/qm Fludarabine and 0.5 mg/kg Campath IH. The initial course was uneventful and engraftment was observed 3 weeks after HSCT with full hematopoietic chimerism. Four months later, he developed recurrent fever, hepatosplenomegaly and increased CRP despite satisfactory CD4 T lymphocyte numbers. An FDG-PET-CT scan revealed multiple active foci in spleen, lungs and lymph nodes. Biopsies of bone marrow and liver showed disseminated epitheloid cell granulomata. After restarting tuberculostatic therapy, all symptoms disappeared 2 months later. Six months after HSCT, he showed signs of Varicella-Zoster (VZV) meningoencephalitis (positive blood and CSF VZV-PCR) without cutaneous signs despite being on oral Acyclovir prophylaxis. Therapy with i.v. Acyclovir during 2 months led to complete clinical resolution. Ten months after transplant, he developed pneumococcal meningitis despite regular IVIG, which recovered after antibiotics. Twenty-two months after transplant, he developed a Hemophilus influencae type B (HIB) pneumonia and bacteriemia despite twice conjugated HIB vaccination, which also recovered. Conclusion: This is the second successful HSCT in a NEMOpatient worldwide and the first HSCT of a NEMO-patient with disseminated mycobacterial disease at transplant. While the cellular immune reaction against mycobacterial residues was expected, the clinical picture of VZV-meningoencephalitis and recurrent invasive infections with encapsulated bacteria despite IVIG and completed T-cell reconstitution was remarkable. In X-rays, osteopetrotic changes of the bones have remained mainly unchanged. He is currently under Amoxycillin prophylaxis. The outcome of haploidentical stem cell transplantation in severe combined immunodeficiencies -a single-centre experience M. Tanyildiz (1) Introduction: Hematopoietic stem cell transplantation (HSCT) is the definitive therapy for a variety of rare primary cellular immunodeficiency (PID) syndrome. As a very few of affected children have a HLA-identical family donor, alternative therapeutic options are either a HSCT from a matched unrelated donor or an haploidentical HSCT. In this study, we aimed at evaluating the outcome of haploidentical HSCT in 8 cases of severe combined immunodeficiency (SCID) in a single center. were transplanted at Ankara University School of Medicine, Department of Pediatric Immunology and Allergy and BMT unit. Three patients received 2 SCT infusions.while the remaining received only one. Three patients with SCID received a conditioning regimen consisting of Busulfan/Cyclofosfamid. The others ( n= 5) none. Stem cells were collected after mobilization with G-CSF at a dose of 10 mcgr/kg/day, sc for five consecutive days. All collections were done using COBE spectra cell seperator. Immunomagnetic bead depletion device (CliMACS) was used for indirect T-cell depletion. Results: Five out of eight cases (62%) were boys and the rest were girls. Mean age at diagnosis was 5.5 months (range:1.5 months-6months). The mean age of the patients at the time of HSCT was 4.6 months (range: 2 months-7.5months). Four out of eigth patiens had CMV antigemia, 5/8 had pnomonia, 1/8 had varicella before HSCT. Five patients developed significant acute graft-versus-host disease (GVHD) in one of those cases likenoid type chronic GVHD also occurred. One patient had veno-occlusive disease. The remaining two patient one unfortunately died due to pneumonia and the other died due to graft failure and sepsis, respectively. Conclusion: Haploidentical Stem cell transplantation is a reasonably successful therapeutic option for children with SCID lacking HLA identical donor. T.H. Terwey* (1), J.-S. Kühl (1), P. Hemmati (1), P. le Coutre (1), S. Neuburger (1), W. Köhler (2), B. Dörken (1), R. Arnold (1) (1)Charité, Campus Virchow-Klinikum (Berlin, DE);(2 X-linked adrenoleukodystrophy (ALD) is a rare inherited neurodegenerative disease caused by loss-of-function mutations of the ABCD gene encoding a peroxisomal ATPase resulting in a beta-oxidation defect with accumulation of very long chain fatty acids (VLCFA). There are two phenotypic variants of the disease: childhood cerebral ALD (CCALD), an acute inflammatory myelinopathy affecting boys <10 years, leading to severe disability, dementia, and vegetative state or death within a few years and adrenomyeloneuropathy (AMN), a non-inflammatory axonopathy of the adult. While hematopoietic stem cell transplantation (HSCT) is accepted as an effective long-term treatment for early CCALD (Peters et al., Blood 104: 861-888), it is not indicated for AMN due to its relative benign nature. However, up to 20-25% of all male patients develop an adult cerebral disease either alone or in combination with AMN, which has a poor prognosis similar to CCALD. Following a 12-month course of progressing polyneuropathy and ataxia, a 46-year old male patient was diagnosed with AMN in July 2005 based on the clinical picture, magnetic resonance imaging, elevated VLCFA levels, and detection of a ABCD gene mutation. Rapidly progressive deterioration of the neuropsychological performance status caused a reevaluation in December 2005, demonstrating an acute inflammatory cerebral disease. Therefore, the patient underwent HSCT from a HLA-identical sibling in January 2006. Myeloablative conditioning consisting of busulfan (4 x 4 mg/kg), cyclophosphamide (2 x 60 mg/kg), and ATG (4 x 10 mg/kg) was followed by transplantation of unmanipulated bone marrow containing 4,8 x 10 6 /kg CD34+ cells and 3 x 10 7 /kg S177 CD3+ T-cells. Additional immunosuppression consisted of cyclosporin A and methyl-prednisolone. The transplantation procedure was well tolerated and, except for fever of unknown origin and mucositis WHO-grade III in the acute phase, no clinically relevant adverse events occurred. Immunosuppression was discontinued 6 months after HSCT and to date, 23 months after HSCT, the patient remains free of graft-versus-host disease, while a mixed chimaerism of 5-33% host cells in the blood persists. Importantly, there are no signs of progressive cerebral inflammation and neurological performance is stable (Table 1) . This indicates that HSCT may represent a feasible and effective, yet experimental treatment option in thoroughly evaluated X-ALD patients with adult cerebral disease. Optimal thalasemia-free survival in patients Pesaro-class I and II coming from developing countries after haematopoietic stem cell transplantation from HLA identical sibling Background: In the developed world, the survival and quality of life of patients with beta thalassemia (B-thal) has dramatically improved with optimization of blood transfusion schedule and iron chelation. By contrast, in countries with limited resources most children with B-thal die before the age of 20 years because of the unavailabilility of safe blood products, expensive iron chelating drugs and because of inadequate management of co-morbidities. For these patients allogeneic haematopoietic stem cell transplantation (HSCT) offers a cure with low morbidity and mortality. Patients and methods: Between June 2005 and November 2007, 19 consecutive patients affected by Pesaro class I (1) and class II (18) B-thal underwent HSCT from an HLA identical sibling in our center. 11 males and 8 females with a median age of 8 years (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) were referred to our center from different developing countries: Lebanon(9), Iraq (3),Palestine(4),Syria (3). Iron chelation was regular (6), irregular(10)or absent (3). Median ferritin level was 1927 ucg/l (956-5698). Median pre-transfusion hemoglobin was 8g/dl (range 5.5-10). 15 patients >4 years old were conditioned with iv Busulphan (dosage according to weight, adjusted from the 5th dose to a target AUC of 4800 mcg/Lt*h) and Cyclophosphamide (200mg/kg); 4 patients <4 years old added Thiotepa (10mg/kg). All patients received cyclosporine, methylprednisolone and short course methotrexate as graft versus host disease (GvHD) prophylaxis. The source of stem cells was unmanipulated bone marrow from an HLA identical sibling; median CD34+ cells were 9x10 6 /kg (2.6-21.7), CD3+ cells 73x10 6 /kg (49.7-117). Results: All patients engrafted, with median ANC take on d+20 (12-25) and median PLT take on d+16 (10-43). Post transplant toxicity was characterized by: hypertension (7), CMV reactivation requiring preemptive treatment(9/19), FUO in aplasia (5), hemorrhagic cystitis (1), reversible neurotoxicity (2) . No VOD or renal failure were observed. The incidence of acute GvHD was 4/19 grade I -II, 1/19 grade III-IV. Chronic GvHD was observed in 2/19 patients. All patients are alive and thalassemia free. Median post HSCT follow up is 356 days (76-748). In conclusion, despite inadequate medical treatment before HSCT, patients affected by class I-II B-thal coming from developing countries have similar clinical outcome to patients from developed countries undergoing HSCT in terms of thalassemia free survival and transplant related mortality. Intensified immunosuppression with ATG reduces graft rejection in class III beta thalasemia patients undergoing BMT from HLA-identical siblings after myeloablative conditioning (1) ( Allogeneic BMT represents the only chance of cure for betathalassemia (B-thal pts). Despite full myeloablation, rejection remains a problem. We describe our series of poor risk B-thal pts undergoing BMT affected by a high incidence of rejection and report improved outcome with intensified immunosuppression. From June 2005 to August 2007, 18 consecutive class III B-thal pts underwent allo-BMT at our centre. Median age was 10(5-15). Origin was Kurdistan (12), Iraq(4), Egypt (2) . Medical condition on arrival in Italy was poor as evidenced by mean pre-transfusion Hb7, HCV Ab (12), median ferritin 4810(302-9157), chelation irregular(12) or absent (6), anti HLA Ab(18). All pts received unmanipulated bone marrow from the HLA identical sibling. Conditioning for first 9 patients (group A) was iv Bu/Cy 160 mg/kg. Preconditioning with HU, aza, flu 100 mg/m² and hypertransfusion according to Sodani et al (Blood 2004) . Intravenous Bu was adjusted from the 5th dose to a target AUC of 4800 mcg/Lt*h. GVHD prophylaxis was cyclosporine-A, MPD and 3 doses MTX. Eight pts engrafted (PMN d19, platelets d19), 1 had primary graft failure followed by autologous reconstitution. The %donor chimerism on bone marrow, peripheral blood and CD3+ at 60 days was: 5pts>95%, 3pts mixed chimerism(MC)<75%, 1pt<5%; at 120 days: 4pts>95%, 4pts MC<75 % and 1pt <5%. All the pts with MC<75% subsequently rejected the graft. None of the patients died. Following this high incidence of rejection, immunosuppression was intensified with flu 150 mg/m², ATG Thymoglobuline 10 mg/kg tot (-7 to -4) and Igs 1,6 g/kg to modulate potential Abs mediating rejection(group B). Eight patients engrafted (PMN d19, platelets d15), one patient died of multiorgan failure following primary graft failure. In group B the %donor chimerism on day 60 was: 8pts>95% (confirmed at 120d) , 1<5%. The reduced incidence of rejection in group B was statistically significant (5/9group A, 1/9group B; p=0.06). Two pts in each group developed grade II acute GVHD and none chronic GVHD. Among other factors analyzed for graft rejection(CD34<6x10 6 /kg, non-splenectomy, refractoriness to platelets and CD3<50x10 6 /kg), only the number of infused CD3 was statistically significant(p=0.04). In conclusion, we report that poorly transfused and chelated class III B-thal pts from developing countries have a high incidence of graft rejection despite myeloablative conditioning. Intensified S178 immunosoppression with ATG results in increased thalassemia free survival. A. Ghavamzadeh*, N. Valizadeh, A. Karimi, G. Bahoush, M. Bashtar, A. Shamshiri, M. Jalili, Z. Shahryari, A. Mosavi, S. Basirpanah, S. Khalilvand, B. Chahardouli, F. Khatami, K. Alimoghaddam University of Tehran (Tehran, IR) Background: Mesenchymal stem cells (MSCs) secrete hematopoietic cytokines, and support hematopoietic stem cells in vitro and have immunosuppressive properties. We aimed to show that co-transplantation of MSCs and HSCs from HLA-identical sibling donors after conditioning regimen is safe and could facilitate engraftment and decrease graft versus host disease (GVHD) and could regenerate damaged organs if exist. Patients and Methods: we coadministered culture-expanded MSCs with HLA-identical sibling-matched HSCs in ßthalassemia patients. Between November 2006 and February 2007, 10 ß-thalassemia patients were enrolled. Patients received Cyclophosphamide-based or Fludarabine -based conditioning regimens and short course methotrexate and cyclosporine as GVHD prophylaxis. On day 0, patients were given MSCs intravenously (1.0-2.24_ 106/kg) 4 hours before infusion of either bone marrow or peripheral blood stem cells. Outcomes of transplantation compared between these patients with 50 matched -historical controls group which were transplanted with HSCs in recent years. Matching criteria were included: recipients' gender, source of stem cell (bone marrow or peripheral blood), thalassemia group (intermediate or major), class of ß-thalassemia major (I, II, III). Results: Chills and fever was only notable toxicity in MSC group. Median time to achieve WBC engraftment ≥ 0.5_109/L was 12.5 days (range 10-20 ) for MSC group and 12 days (range 6-52 ) in matched-historical control group (p-value=0.67). Median time to achieve platelet engraftment ≥ 20_109/L was 18 days (range 12-30 ) for MSC group and 22 days (range 10-81) for comparison group (p-value=0.02). Incidence of acute GVHD was 80% and 76% in MSC and historical-matched group respectively (p-value=1). 3-month overall survival rate was 90% and 91.7% in MSC and matched control group respectively (p-value=0.29). 3month -disease free survival rate was 80% and 87.8% in MSC group and matched historical control group respectively (p-value=0.27). Conclusion: In this study we demonstrated that coinfusion of HLA-identical sibling MSCs with HSCs is seems to be safe. we can´t find statistical significant difference in acute GVHD incidence, severity ,OS,DFS ,Median time to WBC recovery between MSC group and comparison group.most probably explantation is small number of patients in study group.median time to plt recovery was shorter in MSC group (p-value=0.02). The CXCR4 antagonist AMD3100 stimulates the proliferation of myeloma cells H.Y. Kim, S.W. Kim*, H.J. Lee, H.J. Yun, S. Kim, D.Y. Jo Chungnam University Hospital (Daejeon, KR) AMD3100, a small bicyclam molecule, inhibits the binding of stromal cell-derived factor-1(SDF-1) to CXCR4 and induces peripheral mobilization of hematopoetic stem cells cells and progenitor cells. Now, AMD3100 is about to be used clinically, especially for the peripheral mobilization of HSCs in patients with lymphoma and multiple myeloma. However, it has been demonstrated that AMD3100 activates G-protein coupled with CXCR4 and acts as a partial CXCR4 agonist and that SDF-1 is involved in the progression of multiple myeloma. Therefore, it is necessary to address the question of whether AMD3100 functions as a partial agonist for CXCR4 in myeloma cells, before it is released for wide clinical application. In this study, we explored whether AMD3100 affects the proliferation and survival of myeloma cells. As demonstrated previously, AMD3100 and pertussis toxin (PTX) markedly inhibited the SDF-1-mediated chemotaxis of myeloma cells, including three cell lines (RPMI8226, U266, and ARH77 cells) and CD138+ primary human myeloma cells. SDF-1 alone did not stimulate the proliferation of these myeloma cells, nor did it rescue the cells from apoptosis induced by serum deprivation. By contrast, AMD3100 stimulated the proliferation of all three myeloma cell lines (~ 2-fold increases), which was not inhibited by pretreating the cells with PTX (up to 200 ng/mL). This phenomenon was also observed with CD138+ primary human myeloma cells. In addition, AMD3100 enhanced the proliferation of U266 and ARH77 cells induced by interleukin-6 (IL-6) and partially reversed the AG490-mediated growth inhibition of RPMI8226 cells. The signal blocking agents wortmannin and PD98056 did not affect the AMD3100induced proliferation of RPMI8226 cells. AMD3100 partially inhibited the apoptosis induced by serum deprivation, but not that induced by dexamethasone in RPMI8226 cells. The antiapoptotic effect of AMD3100 was further enhanced in the presence of IL-6. AMD3100 partially inhibited the phosphorylation of Stat3 and ERK1/2 induced by SDF-1 in U266 cells. However, AMD3100 on its own induced the phosphorylation of Stat3, Akt, and ERK1/2 in U266 cells. The phosphorylation was also not inhibited by pretreating the cells with PTX. These results indicate that AMD3100 can stimulate the proliferation of myeloma cells, most likely through signaling via other than CXCR4. Despite recent development of new drugs, Multiple Myeloma (MM) is still an incurable disease with a median survival of 5 years. The use of conventional allogeneic hematopoietic stem cell transplantation (SCT) is limited by a high transplantationrelated mortality (TRM). However, allografting with nonmyeloablative conditioning remains controversial. We retrospectively studied a series of 20 patients with relapsing MM treated by nonmyeloablative allograft. Between April 2003 and August 2006, 20 patients with relapsing MM were treated by combining a high dose therapy (melphalan 200 mg per meter square with peripheral blood SCT) followed by a nonmyeloablative allograft. Allograft conditioning consisted of 2 grays total body irradiation, and graft versus host disease (GvHD) prophylaxis consisted of mycophenolate mofetil and cyclosporine. The response rate (RR), toxicity, event-free survival and overall survival were evaluated. Among the 20 patients(eight male; 4 IgG, 4 IgA and 4 light chain MM), 18 were in 1st relapse and 2 were in 2nd relapse. Donors were HLA-identical siblings in 12 (60%) patients. A high beta2 microglobuline (>3 mg/L) was found in 5/16 patients. Median age at allograft was 50 years (range, 29-59 years). At the time of allograft, 4 patients were in complete remission (CR), 9 had a very good partial response (VGPR), 5 had a partial response (PR) and 2 were in progression. Median follow-up after allograft was 26 months (range,1.8-55.1 months). Post allograft responses were CR in 7 patients, VGPR in 8, PR in 3 and progressive disease (PD) in 2. Three patients(15%) died during the year following allograft(2 from PD, 1 from severe GvHD). Acute GvHD occurred in 16 patients (14 grade I/II, 2 grade III/IV) and 10 developed chronic GvHD. Eight patients (40%) relapsed with a median of 9.5 months. Among those, 6 are alive with a median survival of 32.7 months (3 of them are in CR or VGPR under thalidomide or lenalidomide containing regimens). So far, 9 patients (45%) are still alive without relapse with a median follow-up of 31.2 months (6 in CR and 3 in VGPR). These results suggest that nonmyeloablative allograft in patients with MM in first relapse provides a high and durable response rate with a low TRM. The RR in post allograft relapses indicates a synergistic effect between graft-versusmyeloma and new drugs. A comparative matched analysis with non allografted patients is ongoing. These promising results must be further evaluated in clinical trials. Background: POEMS syndrome is an exceedingly rare disorder characterized by a clonal plasma cell proliferation producing a small monoclonal protein usually lambda type restricted. This syndrome is a multisystemic disease, its major clinical feature being a progressive peripheral neuropathy. Single osteosclerotic lesions should be treated with radiation therapy only. However, in cases with widespread bone lesions, the treatment is not well established. In short series of patients, high-dose therapy followed by autologous haematopoietic stem cell rescue (ASCT) has shown to be an effective therapy although respiratory complications are of concern. Patients and methods: Between December 1999 and December 2007, 12 patients (9 F/ 3 M; median age 53, range: 36 -66 years) with POEMS syndrome were treated with melphalan-200 (11 patients) or melphalan-140 (1 patient) followed by ASCT at 4 institutions in Spain. All patients had peripheral neuropathy and a lambda type M-protein (IgA: 9, IgG:3). Eight patients had sclerotic bone lesions. Other systemic features were: organomegaly (9 patients), endocrinopathy (10), skin lesions (12), extravascular volume overload (8), papilledema (3), polyglobulia (2) and thrombocytosis (7). Two patients had pulmonary hypertension and another patient Castleman´s disease. Five patients were previously untreated. The median number of prior therapies was 1 (range, 0-3). Median time from diagnosis to ASCT was 4 months (range, 2-95). Results: No transplant-related-mortality (TRM) or cases of respiratory failure were observed. Of 8 patients evaluable for response, 4 achieved hematologic CR (IF-), 2 near-CR (EP-, IF+) and in 2 the M-protein remained unchanged. All had a significant improvement in neurologic symptoms and other clinical and laboratory features. Three patients restricted to wheelchair are now independent of walking aids. The 2 patients with pulmonary hypertension had significant improvement of the pulmonary arterial pressure and are asymptomatic. Clinical improvement was observed at 4-6 months from the date of transplant. After a median follow-up of 25 months, there are no disease relapses. An update on the 12 patients will be presented at the meeting. Conclusions; In this series, ASCT proved to be a highly effective therapy for patients with widespread POEMS syndrome with no TRM or respiratory complications. PCR negativity predicts a longer PFS after stem cell transplantation in multiple myeloma and is more sensitive Background: Quality if CR can further be assessed by monitoring MRD. A sensitive way to measure MRD in multiple myeloma (MM) is with real-time quantitative IgH-PCR with allele-specific oligonucleotides (ASO-qPCR). Preliminary observations have shown that PCR negativity after high-dose therapy may result in prolonged progression-free survival (PFS). In this study we evaluated the outcome of auto/allografted patients according to their ASO-qPCR and immunoelecrophoretic (IEP) status. Patients and methods: During years 1996-2006 there were 37 patients who after initial debulking therapy received high-dose melphalan (200 mg/m2) and single (n=20) or double (n=10) autografting, or allografting with myeloablative (n=3) or reduced-intensity conditioning (n=4), and who were in CR or VGPR (IEP positive) after transplantation and had a patientspecific ASO-qPCR available for MRD assessment. ASO-qPCR together with serum/urine IEP was performed usually 3-6 months after transplantation, with follow-up measurements in 27 patients. PCR quantification of MRD was based on allele specific primer with consensus TaqMan probe or for some patients on allele specific primers with SYBR Green detection. MRD status is ranked "negative" if at least one ASO-qPCR assessment was found to be negative with the cut-off level for PCR negativity of 0.01 %. Kaplan-Meyer curves for the PCR negative and positive groups and also for the IEP negative and positive groups were produced. Results: The median follow-up time for the living patients is 48 mo. Seven patients have died: 3 in the PCR negative and 4 in the PCR positive group. Median PFS for the PCR negative (N=21) and positive (N=16) groups were 70 and 19 months (P=0.003) while median OS have not been reached in the groups (P=0.101). There were 3 patients who were IEP negative but PCR positive, and none who were IEP positive but PCR negative. Among the patients with negative IEP (N= 21) ASO-qPCR turned out to be positive in 3 (14 %). Median PFS for the IEP negative vs positive groups were 65 and 19 months (P=0.092) while median OS have not been reached (P=0.714). Conclusion: Post-transplant negativity of the patient-specific ASO-qPCR predicts prolonged PFS in MM. The same tendency was found for IEP negativity. ASO-qPCR is a new and promising tool to assess the long-term prognosis after stem cell transplantation in patients with MM, and is more sensitive than IEP. Second autologous stem cell transplantation for multiple myeloma in first relapse A. Chaidos (1) , C. Giles (2) , M. Klammer (2) , I. Gabriel (2) , J. Apperley (2) , E. Kanfer (2) , E. Olavarria (2) , M. Bua (2) , D. Samson (2) , A. Rahemtulla (2) (1)Imperial College London (London, UK); (2) Hammersmith Hospital (London, UK) Background: Induction chemotherapy to maximum response followed by autologous stem cell transplantation (ASCT) is standard practice for eligible, newly diagnosed patients with multiple myeloma (MM). ASCT is not curative and virtually all patients relapse. A second ASCT is usually offered after re-induction chemotherapy, but evidence on the outcome with this approach is limited. Aim: We retrospectively analysed the results of 2nd ASCT after re-induction chemotherapy in 1st relapse. Methods: From July 1994 to May 2007, 283 patients with MM received ASCT using melphalan (255 patients) or melphalan/TBI (28 patients). Thirty-four (12%) achieved complete response (CR) and 200 patients (70.7%) partial response (PR). The transplant related mortality (TRM) of the 1st ASCT was 1.06%. Patients treated with elective tandem autologous or a subsequent allogeneic SCT, and patients who died in remission or received palliation only after 1st relapse were excluded. Relapse occurred in 151 patients. Reinduction and a 2nd ASCT with melphalan 100-200mg/m 2 conditioning was offered in 45 patients. Other salvage treatments were given in 106 patients, who served as control group. Results: Twenty-eight patients (62%) responded to 2nd ASCT. Three patients (6.7%) achieved CR and 25 (55.5%) PR. Eight patients (17.7%) had minimal or no response. Four patients had primary graft failure (8.9%). Five patients died of TRM (11.1%) 11 to 70 days post ASCT. The median progressionfree survival (PFS) after the 2nd ASCT was 17.7 months. The patients with a PFS of <18 months after the 1st ASCT had significantly shorter PFS after the 2nd ASCT (median 6 months) compared to those with a long 1st PFS >18 months (median 18 months, p=0.001). In multivariate analysis, PFS after the 1st ASCT was the most significant variable to predict PFS after the 2nd ASCT. However, the duration of PFS after the 1st ASCT does have significant impact in OS. The median OS after progression from 1st ASCT for patients treated with 2nd ASCT was 53 months and for the control group 13 months (p<0.006). Similarly, OS from diagnosis was 84 and 55 months respectively (p=0.006) Conclusions: A 2nd ASCT after re-induction is effective salvage approach for MM patients in 1st relapse. Most patients will achieve response and despite higher TRM and graft failure rates, a 2nd ASCT may result in longer OS when compared to other salvage treatments. The duration of PFS after the 1st ASCT may predict the PFS after the 2nd ASCT . This single centre analysis assessed the outcome of 186 consecutive multiple myeloma (MM) patients aged > 60 y. treated with autologous stem cell transplantation (auto-SCT), with the specific aim to compare the outcome of the 82 "elderly" (age>65 y.) patients subgroup, with their 104 "younger" mates aged between 60 and 65 years treated in the same period. Except for age, both groups were comparable (P=NS) as for demographic features, disease characteristics (S&D disease stage, monoclonal component), and prognostic factors (b2-microglobulin). "Induction" VAD chemotherapy, was comparable between the "elderly" (87%) and "younger" (94%) group. Prior to auto-SCT, the calculated hematopoietic cell transplantation-specific comorbidity index (adapted from the Charlson Comorbidity Index) was also comparable (77% of the "younger" patients with a 0-1 index, vs. 74% in the "elderly" group; P=NS). The PBSC mobilization procedures were also comparable between both groups. 97% of the patients received high-dose melphalan conditioning. 33% of the "younger" and 28% of the "older" group (P=NS) completed a second auto-SCT. ANC and platelets recovery were comparable between both groups (P=NS), and the median length of hospitalization for the first auto-SCT was not different between the two groups: 19 (range, 2-32) days in the "younger" group vs. 17 (range, 2-39) in the "older" group; P=NS). Infectious and other serious auto-SCT-related complications were also comparable between groups (P=NS). With a median follow-up of 41 (range, 5-227) months after auto-SCT, 120 patients are still alive. Disease progression (n=40; 61%) was the main cause of death, with this being comparable between both groups. Auto-SCT-related mortality was 3.8% (n=4/104) in "younger" and 3.7% (n=3/82) among "older" subjects. Comparing "younger"/"older" subjects, progression-free survival was significantly higher in the younger group (P<10e-4). However, disease response rate after the first auto-SCT was comparable (CR, VGPR and PR rates: 88% vs. 90%, P=NS), and overall survival (OS) was also comparable (57% vs. 54% at 5 years, P=NS; 32% vs. 24% at 10 years, P=NS). In a Cox multivariate analysis model, none of the relevant characteristics was shown to be a critical prognostic features for OS. Of note, age was insignificant for both OS and transplant-related mortality. We conclude that there is no biological justification for an age-discriminant policy for MM therapy. "Physiologic" aging is likely more important than "chronologic" aging. Introduction: The Human Leukocyte Antigen (HLA) system seems to play a role in multiple myeloma (MM) control and could influence disease outcome. This feature has been poorly studied and there are only few data favouring a higher incidence of some HLA specifities such as B18 and B5 in myeloma patients. Aim: To study the relationship between HLA-DRB1 specifities and overall survival (OS) and progression-free survival (PFS) in MM patients who have received an autologous stem cell transplantation (auto-SCT). Patients: A total of 128 patients with a diagnosis of symptomatic MM who were homogeneously treated according to the GEM-2000 protocol (Spanish Myeloma Group/PETHEMA protocol) were analysed. Additionally, 1818 healthy donor individuals from the Castilla y Leon registry for hematopoietic stem cell-transplantation were included as control population. Both populations involved Caucasian individuals. Methods: After genomic DNA extraction, HLA-DRB1 typing at low-resolution level (two digits) was carried out using the PCR-rSSO methodology according to the standards of the European Federation of Immunogenetics. Allele frequencies were estimated by direct counting. Comparisons of allele and genotype frequencies between MM patients and controls were performed with the two-sided Fisher's exact test using GraphPad Prism 4.0 Software. The strength of associations was estimated by the odds ratio (OR), and their 95% confidence intervals (CI) were calculated by Cornfield methods (values of p < 0.05 were considered statistically significant). P-value was corrected (Pc) for the number of valid comparisons made (Bonferroni correction). Log-rank analysis was applied to compare differences between survival curves. Results: DRB1*07 genotypic frequencies were significantly higher in the symptomatic patients as compared to the healthy controls (38.3% vs. 27.6%, p=0.0111, Pc>0.05, OR: 1.63, 95% CI: 1.12-2.36). In addition, symptomatic MM patients showed a lower incidence in DRB1*01 genotypic frequencies as compared to the control population (14.1% vs. 21.7%, p=0.0437, Pc>0.05, OR: 1.69, 95% CI: 1.01-2.82). Patients displaying DRB1*07 allele showed shorter OS (41% vs. 55%, p=0.031) and PFS (15% vs. 29%, p=0.009) as compared to patients with other alleles. Conclusions: The present data suggest that HLA-DRB1*07 genotype is associated with shorter PFS and OS in symptomatic MM patients after auto-SCT. Reduced-intensity conditioning regimen for allografting following autografting has strong anti-myeloma activity and delays disease progression compared to tandem autografting Allografting is a possible curative approach for patients with multiple myeloma (MM). Unfortunately this procedure is only an option for younger patients with HLA-identical siblings. Recently, nonmyeloablative regimens (RICT) based on fludarabine demonstrate stable engraftment of allogenic cells. High-dose therapy/AutoSCT followed shortly thereafter by RICT might improve outcomes in MM as compared to AutoSCT or conventional AlloSCT used alone. In this paper we evaluated toxicity, engraftment, chimerism, graft versus host disease (GVHD) and response to RICT after AutoSCT in 20 patients with advanced stage MM. We compared two retrospective cohort of patients who underwent either tandem AutoSCT (HDT consisted of Melphalan 200 mg/m²) or AutoSCT followed closely by related RICT (patients with HLAmatched siblings). The two groups were matched for pretransplant therapy, disease status at transplant, time from diagnosis to transplant. GVHD prophylaxis for RICT patients consisted of CyA/MTX. The major results are summarized in the Table. In the AutoSCT/RICT group the complete remission rate was higher and the risk of disease progression after transplant was reduced. All patients who reached CR responded after full chimerism and GVHD developed. This finding confirms the existence of a graft-versus-myeloma effect. Since the first clinical signs of response in remitters patients were noted between 70 and 120 days and maximum response between 160 and 200 days after RICT (after DLI in one patient), these responses should be considered immunological responses. These data suggest that an autoSCT followed by RICT significantly reduces the incidence of disease progression. (1) (67%)pts.Failure of G-banding and FISH was universally due to low plasma cell content of the marrow aspirate below 5% of nucleated cells. 59 pts (61%) did not show any chromosomal abnormalities at transplant;37 pts(49%)had chromosomal aberrations, involving the IgH gene locus on chromosome 14q32 and/or deletions of the long arm of chromosome 13(del 13q)in 33 cases. Deletion of the short arm of Chromosome 17(del17p)was only seen in one pt.Our study revealed presence(n=9)of t(4;14)as negative prognostic factor for the outcome of ASCT with median progression free survival (PFS) of 10 months and overall survival(OS)of 34 months compared to pts without chromosomal abnormality who had a median PFS of 19 months and OS of 58 months(p=0.009).Translocation between the IgH gene locus and CCND1, t(11;14), was found in 11 pts with additional del13q in 3 cases.Their median PFS of 19 months was identical to that of pts without chromosomal abnormalities and median OS similar at 62 months. Del 13q alone was found in a small number of pts (n=3) and did not confer an inferior PFS or OS, but the sample size is small. Pts with complex karyotypic abnormalities (n=5) and those with translocations involving Chr 14q32 and unidentifiable partners (n=4) had a poor prognosis with median PFS of 10 months (p=0.001) and 6 months (p=0.01) and OS of 37 months and 23 months (p=0.03) respectively from time of transplant. Hyperdiploidy was not associated with significant survival advantage. 65 pts were ISS stage I at time of transplant with a median TTP of 20 months and median OS of 67 months, while 30 pts scored ISS II/III with inferior TTP of 12 months (p=0.0003) and OS of 37 months (p=0.05). ISS criteria stratify survival in poor risk cytogenetic pts with t(4;14) to a median of 34 months in ISS I vs 14 months in pts with ISS II/III. Conclusion: Chromosomal studies by FISH and G-banding at autologous transplant can identify pts with unfavourable abnormalities, such as t(4:14), who should be considered for clinical trials of novel therapies. High marrow plasma cell infiltration at diagnosis is associated with shorter time to progression following autologous stem cell transplantation for multiple myeloma M. Klammer*, C. Giles, R. Szydlo, I. Gabriel, K. Naresh, D. Samson, J. Apperley, A. Rahemtulla Imperial College NHS Trust (London, UK) Background: The International Scoring System (ISS) has allowed survival stratification of Myeloma patients at diagnosis. We have previously reported that levels of serum albumin and beta-2 microglobulin are not available for the majority of patients referred to our transplant service. In this study we investigate if the plasma cell infiltration at diagnosis, as a marker of disease burden, influences the outcome of subsequent autologous transplantation. 240 patients (152 male, 88 female) with a median age of 59 years (27ys-73ys), referred to Hammersmith Hospital,London, for high dose therapy (HDT) and autologous stem cell transplantation (ASCT) between 1994 and 2007 were treated in a uniform fashion with myeloablative doses of Melphalan. The median OS post HDT/ASCT of all patients was 53 months with a median time to disease progression (TTP) of 20 months. Beta-2 microglobulin at diagnosis was only obtainable from 88 referrals (37%), serum albumin was not stated at all, whereas the plasmacell infiltration in a trephine biopsy at diagnosis was known for 194 patients (81%). In univariate analysis (194 pts) a greater than 60% plasmacell infiltration of the trephine biopsy at presentation was associated with a significantly shorter median TTP (15 months) following HDT/ASCT compared to patients with a lesser degree of marrow plasmocytosis,whose median TTP was 20 months (p=0.003). In multivariate analysis, taking into account age, sex, myeloma type, beta-2 microglobulin levels at diagnosis, ISS score and disease status at tranplant, a high marrow trephine infiltration at diagnosis (>60%) was associated with an increased risk of disease progression (RR 3.2, p=0.001), whereas a high beta-2 microglubulin level (>3.5 mg/l) at diagnosis was associated with a shorter survival (RR 4.3, p=0.01), but did not significantly predict for TTP (RR 1.859, p=0.09). We conclude that the degree of marrow infiltration at diagnosis may play a role in the outcome of later autologous stem cell transplantation, but needs to be validated as a predictive factor in a larger patient cohort. Combinations of cytogenetics and international scoring system can predict poor prognosis in multiple myeloma after high-dose chemotherapy and autologous stem cell transplantation Y. Inamoto (1), S. Kurahashi (2) (1), Y. Kodera (4), I. Sugiura (2) (1)Nagoya University (Nagoya, JP); (2) High-dose chemotherapy followed by autologous stem cell transplantation (ASCT) is performed as a standard treatment for newly diagnosed multiple myeloma (MM). Combinations of recently proposed prognostic factors such as cytogenetics and international scoring system (ISS) may be useful to stratify prognosis after ASCT. We evaluated transplant outcome among 60 consecutive patients with newly diagnosed MM who underwent single ASCT between 2000 and 2007 in 4 institutes. Median age was 57 years old. Median term from diagnosis to transplantation was 6 months. The ISS stages at diagnosis were 1 (n=22), 2 (n=19), 3 (n=17) and unknown (n=2). Cytogenetical analyses of bone barrow at diagnosis detected metaphase abnormalities (including 13 chromosome) in 9 of 51 patients, and interphase abnormalities in 6 of 35 patients (deletion of 17p13 in 2, t(4;14) in 3, and t(14;16) in 1 patient). Patients were defined as high-risk if they had any of these abnormalities or the ISS stage of 3. Twenty-five patients (42%) were classified as high-risk. All patients were conditioned with high-dose melphalan. With a median followup of 2.6 years, overall survival rates 3 years after ASCT in non-high-risk group and high-risk group were 83% and 48%, respectively (P=0.0007; Figure 1 ). Progression-free survival rates were 28% and 11%, respectively (P=0.067). Although 18 patients achieved CR and 10 achieved VGPR after ASCT, 21 of them relapsed. Of these 21 patients, survival rates 1 year after relapse in each group were 100% and 31%, respectively (P=0.036; Figure 2 ). Seventy percent of the patients in highrisk group died within 1 year after relapse, although they received salvage chemotherapy containing thalidomide. In summary, combinations of cytogenetics and ISS can predict prognosis of MM after ASCT. A major problem was a poor prognosis among relapsed patients in high-risk group. Further modalities including new drugs, allogeneic consolidation and combinations of these are subjects for future investigation. Background: Lenalidomide is an immunmodulatory drug, orally administered once daily, which is effective in relapsed multiple myeloma (MM). Here, we evaluated the efficacy and S183 toxicity of lenalidomide in 16 MM patients with relapsed disease after allogeneic stem cell transplantation (allo-SCT). Methods: Median age was 57 (range,37-68) years. The series included 5 females and 11 males. Prior to allo-SCT, all patients were heavily pretreated with multiple lines of chemotherapy, including high dose therapy with autologous stem cell support. Also, prior to lenalidomide treatment, other salvage therapies included donor lymphocyte infusions (DLI) in 93% of cases, thalidomide in 63%, and bortezomib in 88%. NCI-criteria were used to define toxicity. Lenalidomide was given 25 mg orally once daily on day 1-21 every 28 days. No prophylactic anticoagulation was used. Results: The overall median number of completed cycles was 5 (range, 1-10). Very good partial response was achieved in 13%, partial remission in 53%, and stable disease in 27% of the patients. During the follow up period, disease progression was observed in 50% of cases, and death in one patient. The median progression-free survival was 8 months, while the median overall survival was not yet reached. Hematotoxicity was the primarily and major encountered side effect (leukopenia: 6% grade 4, 19% grade 3, 19% grade 2, 38% grade 1; thrombopenia: 19% grade 3, 19% grade 2, 31% grade 1). This myelotoxicity led to dose reduction (usually 10 mg) in 44% of the patients. Infectious complications were observed in 25%. Non-hematological toxicity was also seen in 75% of cases (56% grade 1, 19% grade 2), consisting of cramps in the leg, constipation, symptomatic fatigue, nausea and progressing polyneuropathy (n=1). Thrombembolic complications (cerebral infarction) were observed only in one patient, who was receiving concomitant corticosteroid treatment for acute graft-vs.-host disease (GvHD), but neurological symptoms resolved completely. GvHD of the skin under lenalidomide treatment was seen in 25% of cases (one case grade 2, three cases grade 1), with one case occurring shortly after an additional DLI. Conclusions: Lenalidomide is effective in relapsed patients with MM after allo-SCT. Major toxicity is myelotoxicity, which required dose reduction in a majority of patients. With this background, a dose-finding study to determine the optimal lenalidomide dose as maintenance therapy after allo-SCT has already started. Autologous stem cell transplantation (ASCT) is considered the standard of care for younger patients (pts.) with multiple myeloma (MM). Current evidence indicates that depth of response is an important prognostic factor for long-term survival in MM. Several phase II studies with Bortezomib in combination as induction treatment yelded promising CR rates. We have tested the Bortezomib (B) (1.3mg/mq d 1, 4, 8, 11) , liposomal Doxorubicin (PLD) (30mg/mq d 4) and Dexamethasone (D) (40 mg/mq d1-4, d 9-12)combination for 4 consecutive 21 d cycles before ASCT. Methods: In March 2006 we initiated a pilot study with B-PLD-D as induction treatment prior to ASCT in 22 pts., 14 male, 8 female,median age 58 years, range 51-64 with newly diagnosed MM. Results: Response data are available for all pts. The overall response was 88%including a 31% CR/nCR rate. 20 pts. proceeded to ASCT resulting in an improved response in 10, increasing the ORR to 95% and CR/nCR rate to 54%. The second question was to evaluate the impact of B-PLD-D therapy on stem cell mobilization and collection, our data indicated that adeguate numbers of stem cells can be collected for both single and tandem transplantations, furthermore neutrophil and platelet engraftment were successfull and as expected after ASCT. Conclusion: The results from this ongoins study suggest that the combination of B-PLD-D has promising activity in pts. with MM. Longer follow-up is needed to demonstrate that this better reduction induces longer PFS and OS in MM. VTD (Velcade, thalidomide, dexamethasone) represents an active induction regimen for patients with multiple myeloma J. Drach*, V. Odelga, J. Ackermann, V. Sagaster, H. Kaufmann, N. Worel, W. Rabitsch, P. Kalhs, C. Zielinski Medical University of Vienna (Vienna, AT) VTD was reported to be an active salvage regimen in patients (pts) with relapsed/refractory multiple myeloma (MM). Since initial results with VTD as frontline therapy were also promising, we used VTD as induction treatment prior to autologous stem cell transplantation in MM pts, particularly in patients with poor prognostic features and/or high tumor burden. Velcade was administered at 1.3 mg/m2 on days 1, 4, 8, and 11; thalidomide was given at a daily dose of 100 mg; dexamethasone (20 mg orally) was given on days 1, 2, 4, 5, 8, 9, 11, and 12 . Four to 6 cycles were scheduled every 3 weeks. Concomitant treatment included prophylaxis against deep vein thrombosis (low dose aspirin, 100mg daily) and herpes zoster reactivation. We here report our experience with 11 pts (7 males, 4 females; median age 50 years, range, 36 -67 years) with newly diagnosed MM (two pts had failed 3 cycles of VAD and thal/dex, respectively) treated with VTD. Pts had a high tumor mass(e.g., pt 1: BJ-kappa MM with 95% plasma cell infiltration, serum free-kappa light-chains 4000 mg/l; pt 2: IgG-kappa MM with serum IgG > 9000 mg/dl, bulky plasmacytomas in the iliac bones; pt 3: IgA-lambda MM with 70% plasma cells, IgA 4500 mg/dl, plasmacytoma in the os sacrum > 10 cm in diameter) and/or high-risk cytogenetic abnormalities (del13q in 4 pts; amplification of 1q21 (CKS1B) in 4 pts; t(4;14) in 1 pt). VTD resulted in a rapid response already after 1 cycle in 9 of the 11 pts (82%), 6 pts (54.5%) achieved a CR/near CR, and 3 pts had a PR (including one VGPR). Only one pt experienced disease progression. Rapid tumor mass reduction was not associated with signs of tumor lysis, and one pt had a significant improvement in renal function (serum creatinine from 2.4 to 1.3 mg/dl). 5 pts have already completed G-CSF primed peripheral stem cell collection (2.4 -7.9 CD34+ cells/kg body weight), and to date, high-dose melphalan (MEL200) plus autologous transplantation has been performed in 4 of them (all achieved a CR after completion of high-dose therapy). Toxicity of VTD was mild (Grade 1-2 gastrointestinal side effects and peripheral neuropathy; one episode of deep vein thrombosis). Conclusion: Induction treatment with VTD results in rapid tumor mass reduction and a high remission rate (82%) in MM pts even with poor prognostic features. These preliminary results suggest that VTD is an effective and safe induction treatment prior to autologous transplantation in MM. Influence of comorbidity on outcome of HD-versus non-HD-therapy in multiple myeloma J.R. Novotny*, R. Canitz, H. Nückel, U. Dührsen University Hospital Essen (Essen, DE) Objectives: By definition the results of prospective randomized studies are restricted to those subsets of patients defined by the inclusion and exclusion criteria. They usually do not fully represent all patients suffering from a certain disease. Especially the influence of comorbidity on treatment outcome might not be elucidated. Therefore we chose to retrospectively analyse the role of comorbidities in myeloma patients irrespective of participation in studies or not. Methods: All patients treated in our department for multiple myeloma between 1999 and 2005 were analysed retrospectively. Comorbidity was assessed using both the Charlson Comorbidity Index (CCI) and the Haematopoietic Cell Transplantation-Specific Index (HCT-CI) which is probably more appropriate for modern haematologic treatments, especially HD-therapy with autologous PBSCT. Patients were divided into low-risk and high-risk groups (HCT-CI 0 -1 vs. ≥ 2). Results: Comorbidity was significantly lower in the HD-therapy group (mean 0.9 vs. 2.0). HD-therapy resulted in significantly better survival in the total population (75 vs. 43 months p = 0.009). As expected, survival was significantly lower in highrisk patients in the non-HD-group (32 vs. 54 months p = 0.017). However, survival was not different between low and high risk HCT-CI patients treated with HD-therapy (67 vs. 70 months p = 0.935). Furthermore, survival was not different between HD-and non-HD-therapy in the low risk group (67 vs. 54 months p = 0.548). In the high risk group HD-therapy resulted in significantly better survival as compared to non-HD-therapy (70 vs. 32 months p = 0.010). Similar but less clear-cut results were obtained when the CCI was employed. Conclusion: Our retrospective study showed surprising yet unexplained results. Possibly, different points leading to 2 or more points of the HCT-CI may have completely different effects on treatment outcome. Allthough the HCT-CI was actually developed for allogeneic transplantation its application in this setting does not explain the superiority of HD-therapy in the high risk but not in the low risk group. Further analysis may add valuable information for future treatment allocation in multiple myeloma which cannot be obtained in usual randomized studies. Thalidomide is an active single agent in advanced relapsed or refractory multiple myeloma (MM). The combination of low dose thalidomide with bendamustine and prednisolone might maintain or increase efficacy of the drug while avoiding dose limiting toxicity (DLT). Patients and Methods: The treatment consists of a fixed dose of bendamustine (60mg/qm) day 1, 8, and 15 with prednisolone (100 mg) day 1, 8, 15, and 22 . In addition, thalidomide is given in three escalating doses, starting with 50 mg to a maximum of 200 mg daily. At each dose level 4 to 6 patients with relapsed or refractory MM after high dose therapy with stem cell support were enrolled. Cycles were repeated every 28 days for a minimum of 2 and a maximum of 10 cycles either until a maximal response was achieved, or until a DLT or a disease progression were observed. Between March 2004 and May 2006, 14 patients were enrolled: 4 in the first dose level with 50 mg thalidomide; 4 in the second dose level with 100 mg and 6 in the third dose level with 200 mg. All patients had received a minimum of 2 prior treatment regimens. Three patients had been refractory to the last treatment. Median age was 61 years (range: 40 -69). All patients completed 2 cycles of bendamustine, thalidomide and prednisolone (BPT) -treatment and are therefore evaluable. Response was assessed using EBMT criteria modified to include near complete remission (nCR) and very good partial remission (VGPR). Results: Thirteen of 14 patients responded after at least 2 cycles of chemotherapy with 2 CR, 2 VGPR, 8 PR and 1 MR. One patient was refractory. With a median follow up of 22 months, median progression-free survival was 11 months, and median overall survival was 16 months. The most common side effects were constipation (10 patients WHO grade 1, 2 patients WHO grade 2), polyneuropathy (8 patients WHO grade 1, 2 patients WHO grade 2) and somnolence (2 patients WHO grade 1). None of the 14 patients developed doselimiting hematoxicity as defined by an ANC < 1,0 Gpt/l for > 7 days or an ANC < 0,5 Gpt/l for > 3 days or platelet count < 25 Gpt/l. Transient neutropenia was reported in 9 patients (WHO grade 3 and 4) and thrombocytopenia was observed in one patient (WHO grade 3). Conclusion: BPT with a dose between 50 and 200 mg thalidomide daily is well tolerated in patients with relapsed or refractory MM after autologous stem cell transplantation. Free light chain analysis: Is it useful to monitor patients undergoing haematological stem cell therapy for multiple myeloma? An analysis of "real life" data E. Willenbacher, G. Gastl, D. Nachbaur, W. Willenbacher Innsbruck University Hospital (Innsbruck, AT) Rationale: FLCA is relatively new, highly specific and sensitive laboratory parameter to be used for monitoring MM pts., even in the setting of "non-secretory" disease. Recently it became the basis of a broadened new definition of remission depth (called stringent CR [sCR]) characterized by a normal serum free light chain ratio. We retrospectively analyzed FLCA results in our transplanted MM pts. with regard to early diagnosis of relapse, assessment of treatment efficiency and its impact on clinical decision making, compared to standard M-Protein measurements. Patient characteristics: Between MAR 2005 (when FLCA became available at our institution) and NOV2007, 42 pts. were either alive after or undergoing haematological stem cell therapy (HSCT) for MM. Thissubgroup was identified out of 69 pts. which were treated by either autologous (92 procedures) or allogeneic (19 procedures) HSCT since 1989. For 38 (14 females, 24 males) of this pts. at least two consecutive FLCA were documentedand they were thus considered to be evaluable. Paraprotein subtypes were IgG (15 pts.), IgA (11 pts.), Bence Jones (8 pts.), IgD (2 pts.), nonsecretory (1 pt.) and unknown (1pt.). Light chain subtypes were kappa (22 pts.), lambda (15 pts.) and unknown (1 pt.) . Treatments applied were autologous HSCT (33 pts.), allogeneic HSCT (2 pts.) or both (3 pts.) . 7 pts. were pretreated with "novel agents" (thalidomide, bortezomib), while in 31 classical chemotherapeutic induction therapies were applied. Median time from first diagnosis to last HSCT was 11 months (range, 4-108 months). HSCT was integrated in MM first line (22 pts.), 2nd line (14 pts.), or 3rd line treatments (2pts.). Results: A total of 210 FLCA were performed with a median number of 8 analyses per pt. (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) . With the help of FLCA kappa could be identified as a tumor marker in one pt. with a nonsecretory MM by M-protein measurement. sCR was achieved in 13 pts. on 25 distinct occasions, but was not durable in any of these cases. 14 pts. showed loss of treatment responses documented by FLCA. In pts. clinically judged to be stable frequently a waxing and waning pattern within the reference range was observed. In this subgroup, besides the FLC-ratio absolute FLC values as well as continuous FLC monitoring is needed to draw definitive conclusions on disease dynamics. The clinical relevance of FLCA data for individual MM pts management will discussed and compared to conventional MM biomarkers. An algorithm for the use of FLCA will be presented. Bortezomib treatment under routine care conditions following allogeneic stem cell transplantation in multiple myeloma N. Kröger* (1), C. Gann (2) , W. Blau (3), M. Bornhäuser (4), J. Casper (5) , C. Driessen (6) , U. Hegenbart (7) Objective: To investigate efficacy and safety of bortezomib under routine care conditions in patients (pts) with multiple myeloma after allogeneic stem cell transplantation (SCT). Methods and results: 26 adult pts in 9 German centres (8 female, 18 male; median age 53 years) with at least two prior therapies and disease progression during the last therapy following allogeneic SCT were selected for this analysis after the attending physician had decided to treat them with bortezomib. Bortezomib was given according to label at dose of 1.3 mg/m² i.v. on days 1, 4, 8, and 11 followed by a 10 days rest period for a maximum of 8 three week cycles. All diagnostic and treatment decisions were at the physician's discretion. Most patients (85%) had advanced disease (Salmon and Durie stage 3) and received a median of 5 previous therapies. Median time from allogeneic stem cell transplantation to bortezomib therapy was 28 months (range 3-70). Median bortezomib dose was 1.3 mg/m² (range 0.5-1.3); a median of 4 cycles (range 1-8) were administered. Concomitant medication with dexamethasone was reported in 9/26 pts; in total 34/116 cycles of bortezomib were given in combination with dexamethasone. Overall response rate was 73%. Complete remission was achieved in 15%, partial remission in 42%, and minor remission in 15%. 4 pts had stable disease, 3 pts (12%) showed disease progression. Response to bortezomib occurred rapidly; 77% achieved their best response after the first 2 cycles and 92% after 4 cycles. 4 pts completed the proposed 8 treatment cycles; 22 patients discontinued prematurely, mainly owing to adverse events (7 pts), disease progression (5 pts), and patient's request (5 pts). A total of 299 mostly mild to moderate adverse events (AEs) occurred; 68% were considered related to bortezomib. Grade 3 or 4 toxicities were reported in 18% and most common for thrombocytopenia (33 AEs), leukopenia (4), and anaemia (3). Of the 29 serious adverse events, only 5 considered drugrelated. 1 death, unrelated to bortezomib, occured within 30 days after therapy. Conclusion: Bortezomib was effective under routine care conditions in this small group of heavily pre-treated multiple myeloma patients following allogeneic SCT. Promising results in multiple myeloma patients treated with induction chemotherapy consisting of bortezomib, doxorubicin and dexamethasone followed by autologous stem cell transplantation W. Zinke-Cerwenka*, T. Stojakovic, A. Zebisch, H. Sill, W. Linkesch Medical University (Graz, AT) Background: Standard treatment for patients with multiple myeloma consists of an induction chemotherapy followed by high dose melphalane and autologous stem cell transplantation (ASCT). Incorporation of new agents like the proteasome inhibitor bortezomib (PS-341) in combination with doxorubicin and dexamethasone (PAD) increases response rates in induction therapy significantly and may also contribute to improved results after autologous transplantation. Methods: After 4 cycles of PAD stem cell mobilisation was performed successfully in 23 patients with newly diagnosed multiple myeloma. All patients but one qualified for ASCT by achieving at least SD. Until now, 21/23 patients underwent ASCT after conditioning with melphalane 200 mg/m². The pretransplantation remission status (EBMT criteria) of these patients was: 2 CR, 7 nCR, 10 PR and 2 SD. Evaluation of treatment response was performed 3 months after ASCT. Maintenance therapy after transplantation with thalidomide was determined for all patients who achieved only SD or PR before ASCT. Results: Twenty of 21 patients could be evaluated, one patient did not reach the first evaluation point yet. According to EMBT criteria, 2 patients achieved CR, 12 nCR and 3 PR (total response rate: 85%). One patient died of pneumonia and two patients showed progressive disease. The median follow-up after ASCT is 8 months (range: 3-23). Conclusion: These preliminary data show that improved response rates after PAD induction may translate into improved CR/nCR rates after single ASCT. We have compared 2 consecutive conditioning protocols for multiple myeloma employed in our department between 1994 to 2005. In protocol 1 we used the combination of 3 agents in a sequential manner including etoposide (200 mg/m²/d on days -6 to -4), thiotepa (60 mg/m²/d on days -5 to -3) and melphalan (60 mg/m²/d on days -4 to -2). Protocol 2 was consisted of melphalan 200mg/m² as a single agent given in a single dose on day -1. Harvest methods, supportive care (including the use of G-CSF) and post transplant follow up and treatment did not differ between the groups. Patients that underwent intentional tandem auto-allo SCT were excluded from analysis. Patients: 30 patient (23 males, 7 females) were treated with protocol 1 and 35 patient (21 males, 14 females, p=0.21) were treated with protocol 2. The median age in protocol 1 group was significantly lower (48.2 years, range 34-63 and 58.6 years, range 41-73 (p<0.0001)) representing the change in attitude to age limits in the field of transplantation. Pretransplant disease status and number of plasma cells at transplantation were not significantly different. Results: there was no transplant related mortality in either protocols. With a 79.2 and 41 months median follow up respectively, 18 patients (60%) from group 1 and 21 patients (60%) from group 2 are alive. However, in patients treated by melphalan alone (protocol 2), the median survival was already reached at 71.1 months (figure 1) while the median survival was not reached in patients treated with protocol 1. Conclusion: we conclude that it seems that melphalan augmented by etoposide and thiotepa in a sequential manner may benefit myeloma patients, although results are limited by the age difference between the groups. A randomized study is underway. Longitudinal analysis and prognostic impact of cancertestis antigen expression in multiple myeloma D. Atanackovic (1) Results: We found that CT antigens were frequently and surprisingly persistently expressed, indicating that downregulation of these immunogenic targets does not represent a common tumor escape mechanism in myeloma. We observed strong correlations of CT antigen expression levels with the clinical course of myeloma patients as indicated by the number of bone marrow-residing plasma cells and peripheral paraprotein levels, suggesting a role for CT antigens as independent tumor markers. Investigating the prognostic value of CT antigen expression in myeloma patients after allogeneic stem cell transplantation, we found that expression of genes such as MAGE-C1 represents an important indicator of early relapse and dramatically reduced survival. Conclusions: These findings strongly support the usefulness of CT antigens as diagnostic and prognostic markers as well as therapeutic targets in myeloma. Patients who had received a novel agent were mobilized at similar times but were transplanted later (27/40 vs 16/47, p= 0.002). Among all analyzed, only response during M , timing of M and use of V/T were found to be influential on PFS and/or OS. Evaluation of factors (B2MG, ISS, age, chemo line, timing of M)on response to Induction, revealed only late M to be associated with less response (p=0.05). Among all patients, amplitude of response to VAD(>90% vs others) and novel agents did not prolong PFS or OS. However, among patients who were mobilized later , achievement of >90% response prolonged PFS at 3 yr (85.2 % vs 55.8 %,p=0.01) at 5 yr (85.2% vs 37%, p=0.001) (Fig.1 ) . >90 % responders of VAD had a longer 5 yr PFS compared to <90% response ( 73.5 vs 37.8%). Comparison of patients who had received at least one novel agent during the preASCT, compared to those not , revealed an OS prolongation (3yr:100% vs 83%; 5yr: 100% vs 57%, p=0.007). (Fig. 2) . Conclusions: In this retrospective analysis, we were able to demonstrate a benefical effect of response to induction, earlier mobilization, and use of novel agents, although given selectively to <90% responders. Palifermin reduces intestinal toxicity and length of hospitalisation in multiple myeloma patients receiving high-dose melphalan G. Milone*, P. Murgano, V. Pinto, S. Coppoletta, K. Battiato, A. Di Marco, A. Strano, E. Marturano, E. Mauro, S. Leotta, M. Poidomani Ospedale Ferrarotto (Catania, IT) Introduction: Palifermin has been studied after TBI containing regimen and after BEAM schedule while benefits of this agent has not studied after High Dose Melphalan. Methods: We have employed Palifermin in 20 patients affected with Multiple Myeloma that received high Dose chemotherapy with Melphalan (200 mg/m2) and we compared clinical results obtained using Palifermin with those of a second group of 31 patients affected with MM and treated previously in our Institution using the same High Dose Schedule and the same anti-infectious prophylaxis. The two groups were not different in age (P=0.49), sex (P=0.2), WBC in P.B (P=0.3), and number of lines of chemotherapy received previously (P=0.4). All patients were transplanted using PBSC as hematopoietic rescue and CD34+ dose was not different in the two groups (P=0.9), all patients received G-CSF during aplasia post chemotherapy. Oral mucositis grade was assessed using DMS score. Results: Time for hematopoietic reconstitution and transfusion needs were not different in the two groups. We have not detected significant difference in maximum mucositis score in the two groups, neither duration of oral mucositis resulted different, however Palifermin was associated with a reduction of duration of diarrhea ( 5 days versus 7 days. P=0.06), with a reduction of use of Total Parental Nutrition (P=0.0001) and with a reduction of duration of TPN (15 days versus 4.3, P=0.0002). Length of hospitalization was also significantly reduced with Palifermin in respect to patients that did not received KGF (11 days versus 16 days, P=0.0006). FUO was diagnosed in only 16 % of Palifermin patients versus 34 % of group NO-Palifermin (P=0.1). In conclusion Palifermin in MM patients undergoing Autologous PBSC transplantation determines a reduction of intestinal toxicity and a reduction of hospitalization length. The relapse rate after a tandem transplant program with high dose melphalan and autologous stem cell transplant (AutoSCT) followed by a dose-reduced conditioning and allogeneic stem cell transplantation (AlloSCT) in patients with multiple myeloma (MM) is still considerable. Due to a well documented graft versus myeloma effect, adoptive immunotherapy with donor lymphocyte infusion (DLI) has become a treatment option in patients with relapse after Auto-AlloSCT. To improve the antimyeloma effect of DLI after Auto-AlloSCT in MM, we investigated the effect of low-dose thalidomide (100 mg) followed by DLI in 9 patients with progressive disease or residual disease after the tandem program. The overall response rate was 66.6 % (6/9 patients), including 55.5 % (5/9) complete remission (CR) + near CR. Major toxicity of thalidomide was weakness grade I/II (4/9) and peripheral neuropathy grade I/II (2/9). Only 1 patient experienced mild grade I acute graft versus host disease (aGvHD) of the skin, while no grades II to IV aGvHD and no chronic GvHD was seen. The 2-year estimated overall and progression-free survival were 100% and 89%, respectively. Adoptive immunotherapy with low-dose thalidomide and DLI induces an antimyeloma effect with very low incidence of graft versus host disease. High dose Melphalan (HDM), 200 mg/m², followed by autologous peripheral blood stem cell transplantation (APBSCT) is traditionally restricted to Multiple Myeloma (MM) patients younger than 60 years. This study has investigated the feasibility and the toxicity of a therapeutic program consisting of a tandem HDM and APBSCT in elderly patients. After induction chemotherapy with VAD, 44 elderly pts with MM, stage II-III, median age 63 years (range 60-77), entered a study consisting of two APBSCT. They were mobilized with cyclophosphamide (3-4 g/m²). All patients underwent at least one APBSCT procedure and 27 cases a double.The median time to recycle HDM was 4 months. A rapid haematological recovery was observed in most pts, with ANC >100 and >500/microL and PLT >30,000/microL values reached at a median of 9 (8-12), 10 (9-13) and 13 (9-16) days since autograft, respectively. The median number of units of pocked red blood cells and platelets per transplant procedure was 0 (0-2) and 1 (0-7). Febrile neutropenia occurred in 68.4 % of patients with a mean duration of 2,7 + 1,4 days. The febrile episodes were classified as follow: FUO 76.9%, microbiologically documented infections (CVC-related 15.3%; bacteraemia 7.8%). Mucositis was observed in 78.9 % of cases (WHO grade 3-4 in 26.3%). No difference was noticed between the 1st and 2nd APBSCT in terms of haematological and extra-haematological toxicity. Two patient died because of transplant-related cause (4.5 %). The feasibility and toxicity of progenitor cell mobilization and HDT in the elderly patients were compared with experiences in 86 MM patients <60 years (median 53 years, range 27-59), who received the same mobilization protocol and of whom 44 patients received a double HDM and 9 a triple. No significant differences were observed between these groups in terms of engraftment. HDM appeared to be somewhat more toxic in the elderly patients: a higher incidence of febrile neutropenia (P = 0.08) and longer in-hospital stay (P = 0. 05) were observed. No differences were found in transplant-related mortality (4.7 % of TRM in the younger group) or severe organ toxicity between these age groups except for oral mucositis grade >2, which tended to be more common in the elderly patients (P = 0.07). Conclusion: We conclude that progenitor cell mobilization and HDM supported by APBSCT is also feasible in selected elderly patients with MM. To decrease red blood cell (RBC) transfusion requirements during a tandem high-dose melphalan (HDM) for multiple myeloma (MM) patients (PTS), we conducted a pilot study to assess the effect of high-dose of recombinant human erythropoietin (rHuEpo) started during chemotherapy before the first HDM and autologous peripheral blood stem-cell transplantation (APBSCT). After induction chemotherapy with VAD, 18 consecutive PTS with MM, stage III, median age 58 years (range 41-64), were mobilized with cyclophosphamide (3-4 g/m²) to collect peripheral blood stem cells (PBSC) and entered a study consisting of two HDM (200 mg/m²) with APBSCT. Enrolled patients received rHuEpo (40 000 U subcutaneously one time/week) as soon as their Hemoglobin (Hb) level fell <11 g/dl during induction chemotherapy. rHuEpo was continued at the same dose during PBSC collection and was reintroduced at the time of discharge after the first transplant up to the admission for the second one. If the Hb level exceeded 13 g/dl at any time, rHuEpo was withheld until the concentration decreased to <11 g/dl, at which time it was restarted. Results were compared to those of 20 tandem HDM and APBSCT performed in 15 consecutive historical MM controls matched for hematological parameters. rHuEpo increased the hemoglobin (Hb) level from 10.0 + 2.5 g/dl at diagnosis to 12.9 + 2 g/dl at the time of the first HDM; no major adverse effects occurred. Compared to historical controls (7/15), RBC transfusion requirements were significantly lower for rHuEpo recipients (2/18) (P=0.00001). After the tandem HDM and APSCT, fewer RBC transfusions were needed: 3.3 and 1 RBC units for controls and rHuEpo recipients, respectively (P=0.006). The administration of high dose of rHuEpo during induction chemotherapy and interval beetween the first and second HDM cycle permit the realitazion of tandem chemotherapic program with a reduction of blood product support. Introduction: Autologous stem cell transplantation (ASCT) has been used as a treatment for multiple myeloma. However, the relapse is still frequent in patients with multiple myeloma even after autologous stem cell transplantation. Thus, more effective approach is required to improve the efficacy of autologous stem cell transplantation in multiple myeloma. Bortezomib, a proteosome inhibitor is an effective agent for relapsed or refractory multiple myeloma. A previous study suggested bortezomib might be safely added to high-dose melphalan as a conditioning regimen. Herein, we reported the preliminary results with bortezomib-melphalan conditioning regimen for autologous stem cell transplantation in patients with multiple myeloma. Methods: The conditioning regimen was as follows: On day -4, bortezomib 1.0mg/m² and melphalan 50mg/m² were intravenously (IV) infused, and bortezomib 1.0mg/m² and melphalan 150mg/m² were IV infused on day-1 followed by stem cell support on day 0. In total, seven patients were treated with this conditioning regimen followed by ASCT. Results: Five newly diagnosed patients received 4 to 6 cycles of VAD combination chemotherapy before ASCT.Their disease status at ASCT was as follows: 1 complete remission (CR), 1 very goof partial remission (VPGR), and 3 partial remissions (PR). Two relapsed patients received two kinds of treatments including VAD, thalidomide/dexamethasone before ASCT, and their disease status at ASCT was PR. In all patients, peripheral blood stem cells were mobilized with G-CSF infusion. Median number of collected CD34+ cells was 4.5 x 10 6 /kg (range, 3.5 -7.8). Six patients showed adequate hematologic recovery after ASCT. Median day for absolute neutrophil count over 500/mm³ was 14 (range, 13 -22). One patient died due to pneumonia on day+11. After ASCT, six patients showed CR. Among them, CR was maintained in three newly diagnosed patient, but the other three patients showed relapse (disease-free duration: 222 days, 79 days, and 124 days, respectively). Conclusion: Conditioning regimen with bortezomib and melphalan may be effective for autologous stem cell transplantation in multiple myeloma, however, its feasibility should be further evaluated with larger study populations. A single nucleotide polymorphism (SNP) in a regulatory element of the lactase gene (LCT) has been made responsible for lactase persistence, respectively lactose tolerance. Since lactose malabsorption due to lactase nonpersistence changes the intestinal microflora, we tested whether this SNP 13910 bp upstream of the LCT start-codon influences survival and acute graft vs.host disease (aGvHD) after allogeneic hematopoetic stem cell transplantation (HSCT). We retrospectively typed DNA from donor/recipient pairs in 111 consecutive patients (median age 42 y (19-73); 51/60 female/male; 69 acute leukemia, 20 chronic myeloid disease, 14 chronic lymphoid disease and 8 others; 41/70 early/advanced stage of disease; 70 myeloablative and 41 reduced intensity conditioned; 103 peripheral blood stem cells) transplanted from 90 related and 21 unrelated donors. A lactase-non-persistent genotype (CC) was found in 20 patients (18%) and 30 donors(27%). Median overall survival was significantly shorter for patients whose donor had a lactase-persistent genotype, ie. TC or TT (11.1 months) than for patients whose donor had a CC genotype (median overall survival not reached after 133 months, logrank p=0.0039). Multivariate analysis identified advanced stage of disease (HR 3.37, 95% CI 1.72-6.57, p=0.0004) and a donor LCT -13910 T allele (HR 3.51, 95% CI 1.57-7.87, p=0.0022) as the only two independent risk factors for death. We did not see any influence of recipient LCT-genotype on overall survival. However, patients with CC genotype benefited dramatically from donors with CC genotype compared to donors with any T-allel (OS: 80% up to 12 years vs. none beyond one year). There were no differences in the incidence or severity of aGvHD neither for donor nor for the recipient LCT-genotype. Further analyses showed a significant difference in transplant related mortality (TRM; logrank p=0,0449) with a 1 year TRM of 39.6% for patients with a TC or TT donor vs. 19.6% with a CC donor. Similar, a siginficant lower relapse rate was observed in patients with a CC donor (33.7% vs. 52.7%) at 5 years (logrank p=0.0415). These results point towards an effect mediated by the transplanted cells. Since the LCT -13910 T/C polymorphism is located within an unusually large region of genetic linkagedisequilibrium extending >1 Mb we cannot exclude that this SNP is not causally related to the observed differences but serves as a surrogate marker. Demonstrating the accuracy of the OptiMatch® prognostic system for donor selection H.P. Eberhard, U. Feldmann, A. Gerlach, W Bochtler, C.R. Mueller* ZKRD (Ulm, DE) Most donors registered worldwide are not typed for all relevant loci at allele level. In order to perform a donor search efficiently with regard to time and money required, it is necessary to select those donors for further testing which will most likely turn out to be identical. The prognostic matching algorithm of OptiMatch® is using all partial information available on each donor in combination with high resolution 3locus-haplotype frequencies of the underlying population to calculate the probability of a donor to be allele identical for each of the loci HLA-A, -B and -DRB1 as well as all three loci combined. In order to validate the predictions, we compared the calculated probabilities with the outcomes of over 8000 confirmatory HLA typings performed for German and International patients between January 2006 and June 2007 on German donors where the result was high resolution for the locus/loci considered but the a priory typing was not. We grouped predictions in intervals of 5% and compared the expected and observed number of matches per interval. In most cases the observed numbers were in a 95%-confidence interval around the expected number which is excellent taking into account the number of tests performed. Linear regressions of the observed and expected "hit rates" show R2values between 0.94 and 0.99 for each of the loci HLA-A,-B,-DRB1 individually and an R2-value of 0.98 for all three loci combined. The predictive power of the OptiMatch® algorithm by far exceeds our own expectations since the deviations from the predictions were in the expected range even without appreciation of the variance of the haplotype frequency estimates. With an adequate base of haplotype frequencies OptiMatch® is an excellent tool for the unrelated donor search. In order to facilitate and expedite donor searches, donor lists should be sorted according to the descending probability of being eventually used for the patient considered. This probability may depend on all information available on the donor at any given point of time, in particular HLA data with missing loci or at various resolutions, age, gender and infectious disease markers. Taking into account the evolution of the HLA nomenclature and the heterogeneity of the resolutions provided by the various methods over the last two decades, the HLA part is the major challenge. ZKRD's new OptiMatch® system uses a sound biostatistical approach based on 3-locus high resolution haplotype frequencies of the underlying donor population to determine the probability of allele matches for HLA-A, -B and DRB1. For special cases, it can be tuned down to matching based on allele frequencies only or merely on combinatorial application of the HLA nomenclature. A variety of non-HLA criteria can be taken into account and the granularity of continuous criteria can be selected at several levels. Many details like number, loci, severity and direction of acceptable mismatches, CMV status, donor age and gender including the precedence of the criteria can be selected and interactively applied through a web based interface according to the specific situation of the patient. The complex choices are facilitated by offering pretailored parameter sets for typical constellations. In particular, OptiMatch® implements the entire set of matching preferences available in the European Marrow Donor Information System (EMDIS) and allows searching for donors at the 6/6 (A-B-DRB1), 8/8 (A-C-B-DRB1 or A-B-DRB1-DQB1) or 10/10 (A-C-B-DRB1-DQB1) level. Due to its extensive parameterisation of algorithmic elements, matching criteria and filters, OptiMatch® can provide specifically tailored match lists for each individual patient according to his or her particular needs. Introduction: Bone marrow (BM) contains a rich supply of adult stem & progenitor cells, and may prove to have long-term advantages over PBSC as a source of hematopoietic stem cells for allogeneic bone marrow transplants. Traditional percutaneous needle aspiration methods for harvesting marrow are crude, tedious and expensive and usually requiring over 100 separate iliac bone small volume aspirates, under general anesthesia. A novel device, the MarrowMiner (MM), was developed for the minimally invasive harvest of BM to enable the rapid, convenient, outpatient harvest of large quantities of BM under local anesthesia for use in BMT and an increasing array of regenerative therapies. The MM device consists of a powered handle, which drives a hollow, flexible aspiration shaft through a single marrow entry site, with an atraumatic tip to facilitate movement & aspiration through cancellous marrow. The device was extensively developed & tested in benchtop, cadaver and in porcine studies which found a significant (10 fold) increase in CFU activity in marrow harvested with the MM compared to standard needle aspirates. This report reports the initial, ongoing clinical experience with MM following recent FDA clearance & CE Mark approval for clinical use. Methods: In 9 patients undergoing regenerative autologous marrow derived cellular therapy, marrow was aspirated via the MarrowMiner through a single entry point in the anterior iliac crest under local anesthesia and into 50 ml syringes containing heparin. MM harvests were followed by multiple, standard 6 hole needle marrow aspirates from the posterior ileac crest. BM viability, cell counts, CD34+, and mesenchymal cell panel & CFU assays were subsequently performed on MM samples, and compared to marrow collected from the same patient by standard needle/trocar. Results: The MM was effective for collecting BM under local anesthesia, with no post-procedure pain, and with comparable to superior TNC and CD34+ yields and viability (7AAD) compared to standard aspirates. Volumes harvested were improved with experience from a single marrow entry pass with 30ml aspirated to over 300ml total collected in single or double MM passes. Conclusions: In this initial human use, the MarrowMiner device successfully and safely harvested BM. The results suggest that this novel device may enable significantly improved marrow harvest in a more rapid, reproducible, convenient and less invasive manner for BMT and cell therapy applications. HLA-B*38;DRB1*13 haplotype is associated with >90 birth weight centile in cord blood donors P. Bergamaschi (1) The large employ of cord blood (CB) for unrelated stem cell transplantation is providing worldwide spreading of CB banks (CBBs). CBBs repositories consist of cryopreserved CB units whose characteristics are determined, recorded into databases and managed for stem cell donor search and procurement. By this way, large amounts of data referring to the immunogenetic profile of infant donors are available for further investigation. Certain HLA alleles/haplotypes are known to be associated with protection and/or susceptibility to diseases. HLA markers are also hypothesized to influence some physiological events. Multiple factors affect normal development of the foetus and infant's HLA genotype might represent one of them. In this setting, birth weight (BW) represents a suitable marker of intrauterine growth. DRB1*13 has been recently associated with high BW. We previously reported the protective effect of HLA-B*38;DRB1*13 haplotype against low BW in Turner patients. Assuming that HLA typed infants derived from a CB Bank represent a healthy population, we review the data referring to 1206 CB donors of the Pavia CBB inventory aiming to investigate the association between some HLA markers and newborn's size. BW was corrected according to sex and gestational age by using centiles, whose distribution is depicted in graphic 1. UCB donations and mothers were typed for HLA-A, B and DRB1 by molecular techniques at time of banking. All UCB and maternal haplotype frequencies were obtained by direct counting and unambiguously assessed. We calculated the percentage of infants positive/negative for B*38 and DRB1*13 alleles and B*38;DRB1*13 haplotype and analyzed their distribution according to centiles. Infants carrying the HLA-B*38;DRB1*13 haplotype were found to accumulate in >90 centile group and the association was statistically significant (2-tailed Fisher test, p=0.009), as shown in graphic 2. In our population the association between high BW and DRB1*13 is not statistically significant. Nevertheless, the association between B*38;DRB1*13 haplotype and the highest centile not only supports the hypothetic role for a factor linked to HLA in normal growth and development but also allows to better identify the subregion on chromosome 6 where the unknown marker could be found. In our opinion, despite utility for donor search, data derived from CBBs may provide information about healthy population otherwise not easily available. Cytomegalovirus (CMV) infection significantly affects morbidity and mortality after cord blood transplants (CBT). Previous CMV infection of either CBT donor or recipient negatively influences the outcome. Identification of CMV-safe CB units and prevention of CMV transmission from CB donors represent the major concerns. CB donations are routinely screened for infectious disease (ID) including CMV by testing the maternal serum at delivery and six months after collection. The second testing allows to restrict the infectivity window so that quarantined units are definitively safe or discarded in case of positive results. Direct detection of ID markers on cord blood before issuing for transplant is also recommended. Nucleic acid testing (NAT) is able to detect the genetic material long before antibodies or viral proteins are present and represents the most advanced tool for blood screening. We previously reported the positive effect on CB validation provided with testing the mothers by NAT for HCV, HBV and HIV. Recently NAT has been validated for detecting CMV not only on samples containing cellular DNA but also on CB plasma suggesting us to extend the use of NAT to direct CMV screening of CB. A sample of plasma was taken from each CB at time of banking and after volume reduction performed by centrifugation. Assays of 9 CB donors plus 3 internal controls were set up. External controls were also tested as interlaboratory quality controls. NAT was carried out by Cobas Amplicor CMV Monitor Test (Roche Diagnostics, Mannheim, Germany). In parallel the maternal serum was tested for the detection of CMV IgG and IgM antibodies by traditional serology technique. We review the data referring to the first 150 testing performed. All CB donations were found CMVnegative by NAT; the results of the corresponding antibodies assay were all coherent, as depicted in the table. No false negative was revealed by NAT. CMV NAT permits direct testing of CB, the actual donor. In adjunct to maternal serology allows to identify CMV-safe CB units, bypasses the second serologic testing and shortens the quarantine. Safety of CB is guaranteed also in case of failed attendance of the mother to the second sampling. CB availability is anticipated avoiding costs of storage and maintenance by early disposal of positive units. In our opinion NAT assays as well as direct testing of CB should be reasonably implemented in CB banking. Managing an ethical challenge: a call for guidelines for the management of related donors of haematopoietic stem cells L. Ritchie, B. Jones* on behalf of the EBMT(UK)Nurses and Allied Professionals Group FACT-JACIE (2007) requires that haematopoietic stem cell transplant (HSCT) centres have written criteria for the donation of haematopoietic stem cells to protect the safety of donors. A standardized approach to donor care is already well established in the unrelated setting. In contrast, there are no guidelines regarding the distinct issues surrounding related donors such as the lack of anonymity, independent assessment and confidentiality. Case studies show how ethics impact on clinical practice in the related setting; for example 1. The HSCT donor who is also the favoured live kidney donor 2. The donor with a psychiatric illness 3. The older donor with co-morbidities 4. The donor with a high-risk lifestyle The dynamics of related donor care are clearly complex and there are differences in how individual transplant centres undertake this. Consistency is particularly pertinent following the suggestion of an association between short course G-CSF and lymphocyte genetic abnormalities and possible malignancy. New recommendations outline that the duration of follow-up should arguably be lifelong; donors should be studied longitudinally with cytogenetic analyses of lymphocytes and consideration should be given to long-term insurance cover of donors. Consensus on how to practically implement these recommendations is needed. It has been suggested that the contents of related donor guidelines could be similar to those issued through the World Marrow Donor Association with appropriate modification. Some suggested inclusions are: 1. A clearly identified independent donor advocate with the appropriate mandate to defer a related donor when necessary 2. A clear distinction between the teams managing recipients and donors 3. Thorough preliminary counselling prior to tissue-typing to allow for early deferral of unsuitable donors 4. Access to a hospital ethics committee and timely access to appropriate specialist advice 5. A full donor protocol agreed by the transplant director, audited and managed within a quality system 6. Specified parameters for donor follow-up with sufficient resources to do it properly The EBMT(UK)NAP group suggests that a standardized approach be established in the related donor setting. The call for guidelines is aimed at ensuring a consistent approach in all transplant centres. By establishing a forum, reaching a consensus and adopting an agreed set of guidelines, best practice in the related donor setting may be better assured. Factors influencing the time scale of matched unrelated donor transplant J. Brennan*, S. Kulkarni, M. Dungarwala, R. Saso, M. Ethell, B. Shaw, F. Davies, C. Dearden, J. Treleaven, H. Woods, G. Morgan, M. Potter Royal Marsden Hospital (Sutton, UK) 162 new patients eligible for allograft were analysed to assess the time frame of transplant. Diagnosis was Ac. leukaemia (n=103), Chr. Leukaemia (n=23), lymphoma (n=24), Myeloma (n=6) and other (n=6). 14 patients (8%) died before allograft. 23 cases were not transplanted (standard risk disease=7, patient refusal=5, poor PS= 4, no donor=1 and on waiting list=6). 43 had matched sibling and 37 received the allograft (86%). 71/81 with matched unrelated donor transplant (MUD) who received transplant (88%; RIC= 47, Full=24) were analyzed to establish the time frame for various stages of HSC collection. Diagnosis was Ac. leukaemia (n=43), Chr. leukaemia (n=12), lymphoma (n=9), myeloma (n=4) and other (n=3). Patient's median age was 46yr. (17-67; M=43, F=28). Confirmatory typing (CT) for patient was available at a median of 12 d (4-36 d). National panel search was requested at a median of 0d (0-532) and report was available on the same day in most (range: 0-9d). For 45 patients with potential donor on the national panel CT was requested at a median of 1 d (0-57). Patients who did not have donors on national panel had international search requested at a median of 0 d (0-175) and donor was identified in 22 patients at a median of 8 d . The median time to receive donor CT was 21 d (0-69) for national and 29 d(6-98) for international panel. The donor work-up was requested at a median of 19d (0-194). The interval between the requested and actual date of transplant was 5d (0-75). 49 donors were male and 22 were female. 14/66 donors failed medical (21%). Hence in 9 patients second or third donor and in 5cord blood unit was requested. The median interval between donor work-up request and transplant was 48 d (16-113) and the interval from requesting patient CT and transplant was 134 d (65-365). This interval was 115 days for patients whose donors passed medical and 146 days for those whose donor failed medical (p=0.082). Interval between the donor medical and transplant date was longer for patients whose donors failed medical (15 d vs. 47d, p<0.001). A total of 6 different registries were used and there was no difference in the time required by any of the panels. Donor panels are very prompt in procesing the requests. In conclusion, although asuitable donor can be identified in 85% patients, induction mortality and patient choice are important determinants in performing allograft. One of the important variable for delay in BMT is related to donor issues and not the constraints of the transplant unit. Background: The paclitaxel formulation, Taxol (Bristol-Myer Squibb), is one of the most effective anticancer agents used today, and has the additional advantage of not damaging the stem cell pool. Paclitaxel is effective not only against solid tumors, but has also been proven active in leukemia, lymphoma, and myeloma. Thus, while paclitaxel has been included in mobilization regimens of patients with solid tumors, the efficacy of this agent for mobilizing peripheral blood progenitor cells in hematologic patients has not been investigated. The aim of this study was to assess the S192 mobilizing ability of paclitaxel (170 mg/m²) and filgrastim (rhG-CSF, 8ug/kg), in the presence or absence of cyclophosphamide (4 g/m²), as the initial or salvage regimen in patients with hematologic malignancies. Patients and Methods: Seventy patients (median age 53 years) diagnosed with different hematologic malignancies (25 non Hodgkin lymphoma, 23 multiple myeloma, 12 myeloblastic acute leukemia, 5 Hodgkin disease, 4 lymphoblastic acute leukemia, and 1 chronic lymphocytic leukemia) received paclitaxel-rhG-CSF (P-G; n=46) or paclitaxel-cyclophosphamide-rhG-CSF (P-C-G; n=24). In 25 (36%) this was the first regimen while it was for salvage in 45 (64%) patients, after a prior, unsuccessful attempt with filgrastim. Results: The use of paclitaxel resulted in 73% of patients achieving the threshold number of CD34 > 2 x 10 6 /Kg (72% of treated with P-G, and 75% in the P-C-G group). These regimens allowed successful mobilization in 75% of patients that failed previous mobilization with rhG-CSF. Patients in the P-G group started leukapheresis earlier (day 9), than those receiving P-C-G (day 14), with a median number of procedures performed of 1 (range 1-5). Patients receiving P-C-G exhibited a trend towards both higher number of peripheral blood CD34+ cells (107 vs. 71/mL; p>0.05), and higher yields of CD34+ cells (5.4 vs. 4.1 x 10 6 /Kg; p>0.05) than those in P-G group. The mobilization therapy was generally well tolerated, with grade III and IV leukopenia or thrombopenia significantly lower in P-G patients (p=0.003, and p=0.02, respectively). Thus, only 10% of patients suffered from febrile neutropenia, and 13% required transfusion of platelet concentrates. Conclusions: Paclitaxel is an effective and safe mobilization agent, in patients with hematological malignancies, and may be considered a valuable alternative in patients who have failed standard rhG-CSF mobilization. Bone marrow is a source of hematopoietic stem cells (HSC) in which a high proportion of CD34+ cells coexpressed the B cell antigen CD19. Although the influence of CD34 cell dose and graft T lymphocyte subsets on the outcome after HSC transplantation have been extensively addressed, the potential role of infused B cell subset have not been yet studied. We retrospectively analyzed B lineage-specific Hematopoietic Progenitor Cells (CD34+ CD19+) and B cells (immature and mature B cells, CD34-CD19+) doses on transplant outcome in 96 patients (56 male, 58%) who received an unmanipulated marrow graft from an HLAidentical sibling donor from January 1997 and February 2000. Cell doses were categorized according to samples tertiles and their associations with outcomes were tested in a Cox proportional hazards model. Median age was 31 years (3-58); 81 patients (84%) had hematologic malignancies. All patients received a myeloablative regimen with total body irradiation for 34 patients (35%). Acute graft versus host disease (GVHD) prophylaxis consisted mainly in cyclosporine associated with methotrexate (n=91, 95%). Median number of bone marrow nucleated cells and CD34+ cells infused were 2.5 108/Kg and 5.2 106/Kg, respectively. Median number of CD34+19+ and 34-19+ infused were 0.6 106/Kg and 8.8 106/Kg, respectively. The incidence of acute GVHD grade II to IV was 51%. The number of infused CD34+CD19+ was inversely correlated to the incidence of acute GVHD. Among patients who received less than 0.32 106 CD34+CD19+/Kg (n=32), 24 (75%) experienced acute GVHD. For patients who received more than 0.32 106 but less than 1.05 106 CD34+CD19+/Kg (n=32), 14 (44%) presented acute GVHD compared to 13 patients (34%) who received more than 1.05 106 CD34+CD19+/Kg (p=0.0035). There were no statistically significant association between B cell subsets and chronic GVHD (36 patients, 3-year cumulative incidence 62%) or survival (estimated 3-year survival rate was 53%). Of note, a higher but not significant rate of chronic GVHD was observed for patients who received less than 0.32 106 CD34+CD19+/Kg (p=0.09). In conclusion, a higher graft B lineage-specific Hematopoietic Progenitor Cells (CD34+ CD19+) is associated with a decrease incidence of acute GVHD. Further studies on the specific role of those cells are needed. Pre-apheresis CD34+ cells (PA-CD34) count effectively predicts the yield of the apheresis (Aph); so that a minimum count of PB-CD34 (5-20x10 3 CD34+cells /mL) is required in many institutions for cell collection. The aim of this study is to know if low PA-CD34 should be or not an exclusion factor. We have studied patients candidates to autologous hematopoietic progenitor cell transplantation, in whom Aph was initiated, independently of their PB-CD34 number, at day: +5 in G-CSF, +10 in cyclophosphamide+G-CSF (Cy-G), and between +15 and +20 in other chemotherapy+G-CSF (Ch-G) mobilization regimens. Four times the blood volume was processed (Fenwall CS-3000). A total of 612 Aph were done to 226 patients (108 males and 118 females). Diagnoses were: 61 (27%) MM, 48 (21%) no-Hodgkin Lymphoma, 43 (19%) Breast cancer, 21 (9%) HD, 16(7%) AML, 8(4%) ALL, 8(4%) CML, 7 (3%) CLL, 5(2%) Plasmocytoma, and 9 (4%) other diagnoses. Cy-G was used in 155(69%) patients, Ch-G in 34(15%), G-CSF in 33(15%), without mobilization in 4 (2%). Patients were grouped according to PA-CD34 count: group A CD34≥10x10³/mL (143 (63%) patients); group B with less than 10 CD34 but≥5 x10³/mL (40 (18%) patients), and group C with CD34 <5 x10³/mL (43 (19%) patients). No difference was found in: diagnoses, sex, age, previous treatments, and mobilization among groups. One mobilization was done to 137 (95.8%) patients in group A, to 39 (97.5%) in group B, and to 23(53.5%) in group C (p>0.01, C vs. A and B); Two mobilizations were needed in 5 (3.5%) patients in group A, in 1 (2.5%) of group B, and in 20 (46.5%) in group C (p>0.001, C vs. A and B); three mobilizations were done to 1 patient in group A. One Aph was enough in 46 (32%) patients in group A, and in 1 (2.5%) in group B; two Aph were required for 68 (47.5%) patients in group A, 6 (15%) in group B, and 1(2.3%) in group C; three Aph were performed in 21 (14.7%) patients in group A, 27 (67.5%) in group B, and in 14 (32.3%) in group C; more than three Aph were done to 8 (5.6%) patients in group A, to 6 (15.0%) in group B, and in 28 (65.1%) in group C; statistical differences (p<0.001) were found between A-B, A-C, and B-C. More than 1.9x10 6 CD34/K were obtained in 142 (99%) patients in group A, in 39 (97.5%) in group B, and in 31 (72%) (p>0.01) group C. Enough CD34 for transplant could be obtained in more than 70% of our patients with PA-CD34 <5x10³/mL, when high volume Aph is done. So, low PA-CD34 should not be an exclusion factor. With this knowledge, we may in a safer way evaluate donors for donation. Patients and methods: 22 allogeneic hematopoietic stem cell donors (19 M, 3 F) were consecutively included. The age was 35 years (median; range 27-52). All were given G-CSF 10 ug/ body weight daily for 4 (-5) days with stem cell collection day 5 (-6). In PB, the median number of CD34-pos cells at collection was 97 x 106/L (range 25-172). All collections were performed by Cobe Spectra, and all except one obtained the number of the requested stem cells. 19 donors were harvested one day, 3 donors required two days. The median number of collected CD34-pos cells x 106/Kg body weight recipient was 9.2 (range 3.7-34.7). From the medical records, information regarding thromboembolic risk factors was collected. Concentrations of coagulation factors were measured day 1 before start of G-CSF, day 5 before start of collection, 1 week and 1 month after collection. The F1+2 method was changed during the studied period, see table. A health questionnaire was performed 4 weeks after donation. Results: The medical records reported donors (n) with previous thrombo-embolic episodes (0), hypertension (2), increased cholesterol/triglycerides (1), smoking (2) . No serious adverse events were observed. The majority of donors had increased values of fibrinogen, F1+2, vWF-antigen and PAI-1 day 5 compared to day 1, see table. PT and APTT were not prolonged in any donor. Conclusion: We have shown an increase in plasma levels of different coagulation factors in PB and signs of increased activation of the coagulation system in healthy stem cell donors, treated with G-CSF. This is presumably a result of endothelial cell activation. A larger series of donors would be required for correlation with clinical symptoms. Lower baseline platelet count, but not higher donor age, is significantly associated with poorer PBSC mobilisation efficiency and lower Day 5 CD34+ cell yield in a singlecentre cohort of 56 adult allogeneic PBSC donors J. Sinclair* (1), J. Travers (1), R. Green (2) , K. Douglas (1) ( Aims: Poor peripheral blood stem cell (PBSC) mobilisation in allogeneic donors has adverse consequences for both donor and recipient. We are only aware of one previous study addressing pre-collection donor factors predicting mobilisation efficiency in G-CSF-stimulated healthy allogeneic donors (Suzuya et al., Vox Sanguinis 89:229-235, 2005) , and this study involved a mixed series of adult and paediatric donors. We wished to assess the effects of donor age and baseline donor platelet count (both found to correlate with mobilisation efficiency by Suzuya et al.) in our single-centre experience with exclusively adult allogeneic PBSC donors. Patients & methods: Retrospective audit of donor data for a consecutive series of 57 adult G-CSF-stimulated allogeneic PBSC donors over a 4-year period. Median age was 41 years (range 17-59). Results: One donor had reactive thrombocytosis with raised ESR & CRP, but had also received immunomodulatory drugs for a known chronic inflammatory condition: she did not meet usual donor selection criteria, and as expected she mobilised PBSC relatively poorly: she was excluded from statistical analysis due to the confounding effect of concomitant medication. For the remaining 56 donors who all had platelet count within the normal range (150-400x10 9 /l), baseline platelet count showed a significant positive correlation both with peripheral CD34+ cell count on Day 5 of G-CSF (r=0.465, p<0.001) and with CD34+ cell yield on Day 5 of G-CSF (r=0.42, p<0.01). However, although there was a slight trend towards poorer mobilisation with increasing donor age, this fell short of statistical significance either in terms of Day 5 peripheral CD34+ cell count (r=-0.026, p>0.05) or Day 5 CD34+ cell yield (r=-0.101, p>0.05). Conclusions: There appeared to be no significant adverse effect of increasing donor age in our exclusively adult donor cohort. However, there was a strong positive correlation between baseline donor platelet count and mobilisation efficiency, even though all donors in the series had platelet counts within the normal range. We suggest that baseline platelet count is a surrogate marker for haematopoietic stem cell (HSC) reserve. We suggest that prospective allogeneic HSC donors with thrombocytopenia, even mild, at baseline assessment should not normally donate PBSC, and that donors with platelet counts towards the lower end of the normal range have a somewhat higher risk of poor PBSC mobilisation and should be counselled appropriately. The increase demand for allogeneic bone marrow transplantation resulted in a parallel increase in the size of the registries of unrelated potential donors. However, estimation of the required size of a national registry of potential donors is complicated due to populations' heterogeneity in terms of human leukocyte antigen (HLA) typing in different countries. In order to determine the required size of the potential hematopoietic stem cell registry in Israel, we aimed to study the relationship between the registry size and the probability for an individual matching. The current chance for individual matching in Israel was determined according to the data gathered from two national Israeli registries. The chance for future individual matching was based on applying the following model: New P = 1-([1-current P] to the power of squared X), where current P is the current probability for individual matching, New P is the future probability for individual matching and X is the factor by which the number of the current size of the registry will be multiplied. The model was validated by examining the actual probability for matching in one of the registries in 2006. The size of the two Israeli registries as of the end of 2005 included 258,739 potential donors. The chance for an individual matching in 2005 was 34 and 66% (in two different national registries). According to this model, if the size of the registries will grow to 400,000 potential donors, then the probability for an individual match would increase to 63-92%, and if it will grow to 500,000 then the probability for individual match would further increase to 79-98%. Introduction of the actual 2006 registry size of the large registry into the mathematical model predicted 74% chance for individual matching. The actual observed probability was 77%. Although this model does not take into account the ethnic heterogeneity and the different matching probabilities of specific populations, it may serve for health policy makers as a simple and useful method to estimate the required size of national and international unrelated allogeneic stem cell registries according to different matching probabilities. Peripheral blood progenitor cells (PBPC) are increasingly used as a source of stem cells for hematopoietic transplantation in children. Although technically similar to adult procedures, some technical aspects must be considered in pediatric apheresis due to the size of the patient and donor. A retrospective study of 122 donor; mostly in pediatric age; who had undergone a total of 168 G-CSF mobilized peripheral stem cell apheresis between 1999 and 2007 was studied. The medical charts of all consecutive donors of the pediatric patients who underwent apheresis in our pediatric tertiary center were retrospectively reviewed. The median age (range) and weight (range) of donors were 12 years (2-53 years) and 38.5 kg (11-92 kg), respectively. 78% of the apheresis were applied to pediatric donors (aged less than 17 years). In 21% of the procedures, donor weighed less than 20 kg. All procedures were performed with a commercially available Apheresis System. 46% of the donors underwent more than one collection to reach enough cell count, two of them underwent three. 52% of the procedures were performed via double lumen central venous catheter placed either in femoral or subclavian vessels. Complete blood count parameters of the donors taken at the beginning and at the end of the apheresis are evaluated. Since one of the main limiting factors for apheresis in children is the whole blood volume and the resulting technical problems especially for the ones weighing less than 20 kg, the potential difference of the stem cell products in respect to weight of the donors are evaluated. The apheresis products were compared according to being of the donor 20 kg weight or less or above 20 kg. When two groups are compared, CD34 + cell and WBC counts collected from donors above 20 kg weight are higher (p=0.003, p=0.000), whereas mononuclear cell count is lower (p=0.005). Regarding technical problems, two donors experienced problems related with catheter and one with apheresis system causing cancellation of the apheresis. As adverse events, vomiting was observed in three, paresthesia in one, abdominal pain in one and hypotension in one but none of these events required cancellation of the apheresis. In conclusion; Our findings showed that peripheral stem cell apheresis is an effective and safe way to collect adequate number of stem cell product even in small donors. However, longer follow up is required to assess potential adverse events. We have studied 96 French patients registered into the French haematopoietic stem cell (HSC) donor Registry between 1994 and 2006 for a donor search, and not transplanted because of the explicit reason "no suitable donor found". 92% were adult patients (≥16y) and 8% paediatric patients. 22 were ALL (23%), 14 AML (15%) , 23 CML (24%),1 LLC, 1 other leukaemia , 8 plasma cell disorder (8%), 2 Hodgkin's lymphoma, 3 inherited metabolic disorder, 11 myelodysplasia (11%), 6 non Hodgkin's lymphoma (6%), 3 other malignancies, 2 severe aplastic anaemia. 58 were male and 38 female. Median age was 43 years old (range 2-63). Median time between patient registration and cancelation of donor search was 6 months. Four patients' HLA phenotypes show rare antigenic linkage disequilibrium (LD), 21 had rare high resolution (HR) alleles and rare allelic LD, 37 had rare HR alleles. The 34 remaining phenotypes were for 19 of them incomplete, in particular for patients registered before 2000. We did not find any HLA specificity for the 15 last ones. Rare HLA alleles or LD were mainly specific of Africa (24/62 -39%), then of Asia (13/62 -21%), for both Africa and Asia (6/62 -10). Among the 96 patients, at their registration date, 57 had no French HLA-ABDR low resolution (LR) compatible donors at all worldwide (59%), 14 had between one and five (15%), 7 had between five and twenty (7%). The 17 remaining patients were the first registered, so the number of compatible donors was not recorded at that time, but we have noticed for ten of them a HLA specificity in their HLA-ABDR phenotype that could explain they could not be transplanted because of the lack of suitable donors. So far, 28 patients still have no HLA-ABDR LR compatible donors worldwide, 21 have less then five, 16 have between five and ten, and the other 31 have more than ten. We checked for each patient the availability of a potential compatible cord blood unit in the world. It appears that all of them could receive at least a 4/6 matched unrelated CBU. In conclusion, these patients have not been transplanted because of their HLA phenotypes characteristics which were mainly specific of Africa and Asia. Those results need to be confirmed in a larger population, but it already could lead to a strategy how to improve HSC transplant access for those less represented populations into our donor panel. Unrelated CBU transplant represents an alternative option to treat those patients lacking for a full matched unrelated HSC donor. Unrelated allogeneic stem cell transplantation: is it really worse than related? A single-centre experience L. Yáñez*, A. Bermúdez, M. López Duarte, A. Iriondo Hospital Marques de Valdecilla (Santander, ES) Introduction: Unrelated allogeneic stem cell transplantation is an option in patients who have not a related donor. However this procedure is associated with an increased morbidity and mortality related with graft versus host disease (GVHD) and opportunistic infections. Objectives: Analyze differences between unrelated (UR-SCT) and related allogeneic stem cell transplantation (R-SCT) in patients with acute myeloblastic leukemia, chronic myeloproliferative disease, myelodisplastic syndromes and aplastic anemia. Good prognosis disease was defined by aplastic anemia, primary phase CML, refractory or sideroblastic anemia and acute myeloblastic leukaemia in first remission. Patients and methods: From September 1999 to December 2006, 141 patients underwent allogeneic stem cell transplantation (R-SCT: 87 and UR-SCT: 54) in our centre. To compare both groups, we determined differences between sex, age, prognosis disease, conditioning and stem cell source. Finally we analyzed acute and chronic GVHD incidence, overall survival (OS) and causes of death. Results: We found differences between both groups in age at transplantation and the source of stem cells. Although in UR-SCT the incidence of acute (59% vs 73%) and chronic (46% vs 58%) GVHD was higher than in R-SCT, there were no significant differences. With an eight year follow up, we did not find significant differences either among both groups in the estimated overall survival, (UR-SCT 51% vs R-SCT 48%), considering good prognosis disease (UR-SCT 62% vs R-SCT 60%), age < 50 yrs (UR-SCT 54% vs R-SCT 60%) and age > 50 yrs (UR-SCT 43% vs R-SCT 29%). As previously described in the literature, causes of death were different among both groups with statistical significance (p = 0.042): infections ± GVHD in UR-SCT (82%) and relapse in R-SCT (40%). Conclusions: We have not found significant differences in overall survival among unrelated and related allogeneic stem cell transplantation and it would be related with an elevated number of young patients in the unrelated group. However, when we compared both groups by adjusted age, we did not find any differences either. Allogeneic BMT represents the only chance of cure for betathalassemia (B-thal pts). Often more than one family member is affected, occasionally two affected individuals share a matched healthy sibling. Moreover, a high incidence of transplant rejection is still observed in Pesaro class III pts requiring a 2nd BMT procedure. In these settings, one option is to perform a 2nd BM harvest from the same donor. Though BM harvest is a safe procedure in children, such an invasive procedure must be balanced with ethical issues.We describe our series of 6 paediatric healthy donors (4 females, 2 males), who donated BM twice in favour of their B-thal HLA-identical siblings from June 2005 to Nov 2007. Median donor age 11(7-16). Donor Hb ranging 10-13.8 g/dl (3 were B-thal carriers). Three donors donated BM twice to 2 affected siblings and 3 donors donated twice for the same sib following secondary graft rejection of the 1st BMT. One donor underwent 3 harvests, 1 for the 1st BMT and 2 for the 2nd BMT to increase cell yield. All donors tolerated well the procedure, pain was controlled by paracetamol+tramadol. No side effects occurred. The 3 B-thal carriers required allogeneic blood transfusion after the 1st harvest and 2 of them also after the 2nd procedure. Donors were discharged 2 days after the procedure with iron and folic acid therapy. Median time between the two harvests was 259 days(42-393). The median volume collected in the 1st harvest was 23ml/ donor Kg(14-30) and in the 2nd 18ml/Kg(14-44). Median tot nucleated cells (TNC) in the 1st harvest: 12.5x10 9 /Lt(11-17), CD34 250x10 6 /Lt(150-350); 2nd harvest TNC 16x10 9 /Lt (8-19), CD34 290x10 6 /Lt(120-620). Engraftment occurred in all pts, median day PMN> 500 after 1st BMT 20(13-25) and platelets >20000 17(12-43), after the 2nd BMT PMN 18(15-25) and platelets 20(17-54).The decision to put a paediatric donor through 2 invasive procedures must be balanced after exploring alternative options to protect young donors from this compelled altruism. Discomfort of the procedure and benefits for the recipients were explained. A positive response in the decision to donate was probably influenced by active donor participation and consciousness of high rate of BMT success. Our experience shows that for paediatric donors a 2nd marrow donation is safe and feasible and good cellularity can be obtained. We suggest that when a strong clinical indication to proceed to BMT exists, a 2nd harvest on the paediatric donor can be performed The aim of this study was to evaluate two different strategies to find allogeneic hematopoietic stem cell transplants for patients who did not have an HLA-matched related or unrelated donor. Initially, we used HLA-A, -B, or -DRbeta1mismatched unrelated donors (MM URD) to 14 patients. This was more recently replaced by umbilical cord blood transplants (UCB) given to 27 such patients. Diagnosis, disease stage and age were similar in the two groups. Eleven MM URD transplants were T-cell depleted. The nucleated cell dose and CD34 cell dose were significantly lower in the UCB group (p<0.001). Graft failure occurred in two patients in the UCB group and three in the MM URD group. Time to ANC >0.5 x 10 9 /l was a median of 30 days in the UCB group, compared to 17 days in the MM URD group (p=0.002). Platelet engraftment was also significantly delayed in the UCB group (p=0.03). The UCB patients required significantly fewer erythrocyte transfusions, median three units vs. ten in the MM URD group (p=0.001). At 100 days, complete donor chimerism in the UCB/MM URD examined patients was 63%/44% for CD3, 71%/44% for CD19 and 63%/56% for CD33. Probability of acute graft-versus-host disease (GVHD) grades II-IV was 30% in the UCB group, compared to 21% in the MM URD group. The corresponding figures for chronic GVHD was 9% and 20% in the two groups, respectively. Transplant-related mortality was 30% in the UCB patients and 50% in the MM URD patients. Epstein-Barr virus lymphoproliferative disorder was seen in two patients each in both groups. Three-year survival was 66% in the UCB group vs. 14% in the MM URD group (p=0.006). To conclude, engraftment was delayed, leukocyte chimerism was not significantly different, but survival was superior using UCB compared to MM URD transplants. Transmission of CLL from a blood stem cell sibling donor to the recipient H. Nahi* (1), M. Jansson (2) , B. Sander (2) , P. Ljungman (1) Chromosomal analysis revealed 46,XX,t(11;19)(q23;p13.1). After a reduced intensity conditioning regimen the patient received stem cells from a healthy HLA-identical brother in March 2006. No acute graftversus-host disease (GVHD) developed. Immunosuppressive therapy was discontinued 3 months after transplantation. Bone marrow examination, which was carried out three and six months post-SCT, showed complete hematological remission, but a lymphoid infiltrate with the morphology of CLL was noticed in the BM in the 3-month sample. One year post-SCT a new BM sample was examined and the CLL population now constituted 16% of the total number of cells in the BM. FISH showed trisomy 12 in a chromosome Y-positive cell population. At the same time, the peripheral blood cells showed lymphocytosis. Within a short interval, repeated analysis of the BM and peripheral blood cells showed complete donor-type chimerism. Eighteen months after SCT, a new BM examination was performed and this showed not only a slight increase in the percentage of the CLL clone to 19%, but also a clone with myeloblasts that constituted 16% of the total cell count. Chromosomal analysis showed not only 47XY,+12[7] of donor origin but also 46XX,t(11;19)(q23;p13.1) of recipient origin. A 62-year-old healthy, HLA-identical brother was used as a donor. Medical examination, laboratory test results and BM did not reveal any disease. The donor was reassessed one year after transplantation, including differential counting of white blood cells, which showed no signs of lymphocytosis, and a new medical examination was carried out, which revealed no lymph node enlargement or other illness. Retrospective FISH analysis of the donor's BM at the time of transplantation showed trisomy 12 in 15% of the BM cells. In conclusion, small CLL clones are not uncommon in elderly individuals, therefore, the possibility of an occult hematological disease in the donor raises questions regarding the need for accurate molecular diagnosis of donor cells, especially when healthy elderly people are considered as BM donors. No experience in literature describes possible modifications changes of immune system during G-CSF stimulation in peripheral stem cell healthy donors (PBSC). The objective of this study was to evaluate the changes of lymphocyte subset (SL) in this group. Evaluation of SL panel (CD3+, CD4+, CD8+, CD19+, CD56+ e CD8+CD56+) has been carried out at basal time (before starting G-CSF), at time +3 and +5 since starting G-CSF and at day +30 after the end of the stimulation. Thirty-one sibling donors have been evaluated, HLA-matched, 13 male and 18 female, median age 42 years (range 20-63 years), mobilized with median dose of G-CSF (Lenograstim) as 10 µg/kg/day (range 8.0-14). Apheretic procedures have been performed from day +5 using a continuous device cell separator (Fresenius, COM.TEC system). The majority of donors achieved the PBSC target dose (CD34+ > 4 x 10 6 /kg recipient) with a single apheretic procedure. White cells achieved the maximum value of 40 x10 9 /l (median 23-68). The lymphocyte subsets evaluation showed that during G-CSF stimulation there is an activation of T immune system with pan T growth (CD3+), T-helper/inducer (CD4+) e Tcytotoxic/suppressor (CD8+) with peak value at day +5 post G-CSF (p=0.001) and following immunodepressive scenario + 30 (p=0.001), as regards the basal parameters analyzed. Similar result occurs for the immune system B, with cell CD+19 growth, with peak value at +5 (p=0.001) and subsequent depression at + 30 (p=0.001). The impact that this result could have on the donor clinical risk have to be carefully evaluated. Acquisition and loss of the Janus kinase 2 V617F mutation post allogeneic transplantation L. Schonegevel, A. Butler, N. Patton, R.L. Spearing, P. Ganly* Christchurch Hospital (Christchurch, NZ) The Janus kinase 2 (JAK2) V617F mutation occurs in 50%-60% of patients with myelofibrosis (MF) and essential thrombocythaemia (ET). Although the role of the mutation in myeloproliferative disease is well recognised, the sequence and timing of JAK2 mutation with respect to clinical features of the disease is less clear. Bumm et al [1] showed that transplantation of V617F-transduced bone marrow into mice induces erythrocytosis and granulocytosis and suggested a complex interplay between cell intrinsic and extrinsic factors, implicating additional factors in the manifestation of human disease.We have previously reported [2] on a patient who received a reduced intensity conditioned (RIC) allogeneic transplant for multiple myeloma (MM) using a donor with ET and can now update this report with data on JAK2 mutation status. A 48 year old man presented with stage III MM, received standard induction chemotherapy, and then proceeded to high-dose melphalan conditioning with an autologous stem cell transplant. This was followed by a RIC transplant, with total body irradiation and fludarabine conditioning. The donor has ET with V617F mutation whereas the recipient was initially negative for the V617F mutation using allele specific polymerase chain reaction. Follow up molecular studies at five years post transplant demonstrate that the recipient, who is a 100% donor chimera, has JAK2 V617F without clinical evidence of ET. The donor remains well and maintains a normal platelet count with Hydroxyurea. The alternative scenario is demonstrated by a second patient with ET and multiple cytogenetic abnormalities that transformed to MF. Pre transplant molecular studies demonstrated the V617F mutation. He underwent a myeloablative allogeneic stem cell transplant with Busulphan and Cyclophosphamide conditioning and after three years has no morphological features of MF, normal cytogenetics and no evidence of V617F. These two cases demonstrate the acquisition and eradication of JAK2 V617F positive haematopoiesis with allogeneic transplant. This molecular marker of disease may prove useful for the detection of minimal residual disease or early relapse, and may guide the use of post transplant donor lymphocyte infusions in RIC transplants. Introduction: Allogeneic stem cell transplantation remains the only curative therapy for many patients with hematological malignancies. However, transplant-associated toxicity, particularly graft-versus-host disease (GvHD), remains a major concern that limits treatment efficacy. Naturally occurring regulatory T cells (Tregs) are identified as CD4+CD25highFoxP3+ cells that modulate immunologic tolerance, inhibit primary T cell activation, and have therefore been suggested to protect against GvHD. However, the use of Tregs to prevent GvHD may result in delayed immunological reconstitution, diminished graft-versus-tumor effect, and an associated increased risk of relapse and infection. Objectives: To investigate the characteristics of T cells undergoing allogeneic stimulation, looking for a subpopulation of T cells with a more specific inhibition of host immunity, promoting a specific prophylaxis of post-transplant GvHD. Methods: CD25-peripheral blood mononuclear cells (PBMCs) and naturally occurring CD25+ T cell, generated by negative selection with anti-CD25 magnetic beads, were subsequently stimulated by mature dendritic cells (DCs) in mixed lymphocyte culture for 5-6 days. Following stimulation of CD25-PBMCs, the resultant CD4+CD25+ or CD8+CD25+ populations were isolated. The stimulated T cell populations were assessed for phenotypic markers, cytokine expression profile, cell proliferation, inhibitory capacity and anti-viral response. Results and Conclusion: Stimulation with allogeneic DCs induced generation of CD4+CD25+ and CD8+CD25+ T-cells from CD25-precursors. Similar to naturally occurring Tregs, both populations strongly expressed FoxP3 and markedly inhibited alloreactive T cell expansion. However, induced CD4+CD25+ and CD8+CD25+ T cells did not affect proliferation against the cytomegalovirus (CMV) recombinant protein, suggesting the potential for more selective inhibition of host immunity. The induced CD4+CD25+, CD8+CD25+, produced inflammatory cytokines, such as IL-2 and IFN-a and also preserved the ability to respond to recall antigens (e.g., CMV), thus exhibiting effector properties. As such, these cells are potentially ideal for use as post-transplant GvHD prophylaxis P708 KIR/HLA interactions influence the outcome of haematopoietic stem cell transplantation K. Ludajic* (1), Y. Balavarca (2) , H. Bickeboeller (2) Interactions of donor killer immunoglobulin-like receptors (KIR) of NK and T cells with HLA ligands on haematopoietic cells of patients can, under certain conditions, provoke alloreactivity and influence HSCT outcome. In this study we aimed to determine the relevance of several previously reported models of KIR/HLA interactions, and their effect on the clinical outcome of HSCT in 124 patients diagnosed with various haematological malignancies and receiving HLA matched transplants from unrelated donors. These models include: KIR/ligand mismatching or ‚missing ligand' model (Hsu et al., 2005) , patients HLA-C genotypes (Fischer et al., 2007) , KIR haplotype contents of patients and donors (McQueen et al., 2007) and the effect of single acitvatory donor KIRs (Cook et al., 2004) . Patients missing C1 ligand for donors' KIR2DL2/KIR2DL3 were at the increased risk for developing aGvHD (II-IV) (HR=1.96, 95% CI: 1.07-3.61, p=0.03), while the effect of other missing ligands was not evident. Correspondingly, the analysis of HLA-C genotypes alone, regardless of donor KIR type, revealed that only C2C2 genotypes showed a trend towards higher aGvHD rates (p=0.07). Cox regression analysis of patients' and donors' KIR haplotype contents showed donors' AA hapolotypes being associated with higher incidence of aGvHD (II-IV). When the analysis was extended to include HLA genotype of patients, AA haplotypes in both patients and donors, together with either Bw4-(HR=3.03, 95% CI: 1.25-7.36, p=0.014) or C2Cx HLA genotype (HR=2.82, 95% CI: 1.32-6.02, p=0.0075) also showed significant association with aGvHD (II-IV). In concordance with this result, analysis of single activatory KIR genes (overrepresented in Bx vs. AA haplotypes) revealed that the absence of KIR2DS1 and KIR2DS5 in C2C2 patients was a risk factor for aGvHD, while donor's KIR2DS2 and C1Cx was associated with reduced incidence aGvHD (II-IV). In T cell replete transplantation settings activatory donor KIRs were therefore associated with the improved outcome in C2Cx patients. We conclude that KIR gene effect on clinical outcome must be analysed in the context of the patients' HLA genotype. While the KIR effects analysed were significant for aGvHD (II-IV), we did not find associations with other trasplantation endpoints like cGvHD, relapse, transplant related mortality or overall survival. Background: A low number of antigen presenting cells (APC) in the peripheral blood 1 month after allogeneic HSC transplantation has been previously associated with increased mortality. However, the impact of APC recovery at later time points (3 through 12 months) after transplant has not been thoroughly investigated. Aim: In this study we analyzed the factors affecting APC recovery between 3 and 12 months after allogeneic HSC transplant and determined its impact on the clinical outcome. Patients and Methods: Blood samples from all patients undergoing an allogeneic HSCT in our institution from June 1999 through June 2006 were analyzed. PB numbers of CD11c+ myeloid dendritic cells (mDC), CD123+ plasmacytoid DC (pDC) and CD14+ monocytes were determined based on flow cytometry at 3 (n=161), 6 (n=117) and 12 months (n=80) after transplant. Results: Median numbers of PB APC at 3 months after transplant were: mDC (7.4x106/L), pDC (2.5) and monocytes (254), and then increased gradually at 6 (mDC 9.2, pDC 3.4, monocytes 272) and 12 months (mDC 11.3, pDC 4.6, monocytes 335). mDC recovery was delayed both at 3 and 6 months in patients with advanced disease at transplant (p<0.00001 and 0.003, respectively), and acute GVHD grade II-IV (p=0.00009 and 0.01). Similarly, pDC recovery was delayed at 3 and 6 months in patients with advanced disease (p=0.0001 and p=0.003) and acute GVHD (p=0.0004 and p=0.02). Moreover, both mDC and pDC levels at 6 months were reduced in patients with extensive chronic GVHD (p=0.001 and p= 0.0007 respectively). All other analysed factors did not correlate with mDC and pDC recovery. Also, mDC and pDC numbers at 12 months, as well as monocyte numbers at any time point, did not correlate with any of the analysed factors. Lower numbers of mDC and pDC in the PB at 3 months after transplant significantly correlated with increased overall and transplant-related mortality in univariate analysis. In multivariate analysis low mDC counts correlated with increased overall mortality (p=0.008) whereas low pDC counts correlated with increased TRM (p=0.07). PB Monocyte numbers instead did not correlate with any of the analysed end points. Conclusion: Recovery of mDC and pDC 3-12 months after transplant was reduced in patients with advanced disease at transplant, or with acute or chronic extensive GVHD after transplant. Nevertheless, a delayed recovery of mDC and pDC within 3 months after transplant was an independent factor contributing to a greater mortality. Background: Toll like receptors (TLR) are important constituents of innate immunity cells. They are activated by specific components of microbes and certain host molecules and subsequently influence the Th1, Th2 and NK cell response. SNPs of genes encoding particular TLRs may be therefore involved in heterogeneous reconstitution profile after allo HSCT. Patients/methods: 70 patients with hematological malignancies, aged 32.5 (18-58)y, transplanted from HLAidentical sibling (n=28) or unrelated donor (n=42) were included. The conditioning regimen was myeloablative; based on chemotherapy alone (76%) or combined with TBI (24%). GVHD prophylaxis consisted of cyclosporin, methotrexate, in URD-HSCT-pre-transplant ATG. Donors (D) and recipients (R) were tested for: NOD2/CARD15 8,12,13, TLR4(299), TLR4(399), TLR5(1174) and Il23R (11209026) SNPs. Study end-points included evaluation by flow cytometry of: CD3, CD3+4, CD3+8, CD56, CD19, and CD8 lymphocyte reconstitution in the recipient on day:+28,+56,+100,+180 post alloHSCT in the WT (wild type) vs. mutated variants carriers. Results: The frequency of mutated variants of TLR4(399), TLR4(299), TLR5 and IL23R equaled in the recipients: 6/70 (9%), 11/70 (16%), 3/70 (4%), 4/70 (6%) and in donors: 10/70 (14%), 10/70 (14%), 4/70(6%) and 4/70 (6%) respectively. As presented in the table, there was a significantly higher number of CD3+, CD8+ and CD56+ cells at the regeneration (day +28) in recipients of alloHSCT from TLR(299) carrying donors as compared to WT carrying ones. Similarly, the presence of TLR4(399) in donors resulted in significantly higher number of CD3+. table 1. No significant correlation was found in the late post transplant phase on days +56,+100 and +180. There was also no significant influence of TLR5, IL23R and NOD2/CARD15 variants in both recipients and donnors on immune reconstitution at any of the evaluated time points. Conclusions: In the observed cohort of donor recipient pairs the presence of TLR4(299) polymorphism in donors was found to significantly increase the T and NK cell number on day 28 after alloHSCT compared to WT donors whereas TLR4(399) displayed a similar influence on CD3+ cells only. This results encourage to extended studies. (1) Background: Invariant NKT (iNKT) cells are a subset of immunoregulatory T cells that modify a variety of immune responses including alloreactivity. Central to their function is the interaction of the invariant Valpha24Jalpha18+ TCR with glycosphingolipid (GSL) ligands presented by the nonpolymorphic MHC class I molecule CD1d. Whether iNKT cells, similar to natural killers (NK) and conventional T cells, can directly display alloreactivity is not known. Objective: We investigated whether human iNKT cells can be directly activated in an allogeneic manner and also dissected the molecular requirements in this process. Methods: Monocytes were purified from normal donor buffy coats. iNKT cells were expanded in vitro in the presence of alphagalactosylceramide (aGC) while NKT cell clones were raised by cloning at 1cell/well using flow-sorting. iNKT cells were identified by FACS using specific anti-TCR mAb and tetramers. Cell proliferation was assessed by standard 3Hincorporation assays and cell lysis using the Cytotox96 nonradioactive cytoxicity kit. GSL analysis of dendritic and B cells was performed using a highly sensitive, fluorescent, HPLCbased approach. Results: In mixed lymphocyte reactions involving highly purified populations we show that polyclonal, (i.e., either ex vivo flow-sorted or aGC-expanded), or clonal human iNKT cells and APC can establish a direct cross-talk leading to preferential maturation of allogeneic APC (as determined by surface expression of CD14, CD86 & HLA-DR, secretion of IL-12 and induction of 3rd party MLR) and a considerably higher reactivity of iNKT cells cultured with allogeneic rather that autologous APC (as assessed by proliferation, cytotoxicity and IFNgamma secretion). While the allogeneic activation of iNKT cells is iTCR-CD1d interaction-dependent, GSL profiling suggested it does not involve recognition of disparate CD1d/GSL complexes. Instead, we show that contrary to previous reports, iNKT cells, like NK and T cells, express killer immunoglobulin receptors (KIR) at a frequency similar to that of conventional T cells and that iNKT cell allogeneic activation largely depends on upregulation and function of activating KIR. Conclusions: iNKT cells can display alloreactivity for which they utilise mechanisms characteristic of both NK and conventional T cells suggesting that iNKT cell depletion of the hematopoietic graft might be a means of reducing the incidence and severity of clinical aGVHD. Donor lymphocyte infusions (DLI) provide powerful antileukemic responses, and this approach is often used in combination with reduced intensity conditioning (RIC) to optimize Graft-versus-Leukemia (GVL) effects. However, we and others demonstrated that the efficacy of DLI-induced GVL in long-term complete hematopoietic chimeras is significantly reduced. The aim of this study was to demonstrate that DLI-related cytotoxicity can be induced in complete chimeras by cotransplantation of donor lymphocytes with recipient's hematopoietic cells. Materials and methods BALB/c->C57BL/6 and C57BL/6->BALB/c complete chimeras were obtained by our transplantation protocol including RIC with total body irradiation (150 cGy -300 cGy, day-3), donor-specific cell transfusion (DST, day-2), and selective depletion of DSTactivated residual donor-reactive cells by cyclophosphamide (day -1), followed by donor bone marrow transplantation (BMT) on day 0 (3x10 7 cells). Donor splenocytes alone or in mixture with an equal number of recipient's splenocytes were infused to chimeras 3 months after BMT (4X10 7 cells of each type). Splenocytes were labeled before transfusion with CFSE (to distinguish them from other hematopoietic cells in chimeras). Recipients were sacrificed 1, 3, or 7 days after DLI; their blood and spleen cells were stained by anti-H-2b mAbs (to distinguish between transplanted donor and recipient cells), and the efficacy of DLI-associated cytotoxic response was analyzed by FACS according to the ratio of BALB /c (CFSE positive, H-2 Kb negative) to all CFSEstained cells. Results FACS analysis confirmed that donor cells injected alone to complete chimeras were accepted by recipients but did not proliferate. Transplantation of a mixture of recipientand donor-type lymphocytes to chimeras switched the ratio between donor and recipient CFSE -stained cells to the benefit of the donor within 1 day, while within 7 days most recipient-type lymphocytes were rejected by proliferating donor cells. Naive mice rejected allogeneic lymphocytes within 3 days (Figure) . Conclusions The experimental design presented in this study allows visualization of DLI-induced cytotoxicity in complete chimeras. Our results demonstrate that anti-host cytotoxicity can be restored in complete chimeras by co-transplantation of hematopoietic cells of the host. 46% (p=0,33) . The nonsignificant trend for higher relapse rates in FM group was observed -26% vs. 7%,p=0,18; on the contrary there was no difference in TRM (40% vs. 41%,p=1,0).The incidences of gr.III-IV aGVHD and cGVHD were also similar (FMxPM: 14% vs. 14%, and 44% vs. 44%). Conclusions: For patients undergoing unrelated SCT and older 50 year, equivalent outcome was observed between the 10/10 HLA molecularly matched and 1-2 allele mismatched (8-9/10). It supports our policy to put the accent rather on timely SCT accomplishment (though with mismatched donor) than to often time-consuming identification of perfectly matched donor not only for young patients but for the elders likewise P714 FoxP3 expression and its correlation with graft-versushost disease in allogeneic peripheral blood stem cell transplantation S. Bellesi*, P. Chiusolo, D. De Ritiis, S. Marietti, A. Catalano, F. Sorà, N. Piccirillo, G. Leone, S. Sica Haematology (ROME, IT) Several studies have demonstrated CD4+CD25high Tregulatory cells (Tregs) suppress graft-versus-host disease (GVHD) in animal models. FoxP3 gene encodes a trascription factor that is a key regulator for thymic development and function of naïve Tregs. In humans some authors reported the association between reduced Foxp3 expression and development of GVHD and a low risk of GVHD in patients receiving a graft with high donor FoxP3-positive Tregs. We analized FoxP3 expression and its correlation with GVHD in 6 pts (M/F 4/2, aged 21-55 years) undergoing allo-SCT. Pts' characteristics were: 3AML, 1 CML and 1 ALL submitted to a myeloablative PBSCT from siblings in 4 cases and MUD in 1 case. 1 pt affected by MF/SS received RIC-PBSCT from sibling. PB samples were obtained from donors before mobilization and from pts at days +30, +60, +90, +120, +210 after SCT. 2 pts developed severe aGVHD and were lost to S200 follow-up (FU). 1 pt received DLI for relapse from day +90 to day +120 and developed cGVHD. The pt with RIC-PBSCT developed cGVHD from day +110 onward. Results: 5 donors expressed FoxP3 and only 1 pt developed severe aGVHD. Only 1 donor did not express FoxP3 and the pt developed severe aGVHD. Two pts with no GVHD expressed FoxP3 during FU. The pt who received DLI presented a reduced gene expression at day +90 and +120, with associated cGVHD; at +210 the same pt did not present cGVHD in association with major FoxP3 expression. The pt undergoing RIC-PBSCT developed cGVHD from day +110 post SCT and presented reduced gene expression at days +90, +120, +210. 1 pt with resistant aGVHD presented reduced FoxP3 expression at days +30, +60,+90 and no expression of FoxP3 in the donor. The other pt with aGVHD did not show FoxP3 at day +30, but FoxP3 expression at day +60, after 6 extracorporeal photopheres (ECP) with complete resolution of skin GVHD. In summary 4 pts who did not develop aGVHD expressed FoxP3 at days +30. Two of these pts developed cGVHD in association with reduced FoxP3 expression. Both pts with severe aGVHD presented respectively reduced expression and no expression of FoxP3 at day +30. In conclusion FoxP3 expression decreased in pts with GVHD and its early expression at day +30 in the recipient and in the donor may be associated with low risk of GVHD following alloSCT. According to recent studies FoxP3 expression increased after ECP in the pt receiving this treatment for aGVHD. Induction of Treg cells may be the mechanism required for a tolerogenic shift of immune system post ECP. Antibodies to the donor stem cell population CD34+/VEGFR-2+ are associated with rejection after HSCT but could be decreased with immune absorption A. Nordlander* (1), S. Sumitran-Holgersson (2) Reconstitution of hematopoiesis after hematopoietic stem cell transplantation (HSCT) occurs via a reservoir of donor CD34+ HSC. CD34+/VEGFR-2+ is a primitive, quiescent subpopulation of HSC known to generate hematopoietic or endothelial progeny in vitro and in vivo. We hypothesized that donor-specific antibodies to the CD34+/VEGFR-2+ stem cells may be associated with rejection after HSCT. We studied 19 patients without and 11 with rejections after HSCT and 20 non-transplanted healthy individuals. Ninety-three sera taken pre and post-tx from patients receiving HSCT were studied for the presence of donor CD34+/VEGFR-2+ cell-specific antibodies.We provide evidence that significantly higher numbers of patients with rejections 9/11 (81 %), while 1/19 (5%) (p=0.001) without rejections had antibodies against donor CD34+/VEGFR-2+ cells, but not CD34-/VEGFR-2cells. In eight transplantations, antibodies against donor CD34+/VEGFR-2+ cells were detected already prior to transplantation. Purified IgG fractions from patients with rejections but not controls significantly decreased the ability of these cells to form hematopoietic and endothelial colonies. In multivariate analysis antibodies against CD34+/VEGFR-2+ cells proved to be the most significant risk factor for rejection. In our material rejection of the graft was connected with a very high mortality rate. We have therefore also focused on possible ways to remove antibodies against CD34+/VEGFR-2+ cells by using immune absorption. We treated two patients with plasmapheresis and retuximab prior to transplantation. The treatment was well tolerated with no major side effects. In both patients antibodies against donor CD34+/VEGFR-2+ cells were detected prior to transplantation. Immune absorption decreased antibodies against CD34+/VEGFR-2+ cells and no rejection was seen. However, in one of the patients antibodies against CD34+/VEGFR-2+ cells increased again two weeks after HSCT. Due to the patients poor clinical status immune absorption was not possible. The patient almost rejected her graft with more than 95% CD34+ cells of recipient origin but the patient still had 20% donor T cells. The patient developed acute GVHD grade III which turned the chimerism pattern to complete donor in all cell lineages in two weeks. In conclusion, antibodies against CD34+/VEGFR-2+ cells are associated with rejection after HSCT but rejection may be avoided using immune absorption during conditioning therapy prior to transplantation. Due to the fact that the ABO system is inherited independently from the HLA system, about 45% of allogeneic hematopoietic cell transplants (HCT) are performed across the blood group barrier which does not influence the outcome of HCT in terms of incidence of graft rejection or delayed leukocyte and platelet engraftment. We retrospectively analyzed ABOmismatched (mm) transplant recipients with respect to establishment of donor type blood group (red blood cell (RBC) antigens and isohemagglutinins) and behaviour of blood group in case of relapse. Between August 1985 and October 2007, 283 patients (150 male, 133 female) with a median age of 31.6 years (range, 4 months -70.5 years) underwent ABO-mm allogeneic HCT for hemato-oncological disorders. One-hundred fifteen patients had a major (patients' hemagglutinins against donor RBCs), 121 a minor (donor hemagglutinins against patients' RBCs) and 47 a bidirectional (minor and major) ABO-incompatible donor. The median follow-up (FU) was 27 (range, 0.3-252) months. In 148 of 283 patients (48%) donor-RBC type was demonstrable after a median of 26 weeks after HCT (range, 3 -157 weeks). Corresponding donor hemagglutinins were observed in 90 of 283 patients (29%). One hundred fifteen patients (41%) did not establish donor hemagglutinins, 75 were not evaluable due to early death or missing data. Hemagglutinins of donor blood group type were observed in 82/108 major, 7/56 minor and 1/46 bidirectional ABOincompatible HCTs. Forty-seven patients experienced relapse of their underlying disease. In 42 of them donor RBC type and in 22 complete donor blood group (including hemagglutinins) was demonstrable before relapse. In 13 patients (28%) with relapse the ABO-type switched back to the original blood group and in 24 cases (51%), blood group of the patient did definitely not return to his original phenotype. In 10 cases (21%), blood group typing of the patient was not possible due to the transfusions the patient had received. Our data show that in the majority of major ABO-mm HCT full establishment of donor ABO type is demonstrable. In case of relapse half of the patients presented donor ABO-type and in about a quarter the original ABO phenotype occurred. High concentration of regulatory T-cells in the peripheral blood of patients who have undergone an allogeneic stem cell transplant correlates with reduced incidence of acute graft-versus-host disease and of cytomegalovirus reactivation G.F. Torelli*, B. Lucarelli, A.P. Iori, M.S. De Propris, F. Milano, W. Barberi, V. Valle, E. Iannella, E. Arleo, R. Ricci, A. Guarini, R. Foà Sapienza University (Rome, IT) The immunological reconstitution is ultimately responsible of the clinical outcome of patients who have undergone an allogeneic stem cell transplant (SCT). Aim of the study was to correlate the post-transplant clinical parameters with the concentration in the peripheral blood (PB) of T cells and T-cell subsets, with particular attention to CD4+CD25+ regulatory cells (Tregs), B cells and NK cells. Thirty-nine patients who underwent an allogeneic SCT (21 PBSC, 16 BM, 2 CB; 28 from an HLA identical sibling and 11 from MUD) at our Institute between September 2005 and October 2007 were investigated. Eighteen of these patients were evaluable at 1 year from transplant and the cellular concentrations present in the PB were analyzed at this time point. Four-color immunofluorescence was performed using antibodies against CD3, CD4, CD8, CD25, CD20, CD16, CD56 and CD34. Acute GVHD (aGVHD) was observed in 16 of the 39 patients; it was statistically correlated with reduced OS (27% in patients with aGVHD vs 75% without, p 0.04), increased TRM (52% with aGVHD vs 14% without, p 0.039) and incidence of cytomegalovirus reactivation (CMVr) (89% with aGVHD vs 48% without, p 0.0007). CMVr, that was observed in 23 of 39 patients, was also statistically correlated with reduced OS (20% in patients with CMVr vs 94% without, p 0.05) and increased TRM (48% with CMVr vs 6% without, p 0.002). Considering the 18 patients who were evaluable at 1 year, 33% of them presented aGvHD and 61% CMVr. In univariate analysis, aGVHD correlated with the concentration of Tregs in the PB; it was in fact observed in 56% of patients with Tregs below the median value vs 11% of patients with Tregs over the median (p 0.064). In addition, a high concentration of Tregs in the PB correlated significantly with reduced CMVr (45% of patients with Tregs over median vs 87 % below, p 0.01). A reduced CMVr has been also observed in patients who presented a concentration of NK cells over the median value (29% vs 86% below median, p 0.05). Relapse was not influenced by the concentration of Tregs; nevertheless, we observed a trend of increased relapses in patients with Tregs over median value, but this did not reach significance. Data on OS, DFS and TRM are not evaluable at the moment. These results confirm that the concentration of Tregs in the PB may protect from aGVHD and, as a consequence of less immunosuppressive therapies, from CMVr. The potential antiviral role of NK cells has been also confirmed by our data. /kg ). Diagnoses included standard-risk diseases N= 78 (Chronic lymphocytic leukaemia, low grade lymphoma, high grade lymphoma in first CR, AML/ALL first CR, Refractory anaemia with ringed sideroblasts of myelodysplastic syndrome, chronic myeloid leukaemia first CP, multiple myeloma in CR or partial remission). G-PBSCs have been harvested from 54 matched related and 24 unrelated donors (MRD and MUD). The pts and donors median age was 53 yrs old (23-65) and 46 (24-67) respectively. Eleven myeloablative and 67 reduced intensity conditioning regimens were administered and followed by cyclosporine A alone or ± mycophenolate mofetil or ± methotrexate. The median follow-up was 19 months. 71 pts engrafted. Cumulative incidence (CI) of day 100 grade 2-4 aGVH and 3 years cGVH were 33 % and 61 % respectively. CI of TRM, Relapse, EFS and OS at 3 yrs were 27 %, 33 %, 53% and 59 % respectively. In univariate analysis, low TRM was correlated with low CD34 (p=0,02), low B cells (p=0,047) and MRD (p=0,005). Relapse incidence (RI) was strongly associated with low NK cells number (p=0,007), donor chimerism (p<0,001) and cGVH (p=0,05). Type of donor and cGVH predicted better EFS (p=0,001 and p=0,03) respectively. NK cells, cGVH and type of donor offered better OS respectively (p=0,03; p=0,005 and p=0,01). In multivariate analysis, NK cells number is the only factor affecting independently RI [95% confidence interval, 1-7, RR=2,6 (p=0,05)]. cGVH is affecting independently OS (p=0,04). These results showed role of NK cells in relapse. Graft-versus host disease and infectious complications following reduced intensity conditioning umbilical cord blood transplantation in adults S. Furst*, M. Mohty, C. Faucher, J. El Cheikh, P. Ladaique, C. Lemarie, N. Vey, R. Bouabdallah, A.M. Stoppa, N. Marchetti, C. Chabannon, D. Blaise Institut Paoli-Calmettes (Marseille, FR) Eeduced intensity conditioning (RIC) umbilical cord blood transplantation (UCBT) can represent an attractive treatment modality for pts who lack a suitable HLA-matched donor. The aim of this study was to assess the outcome of 27 high risk pts (AML, n=15; ALL, n=7; NHL, n=3; CML, n=1; and HD, n=1) who underwent RIC UCBT between 2005 and 2007 with a special focus on graft-versus host disease (GVHD) and infectious complications. 23 pts (85%) were in CR (CR1, n=17; CR2, n=5; CR3, n=1), whereas 4 had a more advanced disease.The median age and weight was 43 (range, 17-59) y and 62 (range, 47-125) kg respectively.The RIC regimen included Fludarabine 200 mg/m², Cyclophosphamide 50 mg/kg and low dose TBI (2 Gy).CSA and MMF was given for GVHD prophylaxis.11 pts (41%) received a single CB unit, whereas 16 (59%) received 2 CB units. The median cryopreserved and infused cell doses were 4.9x10 7 TNC/Kg (range, 3.3-7.0) and 3.7x10 7 /Kg (range, 1.9-5.5) respectively.Neutrophil engraftment occurred in 26 pts (96%) at a median of 19 (range, 6-45) days. A sustained platelet recovery (>50000/µL) was observed in 19 pts (70%) at a median of 48 (range, 29-131) days.The overall incidence of grade II-IV acute GVHD was 58% (95%CI, 39-77%; 5 grade II, 9 grade III and 1 grade IV). 16 pts were evaluable for chronic GVHD for an overall incidence of 41% (7 limited and 4 extensive cases).16 pts (59%; 95%CI, 40-77%) experienced at least one episode of a "serious" infectious complication (virus other than CMV reactivation, n=8; bacteria, n=8; fungal, n=4), requiring long-term hospitalization, and of whom 7 pts were in grade III-IV acute GVHD. 6 pts (22%) experienced severe interstitial pneumonia, with 4 pts (15%) developing ARDS. 5 pts (19%) also required transfer to ICU. With a median follow-up of 385 (range, 111-903) days, 5 pts (18%) had relapsed with this being significantly lower in those pts transplanted in CR (P=0.01), but without a significant difference between single and double CB recipients. 8 pts have died (infection, n=4; GVHD, n=2; relapse, n=2; TRM=22%).The KM estimate of OS and EFS was 73% and 58% at 2 years respectively with no significant differences between single and double CBT recipients.There was a trend towards an improved OS and less grade II-IV acute GVHD in pts without HLA-DRB1 antigen mismatch (P=0.05 and P=0.08 respectively).In all, we conclude that RIC UCBT is an efficient therapy for high risk hematological malignancies. However, GVHD and serious infections are still a matter of concern warranting prospective efforts to define optimal prophylactic approaches. Reduced-intensity allogeneic stem cell transplantation is a potential therapeutic approach for adults with high-risk acute lymphoblastic leukaemia in remission S. Lee* (1), J.Y. Kwak (2) Purpose: Reduced-intensity allogeneic stem cell transplantation (RIST) is increasingly being used for patients with hematologic malignancies who are considered poor candidates for myeloablative transplantation because of their advanced age or other concurrent medical conditions. This strategy decreases the risk of nonrelapse mortality while preserving graft-versus-leukemia effect. However, the role of RIST in adults with acute lymphoblastic leukemia (ALL) remains unclear because interpretation of transplantation outcome is mainly limited by the small number of patients studied and by the criteria used to select patients for transplantation. The aim of the present study was to investigate the feasibility of RIST in 31 consecutive adults with high-risk ALL in first or second remission (2001) (2002) (2003) (2004) (2005) (2006) . Patients and Methods: All patients were treated with fludarabine (150 mg/m²) and melphalan (140 mg/m²) followed by transplantation from matched sibling (n=23) or unrelated (n=8; 3 matched, 5 allele-mismatched) donors. Antithymocyte globulin (2.5 mg/kg) was administered to patients who received allele-mismatched unrelated grafts. The indications for RIST were: (1) >50 years 14 (45.2%) and (2) decreased organ function/fungal infections 17 (54.8%). Their median age was 45 years (range, 20-63 years). All patients had high-risk criteria. Twenty-four patients (77.4%) were transplanted in first remission. Graft-versus-host disease (GVHD) prophylaxis was attempted by administering calcineurin inhibitor (cyclosporine for sibling transplants and tacrolimus for unrelated transplants) plus methotrexate. If acute GVHD was absent, the dose of calcineurin inhibitors was gradually tapered from day 60 (for sibling transplants) or day 90 (for unrelated transplants). Results: All but 3 patients who died from early transplantrelated complications achieved successful engraftment. The incidence of acute (grades II-IV) and chronic GVHD was 46.4% (13/28) and 73.1% (19/26), respectively. After a median follow-up of 32 months (range, 10+ to 82+ months) for surviving transplants, the 3-year probabilities of relapse, nonrelapse mortality, disease-free survival, and overall survival were 18.4%, 16.9%, 67.3%, and 70.4%, respectively. The presence of chronic GVHD was found to be associated with lower relapse (P=0.042). Conclusion: Our data suggests that RIST is a potential therapeutic approach for adults with high-risk ALL in remission who are not eligible for myeloablative transplantation. Radioimmunotherapy with yttrium-90-ibritumomab tiuxetan as part of a reduced-intensity conditioning regimen for allogeneic haematopoietic cell transplantation in patients with advanced non-Hodgkin lymphoma: interim analysis of a phase I/II study W.A. Bethge (1) Allogeneic hematopoietic cell transplantation (HCT) using reduced intensity conditioning (RIC) regimens offers a potential curative therapy to patients with advanced NHL. Combined use of radioimmunotherapy (RIT) with RIC may increase anti-lymphoma activity of RIC while HCT provides rescue from hematologic toxicity of RIT. This may allow further dose escalation of RIT. A multicenter phase I/II study of allogeneic HCT combining RIT using yttrium-90-ibritumomab tiuxetan (Y90-CD20) with two RIC regimens for treatment of patients with NHL has been initiated. Patients with indolent NHL (Arm A) receive RIT with Y90-CD20 (0.4 mCi/kg) on day -14 combined with RIC using fludarabine (30 mg/m 2 day -4 to-2) and 2 Gy TBI (day 0). Patients with aggressive NHL (Arm B) receive an escalated dose of Y90-CD20 (0,6-0,8 mCi/kg) on day -14 combined with RIC using fludarabine (30 mg/m 2 day -8 to-4), melphalan (140 mg/m 2 day -3) and campath (20-30 mg day -3 to-2). To date, 35 patients have been enrolled. Diagnoses in Arm A (n=25) were FL (n=13), MCL (n=6), CLL (n=5) and immunocytoma (n=1). Diagnoses in Arm B (n=10) were DLBCL (n=7), transformed CLL (n=2) and blastoid MCL (n=1). Median age was 56 (range, 33-68) years. PBSC grafts were from matched related (n=11) or matched unrelated donors (n=24). All patients were "high risk" with refractory disease or relapse after preceding HCT. Disease stage at time of HCT was CR=6, PR=15, SD=4 (Arm A) and PR=9, CR=1 (Arm B). No additional toxicity due to RIT even with dose escalation to 0.6 mCi/kg in Arm B was observed. Engraftment was rapid and sustained with no graft rejections. In Arm A median time to >500 granulocytes/µL was 13 (range, 0-69) days and to >20000 platelets/µL 2 (range, 0-69) days. In 12 patients platelets never were <20000/µL. In Arm B median time to >500 granulocytes/µL was 13 (range, 9-32) days and to >20000 platelets/µL 13 (range, 3-56) days. TRM in the first 100 days was 3%. Incidence of grade II-IV° GVHD was 40% in Arm A and B. To date, 18/25 (72%) patients in Arm A and 6/10 (60%) patients in Arm B are alive with a median follow-up of 250 (range, 28-466) days. Seven patients in Arm A (infection=5, GVHD=1, relapse=1) and 4 patients in Arm B (relapse=3, infection=1) died. Kaplan-Meier estimate of 1 year survival is 66% in Arm A and 50% in Arm B. In conclusion, the use of RIT in combination with RIC for allogeneic HCT of advanced patients with NHL is feasible and without additional toxicity due to RIT. Reduced intensity conditioning (RIC) regimens are increasingly being used with great success for hematopoietic stem cell transplantation (HSCT) in primary immunodeficiency. Between June 2002 and October 2007 a total of six patients with primary immunodeficiency underwent HSCT in this institution using RIC conditioning.The specific disorders were as follows: chronic granulomatous disease (CGD) 2, familial hemophagocytic lymphohistiocytosis (f-HLH) 1, HLA class II deficiency 1, Interferon Gamma Receptor-2 deficiency (IFG-R2 Def) 1, Chediak Higashi Syndrome 1. The median age was 4.4 yrs (1.8 to 21yrs). Five donors were HLA matched siblings while one was a HLA-matched parent. The conditioning regimen used consisted of iv busulfan (single daily dose x 2 days), fludarabine 30mg/m²/day x 5 days and anti-thymocyte globulin (Fresenius) 10mg/kg/day x 4 days. The first patient received busulfan in four divided doses rather than a single daily dose. In three patients the busulfan dose was pre-determined by test-dose pharmacokinetics to target a steady state concentration (Css) of 800ng/ml. The actual total busulfan dose was 7.8 ± 1.2 mg/kg (181 ± 20mg/m²) and Css was 713 ± 91 ng/ml. Peripheral blood was the source of stem cells in five patients (83%) while marrow was used in one. The only regimen related toxicity was mild fever following the first dose of ATG. Time to reach an absolute neutrophil count of more than 0.5 x 109/l was d +13 (d +11 to d +20). Graft versus host disease occurred in only one patient (limited cutaneous cGVHD). Four of the six patients have stable mixed chimerism while two have complete donor chimerism. All patients are well and free from their original disease with a median follow up of 2 years (0.25 years to 5 years). RIC conditioning using low dose busulfan, fludarabine and ATG (Fresenius) is extremely well tolerated in a variety of primary immunodeficiency states and results in excellent transplant outcome. Background: Several comorbidity indexes applied in allogeneic hematopoietic transplantation include renal insufficiency based on serum creatinine at transplant, which scores 2 points. Few studies have directly evaluated the significance of prior chronic renal dysfunction (CRD) on the outcome of Allo-RIC. The goal of this study was to evaluate the impact of this aspect on the incidence of postransplant acute renal failure (ARF), graft versus host disease (GVHD), as well as overall survival (OS) and non-relapse mortality (NRM). Patients and methods: We included all patients (n=188) who underwent Allo-RIC at our institution between 1998 and December 2006. Prior CRD was defined as follows: estimated glomerular filtration rate (GFR) < 60 ml/min/1.73m², or patients with prior kidney transplantation or nephrectomy from any cause. ARF was defined as a decrease of at least 25% from pretransplant estimated GFR, calculated by modification of diet in renal disease (MRDR) equation. RIC consisted of fludarabine 150 mg/m² in combination with busulfan 8-10 mg/kg (n=61), melphalan 70-140 mg/m² (n=115), cyclophosphamide 5 g/m² (n=7) or with low dose TBI 2 Gy (n=5). GVHD prophylaxis consisted of cyclosporine alone (n=5) or with Methotrexate (MTX) (n=132) or mycophenolate mofetil (n=51). Results: Forty-seven patients (25%) were considered as having a prior CRD; none of them had required dialysis before Allo-RIC. This group of patients had more frequently an advanced disease (85% vs 65%, p=0.01) and had received a greater number of therapies before allo-RIC (2.8 vs 2.4, p=0.09). In our study, prior CRD did not influence the development of ARF, acute and chronic GVHD, relapse, NRM or OS. ARF occurred in 97 patients (62%) over a 1-year period. Multivariate analysis showed an increased incidence of ARF for patients who received MTX based GVHD prophylaxis (HR 1.9, p= 0.02), more than 2 prior chemotherapies (HR 1.8, p= 0.01), suffering from diabetes (HR 2.2, p= 0.007) and with aGVHD (HR 1.5, p= 0.06). Need for dialysis was uncommon (3% of patients and results were compared to those obtained in a group of 28 patients that received standard dose BU-CY2 or BU-CY3. Majority of patients in both groups were affected with AML. No differences in the two groups were found in percentage of MUD donors (25 % in B-F and 28% in BU-CY, p=0.6), there was no difference in HSC source (BM was used in 65% of B-F group and in 64% of BU-CY group, p=0.9), nor in age (mean age was 38 y. in B-F group and 37 y. in BU-CY, p=0.6). Pts defined as in "early disease" were 29% (B-F group) and 30% (BU-CY), p=0.9; Karnovsky score was not different (p=0.4). Mean CD34+ cell dose infused was 4.5 and 5.2 x 10 6 /Kg in B-F and BU-CY groups (p=0.29). GVHD prophylaxis was standard dose CSA + MTX short course while ATG was used only in transplants from a MUD donor. Mean number of MTX dose received were not different in B-F and BU-CY groups (p=0.58). All patients had primary engraftment and mean time to reach N. count > 0.5 x 10 9 /l was not different in the two study groups when evaluated in the strata of patients receiving BM (p=0.7) or PBSC (p=0.3). However in B-F group max score of oral mucositis (DMS score) was lower during days 7-14 post transplantation (p=0.006), B-F group had also a CHE serum level higher than BU-CY group during week 1, 2 and 3 post-transplant (p=0.002). Moreover B-F group had a lower PLT transfusion requirement (p=0.004). A clinical complication was registered S204 in 100% of BU-CY group while in a lower proportion of pts receiving B-F (42%), p=0.01. Haemorrhagic cystitis was recorded in 18% of patients receiving BU-CY and in only 5% of pts treated with B-F (p=0.2). Treatment Related Mortality during all length of study was encountered in 15% of BU-CY and in 5% of B-F group (p=0.3). 80% and 70% of "early disease" pts ( fig.1 ) were projected to be alive in B-F and BU-CY groups (K-M estimate), however advanced phase pts had much lower survival in both treatment groups. The reduced toxicity found in B-F group allowed us to add to this backbone a 3rd drug (TT) and a phase II study employing BUS-FLUDA-TT is now running in advance phase pts. Acute myeloid leukaemia (AML) encompasses a group of chemosensitive diseases, raising concerns that significant reduction of the intensity of the preparative regimen, may have a negative impact on outcome. This report describes the comparative results of 31 AML patients in CR1 receiving reduced-intensity conditioning (RIC) allogeneic stem cell transplantation (allo-SCT) from an HLAidentical sibling in 2 institutions (Nantes, n=13; and Marseille, n=18) using 2 different global treatment approaches (Table below) . After achievement of CR1, the Nantes approach included administration of 1 or 2 courses of consolidation with high-dose cytarabine (HDC), followed immediately by allo-SCT conditioned with a genuine nonmyeloablative, but highly immunosuppressive RIC regimen including fludarabine, low dose busulfan (4 mg/Kg), ATG (5 mg/Kg) and both CsA and corticosteroids for GVHD prophylaxis (FB1A2 group). The Marseille program aimed to deliver after CR1, in addition to HDC, an autologous SCT followed by allo-SCT conditioned with fludarabine, an intermediate dose of busulfan (8 mg/Kg), low dose ATG (2.5 mg/Kg) and CsA alone for GVHD prophylaxis (FB2A1 group). In the FB2A1 group, 12 patients (67%) could actually receive the planned auto-SCT. With a median follow-up of 47 months, the KM estimate of LFS was significantly higher in the FB2A1 group as compared to the FB1A2 group (P=0.01; 72% vs. 31% at 5 years). Overall, 8 patients (26%;95%CI,11-41%) had relapsed at a median of 320 (range,241-707) days after diagnosis, and the significant difference between the 2 groups in terms of LFS was likely due to a higher risk of leukemia relapse in the FB1A2 group (6/13 vs. 2/18; P=0.07). 5 patients died from toxicity, for an overall incidence of TRM of 16% (95%CI, 6-34%), with this being comparable between the 2 groups (2/13 vs. 3/18;P=NS). Such comparable TRM despite a more intensive approach, translated towards a higher OS in the FB2A1 group as compared to the FB1A2 group (72% vs. 42% at 5 years;P=0.07). After controlling for relevant factors, in the multivariate analysis, actual performance of auto-SCT prior to RIC allo-SCT (P=0.04; RR=4.9; 95%CI, 1.1-22.4), was significantly predictive of an improved LFS. We conclude that in order to achieve optimal results in the setting of RIC allo-SCT for AML in CR1, a comprehensive treatment package including some form of high dose therapy prior to allo-SCT and/or intermediate myeloablation incorporated within the RIC regimen is likely necessary to allow sufficient time for the GVL effect. Post-Transplant Lymphoproliferative Disorder (PTLD) is a serious complication of allogeneic HSCT. Most cases are of B-cell origin with reactivation of EBV due to impaired immune surveillance. Although the incidence is generally low (1-2%), higher levels have been reported in T-cell depleted transplants. Both T cell depletion of the graft and the use of ATG or anti-CD3 are independent risk factors for PTLD. Several studies have suggested that selective depletion of T cells as opposed to depletion of both T and B cells increases the risk of PTLD and may explain the higher risk observed in patients receiving pre-transplant ATG. The use of pretransplant Campath, however, has not been associated with an increased risk of PTLD since unlike other methods of T cell depletion Campath also leads to depletion of circulating B cells. Recently an increasing number of allografts have been performed using Reduced Intensity Conditioning(RIC) protocols, which are designed to produce profound immunosuppression rather than myeloablation and are associated with delayed immune reconstitution. Many RIC protocols incorporate Campath antibodies, although the dose of Campath required to optimise engraftment and immune reconstitution, prevent GVHD and yet preserve GvL is unknown. We and others have attempted to de-escalate the dose of Campath in RIC allografts. Since 1999 we have used a total dose of Campath (1G or 1H) of 50 mg with an acceptable GVHD risk and observed a PTLD incidence of 0/115 RIC sibling allografts and 1/96 in the UD setting. Here we report an increased incidence of PTLD after reducing the Campath dose to 30mg. Following this dose reduction we observed 3 cases of PTLD out of 6 RIC allografts. The patients concerned had received sibling allografts for MDS, CML-2nd CP and CLL. All occurred at around 6m posttransplant when withdrawal of immunosuppression was almost complete. The sites of PTLD were cervical, bronchial tract and nasopharynx and all were treated with Rituximab. Two patients made a full recovery whilst 1 proved refractory and died despite salvage therapy. Following this rise in PTLD cases we have increased Campath dose back to 50mg and there have been no further PTLD cases in >6 months. We speculate that the lower dose of Campath has sufficient T-cell depleting activity but allows EBV-infected B-cells to survive leading to PTLD. Based on our experience, we suggest that de-escalatation of Campath dose is performed with caution and monitoring of EBV-PCR. Relapse of AML/MDS after allogeneic stem cell transplantation (SCT) is associated with poor outcome. A subset of patients (pts) can be salvaged with DLI or with a second SCT. It was previously speculated that pts given RIC may have a better chance to be salvaged than pts failing highdose conditioning. We retrospectively analyzed results of 171 SCTs for AML/ MDS with iv busulfan (ivBu)-based regimens. 58 pts were eligible for myeloablative conditioning and were given standard ivBuCy. 57 were given RIC consisting of fludarabine (F) and ivBu (FB2, 6.4 mg/kg) and 56 were given a modified myeloablative conditioning consisting of F and fulldose ivBu (FB4, 12.8 mg/kg). Median age was 40 (17-64), 60 (43-75) and 52 (18-66) years, respectively (p<0.001). 56% had active leukemia at SCT and 47% had unrelated donors with no difference between the regimens. With a median follow-up of 31 months (4-90), 70 pts relapsed, 20 after BuCy, 25 after FB2, and 25 after FB4, cumulative incidence 38%, 49% and 48%, respectively (p=NS). The median time to relapse was 5.3 (1-59), 3.4 (1-34) and 2.7 (1-30) months, respectively (p=NS); 65%,60% and 64% of all relapses occurred within 6 months of SCT, while 20%, 12% and 8% occurred after more than 2 years, respectively (p=NS). 25 pts were treated with immune-suppression withdrawal alone, 12 were given DLI with or without preceding low-dose chemotherapy, 33 were given intensive therapy either salvage chemotherapy followed by mobilized DLI (n=24) or a second SCT (n=9, 5 from a different donor). Treatment types were not significantly different among the regimens. With a median follow-up of 15 months from relapse (0.5-52), 12 pts are alive, 9 in remission. The most important predicting factor for survival after relapse was the duration of remission. Pts relapsing > 6 months and < 6 months after SCT had a median survival of 7.2 and 1.4 months and estimated 2-year survival of 22% and 2%, respectively (p< 0.001). Survival rates were 20%, 0% and 4% after BuCy, FB2 and FB4, respectively (p=0.04). SCT in refractory disease was also predictive of poor outcome after relapse (p<0.001). Multivariable analysis determined short remission after SCT and conditioning with FB regimens as independent adverse factors with hazard ratios of 3.3 (1.9-6.3, p< 0.001) and 2.0 (1.0-4.0, p=0.04), respectively. In conclusion the notion that pts given RIC can be salvaged more easily if they relapse is not substantiated and should not be a rationale to select RIC over myeloablative conditioning. Since 2002 we introduced low dose Alemtuzumab (MabCampathTM) in combination with cyclosporine A for GvHD-prophylaxis in HLA identical sibling or UD alloTX. Within a pro-tocol we used low dose Alemtuzumab in combination with CSA starting with 2x20mg/d (day-2 and day -1, group 40)(10/2002 -06/2005), deescalating the dose to 2x10mg/d (day -2 and day -1, group 20) (07/2005 -05 /2006) and since June 2006 to 10mg/d on day -1(group 10). On a regular basis we collected data on immunreconstitution day + 30 and + 100 after alloHCT. Patients: Overall, 215 consecutive pts. (group 40 n=74; group 20 n= 65; group 10 n=76) received after a mainly fludarabinebased reduced intensity conditioning (99%; 98%; 98%) a graft (PBSC in 99%) from a sibling (40%; 32%; 30%) or MUD (60%; 68%; 70%). Diagnosis were mainly AML/MDS (40%; 78%; 74%) or lymphoma/MM (51%; 23%; 14%). Remission at TX was mainly advanced disease stage/ > CR1 (92%; 86%; 91%), also reflected by the fact that within each group (45%; 22%; 29%) received their second transplantation, respectively. On day +30 and day + 100 in all available pts. the following lymphocytesubgroups were analyzed for immunreconstitution: CD 19, CD3+4+, CD3+8+ and CD 3-/16/56+=NK cells. Results: lymphocytes are shown in median + (range); *** p<0.0001, ++ p=0.0020 Conclusion: Dose deescalation of Alemtuzumab from 40mg to 10mg absolutely does show a significant faster NK -cell regeneration at day +30 in the 10mg dose group compared to 20mg/40mg: this may provide a better Graft versus Malignoma-effect. Further, despite the low dose no other statistically differences in immunreconstitution for CD 4+, CD 8+ and 19+ lymphocytes at days+30/+100 could be observed: CsA and 10mg Alemtuzumab is as sufficient for immunosuppression as higher doses. Allogeneic transplantation following a reduced-intensity conditioning (RIC) has emerged as an alternative to myeloablative transplantation in pts with myelodysplastic syndrome (MDS). Given the uncertainty regarding the most appropriate conditioning regimen, SFGM-TC conducted a retrospective multicenter study in the attempt to evaluate the impact of conditioning on pts' outcome. The record of 61 pts (37 males) with MDS who received a RIC was reviewed. The median age was 55 years (range: 18-70) According to the FAB classification, 11 pts had RA at diagnosis, of whom one had progressed to REAB and one to AML before transplantation. Thirty-two pts had REAB at diagnosis, of whom 2 had progressed to REAB-T and 7 to AML before transplantation. Twelve pts had REAB-T at diagnosis and 6 CMML, of whom 8 progressed to AML before transplantation. The median time from diagnosis to RIC was 12 months . Conditioning consisted of Fludarabin (Flu) plus busulfan (FB; n=29), Flu plus 2-Gy TBI (F-TBI; n=20) and idarubicin plus aracytine and Flu (FlagIda; n=12). Donors were HLAidentical siblings (n=52) and HLA-matched unrelated (n=9). All pts received peripheral blood stem cells. The median of CD34+ infused cell dose was 5 x 106/kg (0.5-17.3). The median follow-up was 44.7 months (21-85). Estimated 3-year overall survival (OS), progression free survival (PFS), relapse and transplant-relapse mortality (TRM) were 35 %, 27 %, 66 % and 30 %, respectively. Neither of the 3 conditioning regimens used (FB, F-TBI and FlagIda) had impact on patients' outcome. In multivariable analyses, while acute III/IV grade GVHD development was the only factor found to adversely influencing OS (HR=3.6; 95% CI: 1.1-12.2), chronic GVHD development was the only favourably influencing PFS and relapse ratios (HR=0.3; 95% CI: 0.1-0.7 and HR=0.2; 95% CI: 0.1-0.6, respectively). TRM was adversely influenced by male sex of recipient (HR=9.2; 95% CI: 1.5-66.6). RIC is an effective treatment in MDS patients irrespective of conditioning type. While acute III/IV grade GVHD appeared to be detrimental, the benefit effect of chronic GVHD was to be bound to GVL effect as demonstrated by the improvement of PFS and relapse rates in patients who developed chronic GVHD. This study shows no impact of conditioning in pts' outcomes. New approaches with focus on immunosuppressive treatment are needed to enhance the GVL effect with an acceptable risk of GVHD. (1) Several studies have shown that the substitution of oral Intravenous Busulfan (Bus) with i.v. Bus appears to provide a better efficacy/tolerability ratio due to predictable linear kinetics with the i.v form. We report the clinical and pharmacokinetics results of a myeloablative conditioning based on the association of iv Bus and Cyclophosphamide (Cy) in adults with hematological malignancies undergoing allogeneic HSCT. 28 patients, with a median age of 41 yrs had AML (18),ALL (2),CML (5), MDS (1),MM (2) .14 were considered as standard risk (1st CR of AL, 1st CP of CML, MDS) and 14 high risk (all the others). Donors were HLA identical siblings (13), unrelated(14) and syngeneic (1); the source of HSC was BM(14) or PB(14).The preparative regimen consisted of i.v. Bus, followed, after a 24 hrs rest, by Cy 200 mg/kg(15) or 120 mg/kg(11) or Melphalan 140 mg/sqm (2) . Unrelated transplants and sibling males with female donors received also ATG-F,15-30 mg/kg. Bus was administered 4 times daily x 4 days at 0.8mg/Kg of ideal body weight (IBW), for those with a BMI < 30; for higher BMI dosing was calculated on the adjusted IBW (AIBW); if actual body weight was less than IBW, the first was used. PK studies, with dense sampling, were conducted after dose 1, 9 and 13. No seizure prophylaxis with phenitoin was given. At a median follow-up of 35 months,TRM for the standard and high risk group were 7% and 29%, respectively, while the relapse risk was 15% and 38%. OS was 86% and 43% at 3 years. AGVHD II-IV occurred in 25% while cGVHD in 43% of cases. With regard to toxicity, severe mucositis occured in 36% of cases; the mean of peak bilirubin levels was 7.8 mg/dl in those receiving the 200 mg/kg Cy and 5.7 in those receiving 120 (p=0.007); statistically significant differences between the two groups were also observed for alkaline phosphatase and ALT levels; only one case of reversible VOD occurred. The median value of AUC after dose1 (AUC1) and dose 9 (AUC3) were 1063 and 1174 microM/L/min; interpatient and intrapatient CV were 30% and 13.8%, respectively. No correlations were found between AUC and toxicity, engraftment, GVHD and relapse. Targeting performance according to AIBW was 67%.This study shows that toxicity from the combination of i.v. Bus and Cy can be acceptable also with doses higher (200) than those published, even if greater but reversible hepatic toxicity has been observed.Finally, the study confirms a good PK profile without dose monitoring expecially when adjusted ideal body weight was used. Use of reduced intensity conditioning (RIC) has enabled larger numbers of patients to benefit from a potentially curative allograft who were precluded due to toxicity of myeloablative conditioning. Our study included 140 (myeloid :71: lymphoid 69) patients with median age of 52 yrs, who received reduced intensity Campath conditioning allografts were analysed. Median follow up of these patients was 17.4 months. Pretransplant parameters considered were age, sex, stem cell dose, type of transplant (sibling vs MUD), stem cell source(BM vs PBSC), CD34 dose, CD3 cell count , previous stem cell transplant, type of disease (myeloid vs lymphoid),conditioning regimen and disease status at transplant (CR vs not CR). There were significantly more patients achieving fully donor T cell chimerism on day 90 with unrelated transplants compared to sibling (43% vs 21%, p:0.01), higher level of CD3 count compared to lower levels (0-4.2 ) and older patients compared to younger age (< 46yrs: 21%FD vs >46 34%FD p:0.09). Multivariate logistic regression of the above baseline covariates found that prior SCT, type of transplant and age group were all significant in predicting fully donor T cell chimerism at day 90. The overall survival in the 1st year was 76%. Univariate analysis of baseline characteristics shows evidence that bone marrow stem cells(p:0.005), unrelated transplants(p:0.02), males(p:0.04), lymphoid disease(p:0.01) and not achieving CR(p:0.001) all significantly increase the risk of death, however in the multivariate analysis the type of transplant, sex and lymphoid patients without CR (or late disease) were found to be predictors for poor overall survival. Disease free survival rate at 1 year is 61% with median disease free survival at 2.9 years. Univariate analysis of baseline characteristics shows evidence that bone marrow stem cells (p: 0.009), lymphoid disease (p: 0.01), males (0.006) and not achieving CR (p: 0.002) all significantly increased the risk of disease progression. Multivariate analyses of baseline characteristics showed type of transplant, sex and lymphoid with no CR as adverse predictors for disease progression. This study has identified factors influencing full donor chimerism as well as overall survival and progression free survival in reduced intensity Campath conditioning transplants and may enable us to optimize our transplant strategies. Clofarabine/Ara-C treatment combined with reducedintensity conditioning for allogeneic stem cell transplantation in patients with high-risk, relapsed, or refractory acute leukaemia S. Buchholz*, J. Krauter, E. Dammann, S. Ehrlich, M. Port, E. Weissinger, M. Stadler, M. Eder, A. Ganser Hannover Medical School (Hannover, DE) Introduction: Allogeneic stem cell transplantation (SCT) is the most effective treatment for high-risk, refractory or relapsed AML and MDS. However, most AML and MDS patients (pts) are older than 50 years and not eligible for conventional myeloablative conditioning. Combined cytoreductive therapy with reduced-intensity conditioning regimen (RIC) is increasingly used for these patients, but limited anti-leukemic activity still results in significant relapse rates. Clofarabine, a nucleoside analogon with properties of fludarabine and cladribine, has shown remarkable activity in relapsed AML and ALL. We therefore evaluate the antileukemic and immunosuppressive effects of clofarabine in the context of RIC allogeneic SCT for pts with high-risk AML, ALL, and MDS. Methods: Up to November 2007 ten pts (median age: 62, range 39 -69, male/female: 8/2) with high-risk, relapsed or refractory acute leukaemia or MDS (7 AML, 2 ALL, 1 MDS; high-risk cytogenetics n=2, relapse n= 4, refractory disease n=4) were treated with clofarabine 30 mg/m² and cytarabine 1000 mg/m² for 5 days for cytoreduction. After 3 to 5 days of rest, RIC was given with 4 Gy of total-body irradiation, 80 and 120 mg cyclophosphamide/kg for related and unrelated donors, respectively, and ATG. All patients were transplanted with unmanipulated G-CSF mobilised peripheral blood stem cells from matched unrelated (n = 7) or related (n = 3) donors. GvHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. Results: Overall, treatment was well tolerated and all but one patient are alive and in haematological remission (follow-up median day + 90, range day +14 to 214). All patients engrafted (median day + 18 for ANC > 0.5 G/l, range: day +12 to 31). So far no acute Graft versus Host-disease > I° has been observed with follow-up > 100 days for 6 pts. Nonhematological toxicity included reversible hepatotoxicity (increase in ALT and AST CTC II -III°) in 8 pts, and mild to severe reversible hand-foot-syndrom in 8 cases. One patient suffered from veno-occlusive disease and infectious complications and died due to multiorgan-failure on day + 33. Conclusions: Antileukemic therapy with clofarabine and Ara-C followed by RIC allogeneic HSCT is feasible in elderly patients with high-risk acute leukemia or MDS with normal engraftment and no increase in GvHD. Non-hematological toxicity of this regimen mainly liver and skin is acceptable. Updated results of this ongoing pilot study will be presented. Most of the reports on RIC suffer from insufficiencies: small populations, short follow-up, heterogeneities in the RIC intensity or the donor source making difficult to draw conclusions for a given population. We report here 100 pts with hematological malignancies (HM) treated with allo-SCT after the same RIC in a single center from 2000 until 2006. All pts received oral busulfan (8mg/m), thymoglobulin (2.5 mg/kg) and Fludarabine (150 to 180 mg/m) (FBT conditioning). All grafts were PBSC from a match sibling donor. All pts received post-graft CSA. Median age was 50 (18-64). Hematopoietic cell transplantation comorbidity index (HCT-CI) was 0, 1-2 and > 2 in 31, 39 and 23 of the 93 evaluable pts. Diagnoses included acute leukemia (39%) (AML=37; ALL=2) (CR1: 32; CR2: 4; Not CR: 3), Myeloid Malignancies (16%) (CML (N=9: CP1: 9); MDS (N=7: CR: 2; Not CR: 5), Lymphoid malignancies (45%) (NHL (N=20: CR=3; PR=13; Prog: 3)); MM (N=17: RC=1; RP=16); HD (N=5: CR=1; PR=1; Prog: 3); CLL (N=3: PR=1; Prog=2). 53, 14 and 33 pts were in CR/CP (CR1/CP1: 45; CR2:8), progression or stable disease. On July 2007, minimal and median surviving patient follow-up were respectively 6 and 34 months. All but one engrafted reaching full donor lymphoid chimerism prior to day 100 in 85% of the cases. 55 pts presented aGVHD (G1: 12; G2: 22; G3: 12; G4: 9) for a cumulative incidence (CI) of G2-4 aGVHD of 43% (33-53); 91 pts were evaluable for cGVHD with a 79% (71-87) CI (Lim= 20%; Ext: 80%). TRM CI was respectively 5%, 14% and 19% (11-27) at 3, 12 months and overall without any impact from age or HCT-CI. TRM was strongly associated with aGVHD occurrence (TRM CI: grade 2-4 aGVHD: 37% (23-51); grade 0-1 aGVHD: 7% (0-14): p<.01). Relapse CI was 15% (8-22) at a median of 169 days (30-769). Disease control was statistically associated with higher cGVHD occurrence (Relapse CI: cGVHD: 10% (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) ; no cGVHD: 42% (14-70): p=.01). 5 year overall survival (OS) and LFS probability estimates are 62 % (50-72) and 60% (48-72) with a plateau starting after 3.5 years. HCT-CI did not show any influence on outcome. Outcome was similar for the pts above or under 50 and for the different diagnoses (5 year OS for AML (N=37: 64% (46-78), NHL (N=20: 63% (41-82), MM (N=17: 44% (22-70). These data confirm that FBT RIC, combining myeloablation (Busulfan) and limited dose of Thymoglobulin, is efficient in a wide population in term of age and diagnosis conducting to sustained long term OS and EFS with limited TRM. Patients and methods: Median age was 56 years (range 19-69). Haematological diseases were: acute leukemia 28, aggressive non-Hodgkin lymphoma 22, indolent lymphoma 18, Hodgkin lymphomas 19, multiple myeloma 21, idiopathic myelofibrosis 8. Pretreatment included autologous SCT (50) or ≥ 3 previous chemotherapy regimens (49). Disease status before SCT was: complete remission or very good partial remission (41), partial response (26 ), refractory disease (49 ). Comorbidity index according Sorror was ≥ 1 in 57 patients (49%). Conditioning regimens were based on thiotepa plus cyclophosphamide and fludarabine ( 83 ), fludarabine plus busulphan or melphalan ( 15 ) and 2 Gy Total Body Irradiation plus fludarabine (18) Stem cells came from sibling donors ( 53) or from matched unrelated donors (63). Ninety-seven patients (84%) received peripheral blood stem cells (PBSC) .We analyzed the influence of patient and disease clinical features before SCT and of transplant procedures on TRM and OS by means of the univariate and multivariate analysis. Results: The cumulative incidence of engraftment was 93%. The estimated 1-and 3-year TRM were 23% and 26% respectively. The estimated 3 year-OS and disease-freesurvival (DFS) were 53% and 48%. Grade II-IV acute graftversus-host disease (GVHD) and chronic GVHD developed in 35% and 45% of the patients. Twenty-one patients (18%) presented a proven or probable mycotic infection after SCT. Unfavourable prognostic factors that significantly decreased OS were: Sorror comorbidity index ≥1, refractory disease before SCT, grade II-IV acute GVHD, chronic GVHD and development of mycotic infections. Sorror comorbidity index ≥ 1 , grade II-IV acute GVHD, chronic GVHD and development of mycotic infections significantly increased TRM rate, whereas refractory disease and diagnosis of acute leukemia before SCT were the only factors associated with a significant decrease of DFS. Conclusions: Our experience demonstrated that, despite the application of RIC regimens, presence of comorbidities and development of GVHD and fungal infections significantly affected TRM. Moreover, refractory disease had a significantly poorer control after transplant. Introduction: The role of anti thymocyte globulin in GVHD prevention is well established, but it is also known that anti thymocyte globulin induces leucopenia and thrombocytopenia and as such might theoretically delay engraftment. Additionally, fast platelet engraftment is a marker for good hematopoietic stem cell graft function. We have prospectively compared the effect of thymoglobulin on engraftment of granulocytes and platelets following a single nonmyeloablative conditioning protocol in a randomized study. Methods: 33 consecutive patients undergoing SCT were conditioned with the NST protocol (I.V.fludarabine 30mg/m²/day (days -10 to -5) and I.V. busulfex (3.2 mg\kg on days -6 and -5), with or without I.V. Thymoglobulin according to randomization (days -4 to -1). Thymoglobulin dosing schedule: a cumulative dose of 7.5 mg/kg r-ATG was given with the following program 0.5 mg/kg day -4, 2.0 mg/kg day -3, 2.5 mg/kg day -2, 2.5 mg/kg day -1. Patients were followed for engraftment of leukocytes and platelets. Results: only one patient rejected the graft (conditioned without thymoglobulin, NS). 28 patients are evaluable for engraftment data (2 patients -conditioning stopped, 2 -underlying disease progression, 1 -toxicity). There was no significant difference with the CD34+ or CD3+ cells graft composite of between the groups. The average time to ANC engraftment was similar between the groups, 14.5±2.8 and 14.8±2.7 days for the thymoglobulin and control groups respectively ( figure 1, p=0.4) . However, the average time for platelet engraftment was significantly shorter in patients receiving thymoglobulin as part of the conditioning. Platelet recovery occurred 10.1±1.2 and 12.2±2.9 days for the thymoglobulin and control groups respectively (figure 2, p=0.018). This was expressed in the number of platelets infused. The patients conditioned with thymoglobulin received only 1.6±0.6 platelet infusions were as the patients in the control group received 6±1.8 infusions (Av±SE, p=0.012). Conclusions: in the non-myeloablative setting, thymoglobulin induces a clinically significant faster platelet recovery and does not delay ANC engraftment. Allogeneic hematopoietic cell transplantation after reducedintensity conditioning (Allo-RIC) is a potentially curative strategy in high risk low grade and aggressive non-Hodgkin lymphomas (NHL). However, the timing and optimal conditioning regimen is still unknown. The aim of this study was to evaluate the long term clinical and molecular responses in 30 consecutive high risk or relapsed/refractory NHL patients (pts) who underwent Allo-RIC transplantation (26 matched siblings, and 4 unrelated donors) between March/1999 and July/2007. Conditioning consisted of S209 antithymocyte globulin combined with: 2 Gy total body irradiation and fludarabine (n=14), fludarabine and busulfan (n=9), 8 Gy total nodal irradiation (n=5), fludarabine and melphalan (n=1) and thiotepa/fludarabine (n=1). The stem cell source was mobilised peripheral blood progenitor cells (n=27) and bone marrow (n=3). The median age was 52.5 years (range: 31-63), 23 pts were male and 7 female and diagnoses were: chronic lymphocytic leukemia (n=13), follicular lymphoma (n=7), mantle cell Lymphoma (n=4), diffuse large cell Lymphoma (n=5) and mycosis fungoides (n=1). The median number of prior regimens was 2 (range: 1-8) with 12 pts in CR, 7 in CRu, 9 in PR and 2 refractory at transplantation. Minimal residual disease was assessed by consensus IgH PCR and multiparametric flow cytometry. Results: All pts engrafted (1 had a secondary graft rejection and underwent a second Allo-RIC) and 93% reached stable full donor chimerism. Four pts received donor lymphocyte infusions for mixed chimerism (n=3) or relapse (n=1). Transplant related mortality was 3%. Overall, after a median follow-up of 38 months (range: 2-106), 26(86.7%)/30 pts are alive with a 5-year progression free survival of 75%. 4 patients died, 1 of acute chronic-graft-versus-host disease (GvHD), 2 of chronic GvHD and 1 of secondary AML. Grade 1-2 and 3-4 acute GvHD developed in 43% and 10% of pts respectively. Limited chronic GvHD occurred in 3(10%)/28 pts and extensive in 6(21%)/28 pts. Chronic GvHD resolved in 4/6 pts and one developed a post-trasplant lymphoproliferative disorder resolved with rituximab. Out of 26 evaluable pts, 23(88,4%) achieved and remain in clinical/molecular remission. In conclusion, our results confirm that Allo-RIC in chemo-sensitive NHL is associated with a sustained clinical and molecular remission in a high percentage of patients, with acceptable GvHD and good performance status Reduced intensity conditioning (RIC) hematopoietic stem cells transplantation (HSCT) has been developed in order to obtain long term disease control using graft-versus-leukemia (GVL) effect in a variety of patients unfit for a standard transplantation procedure. In order to assess the long term disease control after a RIC HSCT we retrospectively reviewed the medical records of 43 consecutive AML and MDS high risk patients transplanted in a single center between 2000 and 2006. 97% of pts were older than 50, median age of 56(range 33-67) or had severe comorbidities -invasive fungal infection (2pts), heart failure (1pt) and relapse after a previous standard HSCT (1pt). High risk criteria were those of IPSS for MDS and in AML pts: failure of first line chemotherapy (8 pts -18.6% in CR2), partial remission (6 pts -14%), poor risk cytogenetics (14pts -32.5%) or molecular biology features(Flt3+ -3pts -6.9%), secondary leukaemia (8pts -18.6%), detectable MRD (1pt -2.3%). 39 pts received a Fludara based conditioning regimen and 4 pts received 6 Gy TBI + cyclophosphamide. Stem cell source was an IS in 29 pts( 67%), a MUD (11 pts-26%) and unrelated cord blood (3pts-7%). Immunosuppression consisted in CSA and/or MMF or short course MTX. Engraftment was achieved in 41 pts (96%). Cumulative incidence of acute and chronic GVHD was 30% (13 pts). With a median follow up of 43 mo (range 12-94) 22 pts (52%) are alive and 18 in CR and 21 died because of disease progression -16 pts (37%), sepsis in the context of severe GVHD -2 pts(4.6%), respiratory failure -2 pts(4.6%), fulminant hepatitis -1 pt (2.3%) psychiatric disease -1pt(2.3%). The estimated OS for the whole group was 52% at 3 years and 48% at 4 years. For the 33 AML patients the 3 years OS was 48%. The estimated 3 years OS was 49,6% for pts receiving a graft from an IS and 64,3% from a MUD. Estimated 3 ys DFS was 75% in pts experiencing GVHD and 29% in the absence of GVHD. Durable CR was achieved in 57,9% of the pts in CR1 (19pts) compared to 28.6% of pts beyond CR1. Conclusion: Our results support the use of RIC HSCT to overcome the high risk of myeloid malignancies for patients which are not candidate for a conventional allogeneic HSCT. When compared to recent reported results (Pautas, Blood Nov 2007; 110: 162) , OS after RIC HSCT is better then for patients receiving chemotherapy alone. Transplant in CR1, GVHD and graft from a MUD seem to be associated with a better outcome. Background: Relapse and graft failure after hematopoietic stem cell transplantation (HSCT) are major problems. Outcomes for patients who have undergone re-transplantation after failure of previous HSCT are generally poor. Although HSCT with reduced intensity conditioning (RIC) has decreased transplantation-related mortality (TRM), it is controversial whether RIC is beneficial for survival after retransplantation. Patients and methods: We retrospectively reviewed the medical records of 231 patients who underwent allogeneic HSCT in our institute between 1988 and 2007. Thirty patients (13%) had a history of autologous (n = 16, auto-group) or allogeneic (n = 14, allo-group) HSCTs. The reason for retransplantation in the auto-group was relapse or continuous disease. One patient in the auto-group had undergone autologous HSCT twice. In the allo-group, 7 patients underwent a second allogeneic HSCT and 2 patients underwent a third allogeneic HSCT due to relapse after previous allogeneic transplantation. One patient needed a third HSCT due to secondary graft failure after bone marrow transplantation. Four patients needed re-transplantation due to graft failure after cord blood transplantation. Results: Median post-re-transplant follow-up time for surviving patients was 950 days (range, 114 to 3,124 days). Five-year overall survival and progression-free survival rates were 49% and 40%, respectively. Causes of death in 14 patients included progressive disease (n = 5), infections (n = 6) and veno-occlusive disease (n = 3). Cumulative incidences of relapse-related mortality and TRM at 2 years after retransplantation were 23% and 36%, respectively. All six patients with follicular lymphoma who have a history of autologous HSCT are alive in remission. Patients with aggressive lymphoma had worse outcomes than those of oatients with other diseases. Univariate analysis showed that no previous allogeneic HSCT (p = 0.03), being in partial remission (PR) or complete remission (CR) at HSCT (p = 0.02), and RIC (p = 0.02) resulted in better overall survival after re-transplantation. PR or CR at HSCT (p = 0.02) resulted in lower risks of relapse and progression. Conclusion: Encouraging results of HSCT with RIC were seen after failure of previous HSCTs, especially for patients with remission at re-transplantation. Myeloablative allogeneic transplantation has been reserved to young well-fitted patients (pts) due to its high toxicity. Nonmyeloablative(NMA) conditioning regimens reduce this toxicity preserving the graft-vs-tumor effect. We present our experience in advanced lymphomas. Patients/methods: Since year 2000, we used iv fludarabine 125 mg/m² (days -7 to -4) and iv melphalan 80 mg/m² (day -2) as NMA conditioning for heavily pretreated lymphomas, aged>55y or with co-morbidities. GVHD prophylaxis consisted in cyclosporine and MTX. Stem cells were obtained from HLAidentical siblings. Cyclosporine tappering from day +50 was initiated if mixed chimerism or persistent disease were present. We evaluate engraftment rate, toxicities, transplant related mortality(TRM), chimerism kinetics, GVHD incidence and disease free and overall survival(DFS and OS). Results: 18 lymphomas(8 follicular, 4 MCL, 4 PTCL and 2 DLBCL) were transplanted (co-morbidities 4, age>55y 6, heavily pretreated 10). Median age was 52y (31-61), 10 were males, median treatment lines were 3(1-5) including 4 autotransplanted. Ten pts (55,6%) were in CR, 4 had chemosensitive PR(22%) and 4 refractory disease(22%). All pts engrafted with ANC>500 on day +14(10-20) and platelets>20.000 on day +12(9-33). Toxicity until day +100 included febrile neutropenia in 6, severe mucositis 2, hepatic toxicity 2, VOD 1, hemorragic cystitis 1 and arrythmia in 1. There were 2 deaths before day +100: 1 refractory CHF and 1 Pseudomonas´sepsis (TRM 11,1%) and 2 deaths beyond +1 year due to infections during chronic GVHD. Complete chimerism(CC) was present on day +30 in 38%, in 93% pts on day +90 and 10/10 evaluated pts were on CC after 1 year. Acute grade II-IV GVHD ocurred in 37,5%, grade III-IV in 25%. Chronic GVHD affected 10/15 pts(67%), extensive in 40%. Only 1/10 CR pts relapsed and 4/4 in PR reached CR after achieving CC on day +90. All refractory pts died (4/4): 2 toxic deaths and 2 lymphoma progressions. Median follow-up was 55 months(28-83) with DFS of 61% and 66,7% OS at 5y. Twelve pts persisted alive in CR with chronic GVHD in 4(33%). Predictors for DFS and/or OS were follicular/mantle cell histology vs others(p<.01) and CR/PR at transplant vs refractory(p<.01). Conclusions: NMA conditioning with fludarabine and low dose melphalan in advanced lymphomas has good toxicity profile, high engraftment rate and long term disease control if pts were chemo-sensitive pretransplant, specially in more indolent histologies. Allogeneic hematopoietic stem cell transplantation (HSCT) for refractory and multiply relapsed Hodgkin's Lymphoma (HL) carries considerable procedure related toxicity and infectious complication risks and many patients do relapse. Furthermore, the use of matched unrelated donors has been limited and sometimes associated with high TRM. We report a small cohort of 8 multiply relapsed (median of 3 relapses) HL patients all having had prior autologous or syngeneic HSCT. Patients were transplanted using a reduced intensity conditioning and a graft T-cell depleted by Campath 1H in the bag followed by T-cell add-back and early donor lymphocyte infusion (DLI). Two of them had chemoresistant disease at time of transplantation. Median time from diagnosis to allogeneic HSCT was 52 months (range 26 to 99 months). Donors were matched related in 3, matched unrelated in 3 and mismatched unrelated in 2. Median follow-up was 704 days (range 76-2198). All patients engrafted and there was no treatment related mortality (TRM). Only one patient developed acute graft-versus-host disease (aGVHD) grade II and none had chronic GVHD before receiving DLI. 5 patients received DLI from 3 months after HSCT for residual disease, progression or mixed chimerism, with a persistent response in 4 of them and a transient disease regression in another. Four of them developed GVHD. Three patients developed chronic extensive GVHD, one limited cGVHD. The two patients with chemoresistant disease died due to disease progression, one despite the development of cGHVD. At last follow-up 6 patients are alive, 5 in complete remission. T-cell depletion with T cell add-back allows for allogeneic HSCT in an increasing number of patients with multiply relapsed HD and may be associated with low TRM risks in spite of a high proportion of unrelated donors. However, patients with refractory disease may not benefit from such strategy. Background: T-cell depletion is effective strategy for prevention of GVHD, but it is associated with an increased incidence of graft failure and relapse after RIST. Alemtuzumab (Campath1H) and antithymocyte globulin (ATG) is the most commonly used in T-cell depletion in RIST. However, the chimerism kinetics about Campath1H versus ATG is not eludicated in RIST. We retrospectively analyzed early kinetics of engraftment following RIST and compared the results of two regimens. Methods: Data were collected retrospectively on 30 patients who underwent RIST (Campath1H, n=15; ATG, n=15) between 2003 and 2006. Donors were HLA-identical siblings in 17 patients, matched unrelated donors in 7 patients and others were HLA mismatched unrelated donors. PB served as stem cell source in 18 patients and BM in 12 patients. GVHD prophylaxis consisted in cyclosporine A or tacrolimus. Results: Patient characteristics were not different between two groups. Neutrophil recovery date was slower in patients treated with Campath1H (p=0.004) although platelet recovery was not different between two groups. CMV antigenemia developed in 11 patients after Campath1H use and four patients after ATG treatment (p=0.011). However, the incidence of CMV disease was not different. Graft failure rate was not different. ATG group showed the increased rate of grade 3~4 acute GVHD and chronic extensive GVHD although there was no statistical difference. Also, even with the difference of infused T-cell dose, T-cell donor chimerism within first month was reported to be delayed in ATG conditioning compared to Campath1H. On day 14, donor chimerism of T cells was 87.4% and 52.5% in patients with Campath1H and ATG, respectively (p=0.012). On the contrary, donor chimerism of non T-cells was 86.8% and 81.2% in patients with Campath1H and ATG, respectively (p=0.64). Similar results were obtained on day 28. T-cell donor chimerism was 78.7% and 50.2% (p=0.06) in patients with Campath1H and ATG, respectively. During 100 days, treatment related mortality (TRM) is 2/15 after ATG treatment because of acute GVHD and infectious cause. Overall survival (OS) and Disease free survival (DFS) at 1 years was 47.2% , 62.5% in Campath1H group, 47.2%, 57.4% in ATG group respectively. Conclusion : In retrospective analysis, patients with Campath1H who received RIST show a decreased GVHD and early donor T-cell chimerism. Our study indicates that Campath1H may offer an effective degree of T-cell depletion without relapse or TRM. Treosulfan has been used in previous studies splitted over 3 days before allogeneic transplantation reaching a maximum of 42g/m² of treosulfan total. Since a prolonged exposure of treosulfan has been shown to be more effective in myeloablation we distributed treosulfan in equal doses over 5 days reaching either a total dose of 40g/m² or 50g/m² in combination with fludarabine 30mg/m² i.v. d-6 to day -2. GVHD prophylaxis consisted of Cyclosporine A and short MTX. Additional ATG was given in recipients of MUD. 21 patients with a median age of 51 years (19-69) were treated with 5x8 g/m² of treosulfan (n=6) and with 5x10g/m² (n=15). Patients suffering from AML or secondary AML (6 each) and NHL (3) were judged to have a high relapse risk. In addition 6 CML pts. were transplanted. The median Sorror score of the whole group was 3 (range 0-6). At the time of transplant 8 pts. were in CR and 13 pts. were not in CR or in chronic phase (CML). Donors were either MRD (5), MUD (10) or misMUD (6) . After a median follow up of 404 days (range 107-635) an overall survival of 61% and an event-free survival of 41% has been reached. The non-relapse mortality at 100 days was 10%, reaching 27% after 1 year. The relapse rate at the same time was 45%. Treosulfan/fludarabine related CTC° 3/4 toxicity was observed for gastrointestinal symptoms (9.5%;2/21), metabolic/laboratory (19%; 4/21), edema and pain (4.8%; 1/21), infection in neutropenia (52.4%; 11/21), and rash (4.8%; 1/21). The distribution of observed toxicities showed no relation to the different treosulfan dose levels. In comparison with the previously observed toxicities and the non-relapse mortality in the 3x10-14 g/m² treosulfan conditioning regimens the 5x8-10 g/m² treosulfan application does not seem to be more toxic or to have a higher nonrelapse mortality. Survival and relapse rate are difficult to compare due to the small patient group and individual risk factors of the pts. However this retrospective analysis may indicate that dose and schedule modification of treosulfan may be possible. Further studies are necessary to define the possibilities of such dose modifications or dose escalation. Allogeneic stem cell transplantation (SCT) can cure patients with therapy related MDS or AML. To predict outcome we analysed 486 patients (pts) with t-MDS or t-AML underwent allogeneic SCT and were reported to the EBMT registry. The median age of the pts was 40 years (r., 3-69) and primary disease were solid tumor (n=167), malignant lymphoma(n=141), myeloproliferative syndrome (n=12), acute leukaemia (n=32), aplastic anemia (n=16) and autoimmune disease (n=1). The median time from primary diagnosis to t-MDS/t-AML was 54 months (r., 1-416). Diagnosis were: RA/RARS (n=28), RAEB (n=49), RAEB-T (n=44) and t-AML (n=308). In a multivariate analysis, being in " non CR" was a significant risk factor for relapse (HR:2.20; 95% CI: Abnormal cytogenetic negatively influenced relapse (HR: 1.78; 95% CI: 1.03-3.07; p=0.04) and EFS (HR: 1.41; 95% CI: 1.01-1.99; p=0.05). Based on these three variables we create a simple risk factor: age > 40y =+2, abnomal cytogenetic=+1 and non CR=+2, which allows to distinguish four risk categories: low (0-+1), moderate(+2), strong (+3 to+4) and high (+5). This risk model separates overall survival from 60% (low risk) 45% (moderate) to 31% (strong) and 21% (high risk) ad non-relapse mortality from 21% (low risk) to 25% (moderate) to 42% (strong) and 44% (high). The impact of transfusion-dependency on the outcome of patients with de novo myelodysplastic syndromes receiving allogeneic peripheral blood stem cell transplantation after myeloablative conditioning U. Platzbecker* (1), U. Germing (2) Introduction: The majority of myelodysplastic syndromes (MDS) patients become red blood cell transfusion-dependent during their clinical course. In fact, the presence or absence as well the extent of red blood cell transfusion-dependency (RBC-TD) has been recently shown to add significant prognostic information for an individual MDS patient treated with supportive care (BSC) only. Given the fact that allogeneic hematopoietic cell transplantation (HCT) is the only curative option for MDS patients, the aim of this study was to elucidate the role of RBC-TD on patient outcome. Methods: We report results of a retrospective multicenter US-German-Austrian study investigating 172 patients with de novo MDS (RA(RS) n=85, RAEB n=52, RAEB-t n=16, MDS/AML n=19) with either IPPS LOW (n=2), INT-1 (n=31), INT-2 (n=37) or HIGH (n=30) corresponding to WPSS very low (n=0), low (n=21), intermediate (n=37), high (n=65), very high (n=10). All of them underwent allogeneic myeloablative conditioning followed by peripheral blood stem cells (PBSC) from related (n=90) or unrelated donors (n=82). Forty-one out of 172 patients were RBC-TD before PBSCT. The median age of the patients was 51 years (range 18-68). Results: With a median follow-up of 37 months the estimated 2-year overall survival (OS) was 56% for all patients and 64 %, 50% and 34% for patients according to IPSS INT-1, INT-2 or HIGH, respectively (p=0.03 for INT-1 vs. HIGH). The OS of all patients was not different when comparing patients displaying vs. not displaying RBC-TD prior to PBSCT (54% vs. 61%, p=0.7). This holds also true when analyzing patients according to IPSS separately. However, when grouping patients according to cytogenetic IPSS groups, there was a trend for a significant better OS in favor of patients being not RBC-TD in the intermediate (83 vs. 54%, p=0.1) but not good or poor group. Additionally, the median OS significantly became worse in patients (n=57) with a ferritin level above 1000 µg/l before PBSCT (57% vs. 38%, p=0.03), whereas donor or CMV status had no impact. Conclusion: These data suggest that the negative prognostic influence of RBC-TD in de novo MDS patients receiving BSC might persist in a subgroup of patients even in the presence of myeloablative allogeneic PBSCT. Additionally, excessive iron overload seems to have negative prognostic impact on patient outcome. We retrospectively evaluated 180 patients with myelodysplastic syndrome (MDS) who underwent allogeneic stem cell transplantation from an HLA-identical sibling donor reported to the GITMO registry from 1996 to 2006. The aim of the study was to compare the post-transplantation outcome of MDS patients according to two types of conditioning: reducedintensity conditioning (RIC, 89 patients) and standard myeloablative conditioning (SMC, 91 patients). Patients in RIC group received fludarabine-based regimens without any addition of in-vivo T-cell depleting agents, while patients in SMC group received busulphan and cyclophosphamide or total body irradiation and cyclophosphamide. No difference was noticed between the two groups as far as WHO categories, cytogenetics, IPSS risk, time from diagnosis to transplant, disease status at transplant and pre transplant transfusion dependency are concerned. As compared to patients in SMC group, patients receiving RIC were older (medina age 52 years vs. 41 years P<0.01) and more frequently transplanted with peripheral blood stem cells (P=0.01). Moreover, patients in RIC group showed a trend to an increased Sorror's comorbidity score (P=0.07). The twoyear overall survival (OS), relapse incidence (RI) and transplant-related mortality (TRM) were similar in the two groups (RIC vs. SMC: OS 33.9% vs. 42.9%, RI 40.1% vs. 32.4%, TRM 26.7% vs. 31.3%, P=ns). In multivariate analysis, type of conditioning, stem cell source, age and Sorror's score did not show any significant impact on OS, RI and TRM. The introduction of RIC for patients with MDS at high risk for TRM (advanced age, high Sorror's score and non T-cell depletion) leads to a reduction in TRM and an acceptable relapse incidence. These results may set the grounds for prospective trials comparing RIC with standard myeloablative regimens from an HLA-identical sibling donor in younger patients with MDS. Optimized transplant care allows more myelodysplasia (MDS) patients access to potentially curative stem cell transplantation. Early relapse complicates 30% of advanced MDS. Pre-transplant induction chemotherapy reduces relapse incidence but with increased transplant related mortality (TRM). 5Azacytidine (5Aza) in advanced MDS provides a survival advantage in part by an early marrow blast response, delay in AML progression, and is well tolerated. We examined the use of pre-transplant cytoreduction with 5Aza in 25 patients with IPSS Int-2 or High risk MDS and secondary/therapy-related AML to determine its impact on overall and transplant outcomes. Patients received at least 4 cycles of 5-Aza. Patients with improving marrow blast counts continued on 5Aza(n=13), those with greater than 30% blasts received FLAG (n=7), and 5Aza patients with progressive blast counts crossed over to FLAG (n=5). Patients achieving a marrow CR (IWG 2000) proceeded to transplant. 25 patients (median age 58) were followed from the initiation of treatment. With a median follow up of 605 days (range 42 to 1898), event free survival(EFS) of both 5-Aza and FLAG treated patients was comparable (MS not yet reached) in contrast to shorter EFS of 5-Aza patients crossing over to FLAG (MS 18 months). Tests of equality over groups are not significant, although data suggest better early EFS in the 5-Aza alone group. Of the initial 25 patients, 21 underwent allogeneic transplant. Four were ineligible due to refractory disease or comorbidities. Patients received targeted busulfan-based preparative regimens; 9 received matched related and 12 matched unrelated donor transplant. All engrafted at the expected timepoints, and were 100% donor at day +30. With median follow up of one year, EFS of the 5-Aza and FLAG groups have not yet reached MS; both are far superior to the 5-Aza to FLAG group (MS 7 mo). All patients in both the overall and transplant cohorts receiving chemotherapy had a higher rate of early TRM, relapse and overall mortality compared with those receiving 5-Aza alone. The inferior EFS of the 5-Aza to FLAG cohort identifies patients at higher risk for early relapse. This pilot study represents the longest continuing follow up of patients treated with this strategy, and their survival, relapse and TRM rate compare favorably with historical outcomes for MDS. These promising results will be confirmed in planned larger multicenter trials in the US. (2), R. Trenschel (2) , L. Kordelas (2) , M. Dischkowski (2) , N. Steckel (2) Treosulfan (Treo) based conditioning prior to allogeneic blood stem cell transplantation has been recently been introduced with the intention to develop of a dose-intense but at the same time well tolerable conditioning regimen. We have retrospectively analyzed Treo-based compared to standard myeloablative preparative regimens in MDS patients. Methods: In a bi-centre retrospective analysis we report the outcomes of 48 pts. with MDS. 29 pts. (60%)received a TBI and 19 pts. (40%) a treosulfan based conditioning. The median age was 47.5 years (range 18-68). 37 pts. (77%) suffered from a primary and 11 pts. from a therapy-related MDS. In the TBI group only 2 pts. had a therapy related MDS while in the treosulfan group 10 pts. had a primary and 9 pts. a therapy-related MDS. 18 pts. were transplanted from a MRD and 30 pts. from a MUD. Treosulfan was combined in 16 pts. with fludarabine and in 3 pts. with cyclophosphamide. After a median follow up of 4 years (range 1.5-7) a significant superior survival was obtained after Treo. The overall survival (OS) after Treo was 74% after 1 and 3 years vs. 62% and 37% after TBI (p=0.0417). Relapse free survival (RFS) after Treo was 74% after 1 and 3 years vs. 55% and 31% after TBI (p=0.0105). The relapse incidence (RI) after Treo was 0% after 1 and 3 years vs. 14% and 34% after TBI (p=0.0067). Non-relapse mortality (NRM) at 100 days, 1 and 3 years after transplant with Treo vs TBI was 5 vs. 14%, 26 vs. 31% and 26 vs 35% (n.s.). The significantly better results of Treo vs. TBI may be in part due to an inhomogeneous distribution of primary and therapy-related MDS. When primary MDS pts. were compared a trend but no significant better results for Treo could be shown (at 3 years OS Treo vs. TBI 60% vs 32% (p=0.303); RFS 60% vs 29% (p=0.2093) and RI 0% vs 33% (p=0.0558)). The results strongly suggest that Treo-based conditioning might have a significant advantage over conventional TBI-based conditioning in MDS pts. Further prospective, randomized studies are necessary to confirm this observation. Background: The incidence, risks and timing of t-MDS and t-AML vary among studies and different diseases. Only few prospective reports are available on cytogenetic results of patients undergoing HDC and ASCT. OBJECTIVES: Primary objective:1. Identify t-MDS and t-AML, both pre-and post-ASCT and 2. Identify clonal cytogenetic abnormalities. Secondary objective: 1. Prescreen patients prior to ASCT, 2. Use results in clinical decision making, 3. Preserve these specimens for future research 4. Evaluate the economic impact and feasibility of this testing as standard pre transplant workup. Patients and methods: Patients with relapsed or refractory diffuse large cell lymphoma (DLCL) and Hodgkins lymphoma (HL) who were potential candidate for HDC ASCT were eligible in this prospective trial. Unilateral bone marrow biopsies and aspirates were performed and analysed by conventional GTG-banding and fluorescence in situ hybridisation (FISH). In selected cases, FISH was used to detect abnormalities involving chromosomes 5, 7, and deletions of 20q and 11q23. Results: From April 2005 to November 2007, 95 patients were enrolled. Forty-five patients did not have HDC ASCT due to progression (9), refusal (4), noncompliance (4) or other reasons (28) Conclusion: Routine cytogenetics analysis in patients with minimal pre-treatment and short follow-up is unlikely to detect the target objectives. Cost-saving and financial impact cannot be evaluated as target population need to be selected more carefully. Large number of patients with no HDC ASCT also influenced the financial analysis. Objective: to evaluate factors associated to the development of chronic graft-versus-host disease (cGvHD) after hematopoietic stem cell transplantation (HSCT) from unrelated donor (UD) and to assess prognosis of patients with cGvHD. Patients and methods: data were retrospectively analyzed from patients (pts) aged ≥ 18 years old who underwent HSCT from UD in Italy between June 1999 and June 2006 for a haematological malignancy. HLA and clinical data were collected from IBMDR and GITMO registries, respectively. Informed consent was obtained from each participating centre. Patients who survived more than 100 days were evaluable for cGvHD. Following factors were analyzed: pt's and donor's age, gender, CMV serology; year of HSCT, disease status at HSCT, source of stem cells, conditioning (myeloablative vs. reduced-intensity), use of TBI, ATG as GvHD prophylaxis, aGvHD grade II-IV, HLA-matching between pt and donor. Factors significantly associated to cGvHD (p<0.20) were included in the final multivariable model. Univariate timedependent Cox regression analysis for survival (OS), relapserelated death (RRD) and non-relapse mortality (NRM) was performed. Semi-landmark plot was constructed to illustrate effect of cGvHD on survival (figure 1). Results: from a total of 1271 pts, 959 pts were alive at day +100 after HSCT, therefore evaluable for cGvHD; median age at HSCT was 38 years (range 18-66); median follow-up since HSCT was 508 days (range 100-3100); median day of onset of cGvHD was +144. Factors significantly associated to cGvHD occurrence in multivariate analysis were: donor's CMV serology, year of HSCT, TBI-conditioning regimen, aGvHD grade II-IV and use of ATG as GvHD prophylaxis. In timedependent Cox regression analysis, hazard ratio (HR) of mortality for limited cGvHD compared to pts without cGvHD was 0.43 (95% C.I. 0.31-0.61, p<0.0001); no survival difference between pts with extensive cGvHD and those without cGvHD was observed (HR= 0.90, 95% CI: 0.66-1.24, p=0.90). Analysis for RRD and NRM between pts with extensive cGvHD and those without cGvHD showed HR= 0.47 (95% CI: 0.24-0.75, p=0.003) and HR= 1.79 (95% CI: 1.20-2.67, p=0.004), respectively. Conclusion: occurrence of limited cGvHD is associated to better OS compared to pts not developing cGvHD or those with extensive cGvHD. Extensive cGvHD is associated to less RRD but more NRM than pts without cGvHD, and finally comparable OS. HLA matching between donor and pt did not affect cGvHD occurrence. Background: Bone marrow-derived mesenchymal stem cells (MSCs) modulate immune responses in vitro and in vivo. Methods: Within the European Group for Blood and Marrow Transplantation (EBMT) expansion consortium, we have adopted a common MSC ex vivo expansion procedure and performed a multicenter, phase II study to treat steroidresistant, severe acute graft-versus-host disease (GvHD) in 55 patients. Findings: The median dose of bone-marrow derived ex vivo expanded MSCs was 1.4 (range 0.4-9) x10 6 cells/kg. Twentyseven patients received 1 dose, 22 patients received 2 doses and 6 patients received 3 to 5 doses of MSCs obtained from HLA-identical sibling donors (n=5), haplo-identical donors (n=18) and third-party HLA-mismatched donors (n=69). No side effects were seen during and immediately following MSC infusions. Thirty patients showed a complete response (55%) and 8 showed improvement, the overall response rate being 69%. The response rate was 80% in children and 60% in adults (p=0.28). Response rate was not influenced by MSC donor HLA-match. Three patients had recurrent malignancy and 1 developed de novo acute myeloid leukemia of recipient origin. Patients with a complete response had a lower transplantation related mortality 1 year after MSC infusion as compared to partial or non-responders (37% versus 72%; p=0.002) and higher overall survival 2 years after hematopoietic stem cell transplantation (52% versus 16%; p=0.018). Twenty-one patients are currently alive. Eight patients have chronic GvHD. Interpretation: Infusion of in vitro expanded MSCs, regardless of the type of donor, may be an effective therapy for patients with steroid-resistant acute GvHD. The IL23R gene on chromosome 1p31 encodes a subunit of the receptor for the proinflammatory cytokine interleukin-23. Recently, IL23R could be identified as a novel inflammatory bowel disease susceptibility gene. The exchange of arginine with glutamine at position 381 of IL23R causes a blockade of the IL-23 signaling pathway which is associated with a reduced risk of both Crohn ´s disease and ulcerative colitis. IL-23 is a pivotal cytokine in the differentiation of T cells into inflammatory, IL-17-producing T cells. Because of the requirement for IL-23 in autoimmune disease we hypothesized that IL23R Arg381Gln reduces the risk of graft-versus host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). 182 donors and 182 children (median age, 12 years) who underwent allogeneic bone marrow (n=114) or peripheral blood stem cell transplantation (n=68) in a single center were genotyped of IL23R for rs11209026 (c.1142G>A, p.Arg381Gln) using TaqMan real-time polymerase chain reaction. The donor was HLA-matched unrelated in 60% of transplants and HLA-identical related in 30% of transplants. Conditioning regimen was myeloablative in all cases. Two forms of post-transplant immunosuppression predominated, cyclosporine A and methotrexate in 60% of transplants and cyclosporine A alone in 27% of transplants. The IL23R Arg381Gln variant was present in 20 of the 182 donors (11.0%) and in 18 of the 182 patients (9.9%). Interestingly, we found a significantly reduced incidence of acute GVHD grade II-IV in patients who were transplanted from a donor with this specific single nucleotide polymorphism (5.0% versus 33.3%; p=0.009). Furthermore, there was no severe acute GVHD grade III-IV if the polymorphism occurred in the donor (0% versus 14.2%). In five of the donor-patient pairs the variant was present in both individuals and no acute GVHD was observed. The occurrence of the IL23R polymorphism, in either donors or recipients, had no significant impact on chronic GVHD, relapse rate, treatment related mortality, and overall survival. In conclusion, the single nucleotide polymorphism of the IL23R gene in the donor is associated with a significant decrease of acute GVHD after allogeneic HSCT in childhood. Therefore, the IL23 signaling pathway seems to play an important role in the pathogenesis of acute GVHD. Blockade of the IL23 receptor by a monoclonal antibody could be a rational therapeutic strategy for acute GVHD. Purpose: The aim of this retrospective study is to assess the usefulness of endoscopic evaluation and factors predicting the clinical outcome of gastrointestinal acute GvHD (GI-GVHD) in Japanese transplant recipients. Patients and Methods: 294 patients who underwent allogeneic stem cell transplantation at Keio BMT Program from November 1998 to December 2006. Patients experienced rejection, the second or subsequent transplantation, or death before engraftment were excluded, and 230 were eligible for analysis. Both upper and lower GI endoscopies were performed in all recipients with persistent GI symptoms. Biopsies were taken from regions of mucosal abnormalities as well as normal appearing mucosa. Immuno-staining for CMV was also performed in all biopsy specimens. GVHD prophylaxis was attempted with MTX and cyclosporine (for sibling transplant) or tacrolimus (for unrelated transplant). Grading of acute GI GVHD was done according to the consensus criteria. Results: Overall incidence of AGVHD with GI involvement was 32.8% (Grade II with GI involvement 20.3%, Grade III 6.1%, and Grade IV 6.4%). Most frequently observed endoscopic finding of upper and lower GI-GVHD was mucosal edema and derangement of vasculature (88%) followed by redness (64%) and erosion/ulcer (44%). Mucosal edema was not observed in CMV gastroenteritis without GI-GVHD. 10 patients with typical endoscopic and histological findings of GI-GVHD had biopsy specimen positive for CMV. In multivariate analysis, an early dose reduction/discontinuation of carcineurin inhibitor due to its side effect was identified as a risk factor of developing GI-GVHD. The factors associated with GI -GVHD not responding to 2 mg/kg/day steroids included patient age over 40(P=0.03), female to male transplant(P=0.04), incomplete immunesuppression with carcineurin inhibitors(P=0.01),Grade III/IV GVHD(P=0.001), lower GI involvement(P=0.004),and noninfectious fever at the diagnosis of GI GVHD (P=0.0001). Presence of CMV positive epithelial cells did not adversely affect the treatment outcome. Nine out of ten those patients were successfully treated with steroids and DHPG. In multivariate analysis, only the non-infectious fever at the diagnosis remained as a significant factor (P=0.01). Conclusion: The results of this study suggested that endoscopic findings are useful to diagnose GI-GVHD, and some clinical features could identify GI-GVHD requiring more intensive immunosuppression as a front-line therapy. Chronic Graft Versus Host Disease (cGVHD) is associated with high morbidity and mortality. We have previously shown that patients with scleroderma-like cGvHD have agonistic antibodies activating Platelet Derived Growth Factor Receptor (PDGF-R); The up-regulation of PDGF-R intracellular pathway leads to increased collagen synthesis, contributing to pathological lesions observed in scleroderma-like cGVHD. In vitro models show that imatinib strongly inhibits basal or PDGF-induced synthesis of collagen in Scleroderma fibroblasts, suggesting a potent antifibrotic effect in vivo. We have thus opened a pilot study for the treatment of refractory cGvHD with low dose imatinib. All patients had severe cGVHD with scleroderma-like features, having failed at least 2 lines of treatment, with active disease and measurable involvement of skin or other districts. The association with ongoing low dose steroids or low dose cyclosporin was allowed. Patients received Imatinib, starting from 100 mg/day, increasing to 200 mg/day in absence of severe adverse reactions. Toxic effects and response were evaluated after 3 and 6 months of therapy with Imatinib. Fifteen patients have been treated (9 male, 6 female; median age 29); 8 had Acute Leukemia, 2 CML, 2 MM, 2 thalassemia, 1 breast cancer; 11 received transplant from identical sibling and 4 from alternative donor; stem cell source was BM in 8 ,PBSC in 6,CB in 1. Median duration of cGVH 37 was months .; main GVH target were: skin in 14, bowel in 5 and lung in 4. Four patients experienced minor extrahematological toxicity and one patient a grade 3-4 toxicity; no relevant haematological toxicities have been observed; anemia was seen in a patient with CML. No TRD were observed and treatment was interrupted in one patient. With a median Follow up of 8 months, 14 patients are evaluable for response on day +180, with an ORR of 86% . Improvement was seen both in sclerodermatous GvHD and chronic bronchiolitis. Two patients with osteo-myalgia had complete resolution of the symptom. A general amelioration of subjective was also seen in most patients. In conclusion: significant responses can be obtained with low dose imatinib in patients with advanced refractory chronic GvHD. Tolerance of Imatinib therapy was good, both in children and adults. A GITMO polycentric prospective study is S216 ongoing in order to confirm these data in a larger number of patients. (1) Introduction: The biological role of host and graft B-cells during allogeneic stem cell transplantation (allo-SCT) has so far been only partially elucidated. Based on preclinical data which suggest that residual host B-cells serve as antigenpresenting cells after (allo-SCT) and mediate GVHD rather then graft versus leukemia effect (GVL), we tested whether depletion of host B-cells prior reduced intensity conditioning (RIC) allows to reduce a major complication of RIC: chronic GVHD. Patients and methods: 150 patients treated with nonmyeloablative allo-SCT for various haematological diseases were included in a retrospective, single centre, cohort study. We compared outcome, in terms of overall survival, progression free survival and the incidence of acute and chronic GVHD, between patients who were treated with rituximab within 6 months prior to their allo-SCT and patients who did not receive rituximab. Results: Twenty patients (13%) received rituximab prior to allo-SCT. The follow up in both groups was at least 48 months. No significant differences in factors that could influence the occurrence of GVHD, such as donortype or HLAmismatching, between both groups were detected. Rituximab prior to allo-SCT significantly reduced extensive chronic GVHD in a multivariate analysis (20% decrease; p=0.04). Surprisingly, there was no correlation with the incidence of acute GVHD grade II-IV or grade III-IV. Rituximab prior to the allo-SCT did furthermore not hamper GVL effect as measured by progression free survival. Conclusion: Depletion of host B cells with rituximab prior to allo-SCT decreases the incidence of chronic GVHD and does not impair the GVL-effect. These results support to further explore B-cell depletion in RIC. Mesenchymal multipotent stromal cells (MSC) are post natal stem cells that differentiate into many cell lineages and can control allogenic T lymphocyte (TL) stimulation. Such a property has been used to inhibit steroid-resistant graft versus host disease (GVHD) that may occur after allogenic hematopoietic stem cell transplantation (HSCT). Recently we showed that MSC-driven inhibition was associated, in vitro, with TL transmigration through the MSC layer (J Cell Physiol ahead pub, sept 2007). We further investigated this issue to assess whether other mechanisms were involved in TL inhibition. CFSE-labeled, highly purified TL were seeded over allogenic MSC previously plated in regular 24-well plates, or in chambers whose basement consisted in 0.4 or 3.0-micron pore-sized membranes (mm), in presence or absence of TL mitogens (PHA+IL-2, anti-CD3 and -CD28 mAb immobilized on beads). TL proliferation was assessed after 5 days by monitoring CFSE dilution. In coculture with 3mm, TL migrated from 48 hours on through the MSC layer and the membrane. After 5 days of culture with mitogens, TL recovered in the lower chamber had significantly proliferated, and were only marginally inhibited by MSC. When 0.4mm were used, TL did not cross the membrane and remained tightly associated with MSC, as trypsination was necessary to harvest them. Despite their localization nearby the MSC, these TL responded efficiently to mitogens, and were only marginally inhibited by MSC. By contrast duplicate cultures performed in regular hard plastic-bottomed wells showed that MSC were fully inhibitory in such a setting. These observations demonstrate that TL inhibition occurs only when transmigration targets TL under MSC bound to a waterproof surface. When TL transmigrate under MSC that adhere to a membrane allowing metabolite exchange (0.4mm), or when TL can run away from MSC after transmigration (3mm), no significant inhibition ensues. This suggests that TL have to be sequestrated for at least 48 hours in a hermetically sealed volume under MSC in order to be inhibited. This is consistent with the observation that MSC induce TL inhibition via the activation of the intracellular enzyme indoleamine 2,3 oxygenase that metabolizes Ltryptophan. Small sealed volumes under -or engulfed in -MSC certainly represent a privileged environment to induce efficient L-tryptophan depletion and LT inhibition. These data suggest that infused MSC may transiently build up a similar microenvironment to inhibit GVHD in leukemic patients. Background: On day +100 after an allogeneic HSCT, most patients are ready to be definitively discharged from a transplant center to return home. However, transplant mortality (TRM) is not over yet . Predicting chronic graft versus host disease (cGvHD) and TRM would be clinically useful, and has been reported using platelet counts. Aim of the study: To identify laboratory values predicting chronic GvHD and TRM on day+100 after an allogeneic HSCT Patients and methods:. We studied 808 patients with hematologic malignancies and with laboratory data available on day+100 . The diagnosis was acute leukemia (n=326), chronic myeloid leukemia (n=274), lymphoma (n=51), mylodisplastic syndromes (n=75) other (n=82). Patients were allografted between 1990 and 2006. Donors were HLA identical siblings (n=543) or alternative donors (n=265). Variables analyzed where blood counts and full biochemistry. Univariate analyses were performed using each of the laboratory data. Significant variables were entered in multivariate COX analyses, together with donor/recipient gender and age were then performed, with TRM as an end point. Results: The cumulative incidence (CI) of transplant related mortality was overall 17%. In multivariate analysis the following laboratory values on day+100 were independent predictors of TRM: platelet counts (Plt) , gamma glutamyl transferase (gGT) and cholinesterase (CHE). We then cut these variables at the median values: Plt 100x10 9 /L, gGT 50 IU/L, CHE 3500 IU/L, and assigned a score of 1 for unfavourable values (Plt<100; gGT>50; CHE<3500). Patients were thus scored as 0,1,2,3. The CI of chronic GvHD and TRM were significantly (p<0.0001) different in the 4 groups, as shown : chronic GvHD was 27% in patients with score0 and 52% in patients with score4. Conclusions: This study confirms that transplant mortality beyond day+100 can be predicted by simple laboratory tests. Patients with a Score 0-1 have a low risk of developing cGvHD / TRM , and could be eligible for early discontinuation of immunosuppressive therapy. Patients with Score 2-3 are at high or very high risk of TRM and may be considered for intensified immunosuppression. The involvement of mediators of the innate immune response in immunological processes after allogeneic haematopoietic stem cell transplantation (HSCT) has been an important finding. Toll-like receptor (TLR) signalling plays a key role in the innate response to pathogens. More recently, endogenous TLR ligands released upon cell damage were discovered and TLR4 influence on cross-presentation and anti-tumor responses has been demonstrated. The MyD88 adapter-like (Mal/TIRAP) protein is critically involved in TLR 2 & 4 signalling. A polymorphism leading to a non functional variant (Ser to Leu180) has been reported to be protective in infectious conditions due to a reduced inflammatory response. As bacterial products like LPS and possibly endogenous TLR ligands contribute to initiation and maintenance of graft versus host disease (GvHD), we hypothesised that one or two non functional Mal variants present in donor or recipient cells might affect outcome after allogeneic HSCT. Mal genotyping at position Ser180 was performed in 611 donor recipient pairs from the Eurobank cohort receiving full or reduced intensity conditioning transplants. Allele frequencies were in line with published data; 72% of recipients and 71% of donors were homozygote wild type (wt) Ser180/Ser180, 26% and 25% were Ser180/Leu180 and 2% and 4% Leu180/Leu180. There were no significant differences between the cohorts in age, gender mismatch, immunosuppression, conditioning and transplant center. Unexpectedly, 70% of recipients homozygote for the mutant allele relapsed (HR 6.1, p=0.016 multivariate analysis). Cumulative incidence of relapse was 60% and 75% at 2 years and 5 years respectively for this group of patients as compared to 24% and 28% at 2 years and 5 years for patients with Ser180/Leu180 or Ser180/Ser180 genotype. The overall incidence of chronic GvHD but not acute GvHD was reduced (58% vs 46%) when the donor possessed the mutant allele. This finding was significant in univariate (p=0.02) but not multivariate analysis (p=0.12). In multivariate analysis, donor possession of the mutant allele was associated with decreased TRM(HR=0.48, p=0.038). However, 2 and 5 year projected survival was not different, probably because of the higher incidence of relapse (see table) . Our findings suggest an important role for TLR signalling in GvH and GvL processes after allogeneic HSCT. Moreover, the recipient genotype might be useful to identify patients being at high risk for relapse. For thirty years corticosteroids have been the first line treatment for Acute Graft Versus Host Disease (aGVHD). High doses of steroids after Haematopoietic Stem Cell Transplantation (HSCT) have detrimental effects on opportunistic infections and loss of bone-and body-mass. At our center 115 patients with 116 grafts have been treated with oral 8-methoxypsoralene and ultraviolet light type A, (PUVA) for aGVHD, (one patient was retransplanted after two years due to relaps). Steroid resistant GVHD was present in 73% of the cases when starting PUVA-therapy. The median time from onset of aGVHD to PUVA treatment was 14 days and the median time to PUVA treatment was 38 days post HSCT. Judging the response up to fourteen days after end of PUVA treatment, 90% had complete or partial response in the skin while 6% progressed. The aGVHD responded completely in 57% of the cases, and progressed in 16% of the cases. In 59% of the cases, a corticosteroid taper with over 50% was due fourteen days after PUVA treatment. In a multivariate analysis for siblings, a higher nucleated cell dose, and for matched unrelated donors, high risk disease was significant for complete response in the skin. A lower aGVHD grade at start predicted a complete response in the skin by both groups. In the whole material, low aGVHD grade at start of PUVA, high risk disease, male donor sex and malignancy predicted for total response in the skin. Patients starting with less than 50% of the body surface (BSA) involved in cutaneous aGVHD, stage + to ++, had significant better oneand two year survival than patients with over 50% of BSA affected at start of PUVA-therapy. Recipients of sibling grafts did not have better survival than recipients of unrelated grafts. Complete response in the skin and of the aGVHD in total, predicted for better overall survival. To conclude, PUVA used as an early addition to corticosteroids for the treatment of cutaneous aGVHD, spared steroids and induced a complete response in the skin of 74%. Microbial components activate innate immune responses via Toll-like-receptors (TLR) and NOD proteins. In previous studies, we have shown a decreased severity of intestinal GvHD with increased survival in mice lacking TLR2, but not TLR4. Furthermore, increased faecal E. coli loads within 11 days after BMT were associated with more severe GvHD. The present study was undertaken to elucidate the underlying mechanisms. Methods: After conditioning with treosulfan and cyclophosphamide, C57BL/10 wildtype (Wt) and C57BL/10 TLR2-/-mice (H2-Db) received a MHC-mismatched (H2-Dd) BM graft and T cells from Balb/c donors. Animals were sacrified and the following parameters were assessed in the colon at close intervals during the first 20 days by in situ immunohistochemistry: macroscopic and histologic signs of intestinal GvHD, E. coli numbers in faeces, amounts of CD3+ T cells, FOXP3+ TREGs, POX+ granulocytes, CASP3+ apoptotic cells. Results: The time sequence of intestinal GvHD was as follows: (1) Conclusion: Our data suggest that three factors are critical for epithelial damage during the induction phase of GvHD: early apoptosis of epithelial cells induced by CD3+ cytotoxic effector cells, presence and translocation of pro-inflammatory bacterial species (in particular E. coli), and, finally, the attraction and infiltration of granulocytes after activation of the innate immune system (in particular via TLR2). These findings might point the way to the modulation of the intestinal gut flora and the innate immune system in order to prevent severe GvHD in humans. Introduction: Allogeneic hematopoietic cell transplantation (aHCT) proves as an effective and often life-saving treatment for a broad array of hematological malignancies, but its major limitation remains acute graft-versus-host disease (GVHD). Utilizing a murine aHCT model of bioluminescence imaging we have demonstrated that acute GVHD can be separated temporally and anatomically in a GVHD initiation phase confined to secondary lymphoid organs and a subsequent GVHD effector phase in peripheral target tissues (intestinal tract, liver and skin). It has been proposed that host conditioning is crucial in the activation of alloreactive T cells determining the tissue tropism in acute GVHD. To evaluate the impact of host conditioning on the selective migration of alloreactive T cells we transplanted non-myeloablative, myeloablative conditioned, and non-conditioned recipients. Methods: Balb/c wild type or Balb/c Rag-/-or Balb/c Rag-/-cGC-/-or NK cell deficient F1(Balb/c x FVB/N) recipients (H-2d or H-2dxq; no conditioning, non-myeloablative: 4Gy or myeloablative: 8Gy respectively) received allogeneic luciferase transgenic FVB-L2G85 T cells (H-2q) and bone marrow cells. In vivo bioluminescence imaging visualized the proliferation and migration of alloreactive T cells and guided flowcytometric, immunohistochemical, immunofluorescence and real-time PCR analyses. Results and conclusions: Host conditioning exacerbates GVHD target tissue infiltration by alloreactive T cells correlating with the degree of irradiation. Non-conditioned recipients showed less GVHD development. Host conditioning did not significantly influence alloreactive T cell activation, expansion and homing receptor acquisition during GVHD initiation. However, T cell recruitment by GVHD target tissues emerged to be important during the effector phase in aHCT recipients. In secondary cell transfer experiments we found that in vivo primed alloreactive T cells migrated to GVHD target tissues in conditioned secondary recipients irrespective of the lymphoid priming sites. In non-conditioned secondary recipients few alloreactive T cells migrated to GVHD target tissues and homed preferentially to secondary lymphoid organs. We show that recruitment by inflamed GVHD target tissues during the effector phase dominates the migration of alloreactive T cells whereas homing instruction of alloreactive T cells during the initiation phase plays a minor role in GVHD pathogenesis. , 39 males and 26 females. The underlying diseases were CML (n = 37), AML (n = 11), ALL (n = 8), SAA (n = 6), PNH (n = 1) and MDS (n = 2). The sources of HSCT used were bone marrow (n = 37) and peripheral blood (n = 28). The histopathological specimens were blindly and independently examined by two observers and consensus results were considered for statistical processing. Results: A significant correlation was founded between the histological grades proposed by Horn and Shulman et al. (P <0.0001). The criteria "Peri-ductal lymphocytic infiltrate with exocytosis", in Shulman's classification, was correlated with survival (P = 0,004). "Peri-ductal infiltrate" showed significant correlation with the localized or extensive clinical cGvHD (P = 0.04). Conclusion: Our results suggest similarity between final diagnoses obtained by both Horn and Shulman's classification. The periductal lymphocytic infiltrate was the most important histological criteria for the severity of oral cGVHD. The lymphocytes migration through ductal epithelium could have negative influence in outcome. Our results may be useful for understanding oral cGvHD etiopathogenesis and severity damage of the cGVHD in MSG. Introduction: Chronic graft-versus-host disease (cGVHD) is the most common cause of poor outcomes after haematopoietic stem cell transplantation (HSCT), while the pathophysiology of cGVHD remains poorly understood. Since both cGVHD and autoimmune disease share clinical features, we speculated that autoimmune disease related genes may be candidate cGVHD-related genes. IL-17 producing T lymphocytes have been recently shown to comprise a distinct lineage of proinflammatory T helper cells, termed Th17 cells, that are major contributors to autoimmune disese. IL-17 has been designated IL-17A to indicate that it is the founding member cytokine family, which now includes IL-17A-F. IL-17A and IL-17F were originally associated with memory CD4 T cells and have more recently been specifically linked to the Th17 lineage. The human IL-17A and F gene is located on 6p12. Here we attempt to determine whether IL-17A and F gene polymorphisms influenced the cGVHD of patients receiving an allo-HSCT from an HLA-identical sibling donors. Material and method: This study investigated the association between polymorphisms of IL-17A and IL-17F genes and the incidence of cGVHD. We analyzed 111 cases of Japanese HLA-matched sibling recipients and their donors who underwent HSCT. Four single nucleotide polymorphisms (SNPs) in the IL-17A and F gene (rs12204016, rs721430, rs763780, rs602670,) were selected from public databases using the International Hapmap Project Generic Genome Browser. Result: The IL-17F rs721430 C/C genotype was less frequent in patients with cGVHD (C/C vs C/T+T/T = 25.0% vs 75.0%) than in those without cGVHD (C/C vs C/T+T/T = 44.1% vs 55.9%; odds ratio, 0.42; P = 0.0357). We investigated the association of acute GVHD and this polymorphism, and there was no association between recipient or donor genotype and acute GVHD. There is no relationship between cGVHD and IL-17A SNP (rs12204016) in pt and Donors. Paitent's haplotype of IL-17F(rs602670-rs763780-rs721430)T-C-G+T-T-G has been correlated with low incidence of cGVHD(p= 0.0053). There was no association between donor haplotype and cGVHD. Conclusion: The current findings indicate that the IL-17F rs721430C/C genotype and T-C-G+T-T-G haplotype have a protective effect against cGVHD. Background: Modern approaches to predict the occurrence/severity of acute graft-versus-host disease (aGvHD ) are needed. This study aimed to identify a molecular signature correlating with occurred early aGvHD. Therefore, we performed a longitudinal prospective study comparing expression changes of a 48 genes pattern associated with immune alloreactivity. Methods: We used a TaqMan® Low Density Array based on comparative CTdd CT method on Applied Biosystems 7900HT to perform relative quantification of cDNA. We obtained peripheral blood mononuclear cells (PBMC) from 20 patients with haematological malignancies before and sequentially after allogenic stem cell transplantation (A-HSCT) with HLAidentical sibling (n=18) or MUD donors (n=2). PBMC serial samples were collected between 10-90 days after SCT from patients who developed aGvHD (+) and from those did not (-). Four patients did not experience aGvHD, others had II-III grade one. In this case, molecular profiling before and after initiation of steroid therapy was investigated. Results: Pattern of gene expression changes following HSCT. We divided genes that were altered at onset of aGvHD into 3 different categories. In the first group the largest number of genes was heavily down-regulated. Second group included genes lightly down-regulated; in the third group genes exhibited unclear behaviour. First group included a pattern genes that became undetectable: multiple chemokines, inflammatory factors, IFN-gamma inducible genes, such as signal transducer, activator of transcription-6, interferon regulatory factor-1 and 8. Following steroid therapy, expression level of all these genes was subsequently increased. In the second group, expression profile of IL-12A, IL-4, IL-10, IL-18, IL-6 cytokines showed mild reduction but were however over-expressed than normal control. On the contrary, in patients aGVHD(-) neither type 1 nor type 2 cytokines could be detected as compared with their baseline values. In all patients aGVHD(+), Fox-p3 showed negative expression level together to inducible T-cell-co-stimulator factors such as CD52 and CD83. Conclusion: Comparing normal and aGvHD+ group, significant gene expression changes could be identified. We believe that our array system may be used in future to identify transplant recipients not at risk for developing aGVHD. These results are encouraging for the establishment of a diagnostic tool useful to pre-emptive therapy of aGvHD. Using HLA-matched unrelated donors (URD), outcome following hematopoietic stem cell transplantation (HSCT) is approaching that using HLA-identical sibling donors. Do some leukemia patients benefit from an URD transplant, because of a stronger graft-versus-leukemia (GVL) effect? We analyzed the outcome in 941 URD (8/8 HLA-allele matched) transplants, compared to 3158 HLA-identical sibling transplants for acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML), receiving myeloablative conditioning between 1995-2004 and reported to the CIBMTR. Transplant-related mortality (TRM), relapse and leukemia-free-survival (LFS) were compared for each disease. In a Cox proportional hazard regression model including variables important for outcome, acute and chronic GVHD were added as timedependent variables. Using URD compared to HLA-identical sibling transplants, TRM was increased in early AML (40% vs. 24%, p=0.001), CML (38% vs. 31%, p=0.02) and advanced AML (44% vs. 31%, p=0.01). In multivariate analysis, TRM was associated with URD in AML and CML, but not ALL (p<0.001). Relapse was similar in univariate analysis using URD or HLA-identical sibling transplants for all diagnoses in early, intermediate and advanced disease. In the Cox model, relapse comparing URD and HLA-identical sibling transplants was the same in ALL and CML. For AML, relapse was increased in the URD recipients (p=0.0015). In patients without acute or chronic GVHD, multivariate analysis showed similar probability of relapse, but decreased LFS for URD transplants for all three diagnoses. In multivariate analysis, LFS was decreased in patients with AML and CML using URD, but comparable to HLA-identical sibling transplants in patients with ALL. In conclusion, relapse was the same using URD compared to HLA-identical sibling transplants, suggesting similar GVL effect. TRM was increased and LFS was decreased using URD in patients with AML and CML, but not ALL. Therefore, an HLA-identical sibling donor should be the first choice in AML and CML. Introduction: Chronic graft-versus-host disease (cGVHD) is a major complication of allogeneic stem cell transplantation that shares similarities with autoimmune diseases. We have studied the pathology of both clinical and experimental cGVHD. In particular, we investigated the extent of fibrosis in internal organs of both patients and mice with cGVHD. Methods: Histology sections of 20 patients with cGVHD were analysed. The extent of collagen deposition in spleen, liver, lungs, gut and skin was measured quantitatively by Sirius red staining and compared to controls. In the mouse model cGVHD was induced through injection of human PBMC into immune deficient RAG2-/-ãc-/-mice. Collagen depositions in sections of skin, spleen, liver and lungs were quantitatively measured in mice with symptoms of acute or chronic GVHD and with a control group. Results: In cGVHD patients moderate to severe fibrosis was observed in skin, spleen, lung and gut. The liver was not affected. In cGVHD collagen constituted 14.7% of lung tissue while this was 2% in controls (p=0.011). For the spleen this was 13% in patients with cGVHD and 2.4% in patients in the control group (p=0.039). For the gut this was 24% in patients with cGVHD and 11% in controls (p=0.038). In the experimental models fibrosis was most prominent in the spleen and to a lesser extent in the skin and lungs of mice with cGVHD. In mice with cGVHD collagen constituted 15% of lung tissue, while this was 3% in control mice. For the spleen this was 33.0% in cGVHD, 16.4% in acute GVHD and 13.7% in controls (p=0.002). Conclusion: The fibrosis seen in chronic GVHD appears to be much more generalized than previously thought. In patients skin, lungs, spleen and gut may all be affected. These symptoms are shared by the RAG2-/-ãc-/-cGVHD model. We conclude that systemic fibrosis is a hallmark of cGVHD and that the mouse model offers a relevant platform to study its pathogenesis. This study was supported by the Dutch Cancer Society (KWF 2006-3685) The Complement system consists of several serum proteins and cell membrane receptors that belong to the innate immunity. Its activation leads to the production of proinflammatory and cytotoxic factors. We investigated here its role in GVHD in both humans and a mouse model. Thirty nine patients allografted for diverse haematological malignancies in our institution entered prospectively the study. Extensive exploration of the Complement system was carried out before and sequentially after allogeneic haematopoietic stem cell transplantation (HSCT). We then analyzed Complement activation in a parent (C57BL/6, H-2b) to F1 [(C57BL/6xDBA2), H-bd] GVHD mouse model. Serum C3 Complement fraction and serum albumin protein (SAP) were measured twice a week up to Day 28 post-HSCT in lethally irradiated (9.75 Gy) mice reconstituted with either syngeneic or allogeneic bone marrow cells and splenocytes (10E7 and 13x10E6 cells/recipient, respectively). An activation of the classical pathway was observed in 10/39 patients. Complement activation was found in 9 of the 22 patients who received myeloablative conditioning (40%) and in one of the 17 patients allografted with RIC (5.8%), mainly within the first month following HSCT. We observed a strong association between Complement activation and the development of acute gastrointestinal GVHD. Nine of the 10 patients(90%)who activated the Complement system developed gastrointestinal (GI) GVHD within two weeks following the activation, whereas only 4 of the 29 (13.8%) who did not activate the system, all of whom had received a RIC, did so (p<0.001). No correlation was found with the use of monoclonal antibodies, the degree of HLA and sex disparity between donor and recipient, the CMV status before the graft or the development of isolated acute cutaneous GVHD. In the mouse model, despite an important inflammatory response due to the conditioning, the C3/SAP ratio was significantly decreased from Day 7 to 28 after HSCT in allogeneic recipients of HSCT in comparison to that of naïve (p=0.0005) and of syngeneic recipients (p=0.0002), indicating an activation of the Complement system in allografted mice that uniformly developed GI GVHD by Day 20 after HSCT. Our results show that allogeneic HSCT induces Complement activation in both humans and mice, as a consequence of the immune alloresponse and suggest that Complement C3 and C4 dosage might be useful in early prediction of acute gastrointestinal GVHD in humans. G-CSF-mobilized donor graft for peripheral blood stem cell transplantation (PBSCT) that contained a large number of myeloid precursor cells, which suppress alloreactive donor T cells resulting in inhibition of acute graft-versus-host disease (GVHD). However, the molecular mechanism remains controversial. Here, we investigate whether indoleamine 2,3dioxygenase (IDO) could be responsible for downstream molecular mechanism. We first evaluated the effect of G-CSF on acute GVHD mouse model. We isolated splenocytes as a donor graft from control and G-CSF injected B6 mice and transplanted into irradiated B6D2F1 recipient mice. Compared with controls, lethal acute GVHD was completely prevented by G-CSF injected donor graft. We found that CD80highGr-1+CD11b+ cells were highly induced in G-CSF injected donor graft. We assessed the suppressive activity of CD80highGr-1+CD11b+ cells in acute GVHD. CD80highGr-1+CD11b+ cells significantly inhibited alloreactive donor T cell expansion in vitro and in vivo resulted in prevention of acute GVHD lethality. We next determined IDO expression in CD80highGr-1+CD11b+ cells. However, IDO was slightly expressed and did not significantly higher than bone marrow Gr-1+CD11b+ cells, which promote alloreactive T cell responses. In addition, IDO inhibitor, 1-methyl tryptophan (1-MT) was not able to restore alloreactive T cell expansion in vitro allo-MLR. Subsequently, we examined whether IDO could be responsible for inhibiting acute GVHD after transplantation. Administration of 1-MT to recipient mice was no effect on CD80highGr-1+CD11b+ cells-induced prevention of lethal acute GVHD. Thus, there was no change CD80highGr-1+CD11b+ cells-induced survival between WT and IDOdeficient donor graft. Taken together, our data suggest that administration of G-CSF induces generation of CD80highGr-1+CD11b+ myeloid suppressor cells in donor graft prevent lethal acute GVHD through an IDO independent mechanism. The therapeutic efficacy of allogeneic hemopoietic stem cell transplantation (SCT) for haematological diseases largely relies on the graft-versus-leukemia (GvL) effect exerted by donor T cells, but an uncontrolled graft-versus-host disease (GvHD) bears a risk of complications. CD4+CD25highCD127low regulatory T cells (Treg) -a subset of CD4+ T helper cells (Th) -are believed to maintain tolerance and to inhibit GvHD after SCT. It remains controversial whether the frequency of Treg in peripheral blood correlates with GvHD. Methods: The frequency of CD4+CD25highCD127low Treg in the peripheral blood was determined by flow cytometry in 85 patients (median age 47 y, range 16-65; 49 male) with a median follow-up of 564 days (range 164 -3606) post SCT, and compared to 11 healthy controls. Results: Patients received sibling (n=41) or unrelated donor (n=44) allogeneic SCT after myeloablative (n=34) or reduced intensity (n=51) conditioning. At the time of the study all patients had a full donor chimerism, 18 still received immunosuppression, 44 had signs of chronic GvHD, 25 suffered from extensive chronic GvHD. Although similar mean lymphocyte counts were found in patients (1511.5/µl ± 843/µl) and controls (1555/µl ± 411/µl) the CD4+Th counts were significantly lower in patients (336.8/µl ± 226.4/µl) vs controls (798/µl ± 212/µl), p<0.0001. Treg counts were also lower in patients (27/µl ± 20/µl) than in controls (64/µl ± 20/µl), however no significant difference was found in the proportion of Treg among Th cells with 8.6% in patients vs 8.1% in controls. Treg counts and Treg proportions among Th cells were similar in patients receiving or not receiving immunosuppressive treatment (28/µl ± 24/µl vs 26.7/µl ± 18/µl; and 8.2% vs 8.8%; n.s.), in patients with or without chronic GvHD (25/µl ± 18/µl vs 29.3/µl ± 22/µl and 9.1% vs 8.1%; n.s.) as well as in patients with (n=9) or without subsequent relapse (19.8/µl ± 14/µl vs 28/µl ± 20.4/µl and 7.3% vs 8.7%; n.s.). Conclusion: This data indicate that the recovery of the Treg subset in peripheral blood closely follows the CD4+ Th cell recovery after allogeneic SCT. Moreover, levels of CD4+CD25highCD127low Treg in peripheral blood do not allow predicting chronic GvHD or relapse after allogeneic SCT. The Treg subset paucity as a possible contributor to chronic GvHD has to be assessed in target organs and is not evident in peripheral blood. Introduction: Cytotoxic-T-Lymphocyte-Antigen-4 (CTLA-4) is an inhibitory molecule that downregulates T cell activation. The C/T60 Single Nucleotide Polymorphism (SNP) has been shown to affect CTLA-4 function in vitro and has been correlated with the development of different autoimmune disorders and Graft-versus-Host-Disease (GvHD) after human allogeneic hematopoietic cell transplantation (allo-HCT) in vivo. However, several allo-HCT related factors may have influenced these results. Objective of the study: To analyse in a group of allotransplanted pts who received Donor Lymphocyte Infusion (DLI) for molecular or overt relapse the impact of the C/T60 SNP (AA, AG, GG) of the donor and/or recipient on the response and the GvHD development post-DLI. Patients/Methodology: By using PCR and pyrosequencing analysis, we genotyped the C/T60 CTLA-4 SNP in a group of 98 healthy subjects (controls), 43 allotransplanted recipients and their matching DLI donors. We compared with Fisher's test the distribution of the alleles between the controls, the recipients and the donors accordingly to response (any response vs no response) and GvHD development (any grade GvHD vs no) after DLI. Results: In the control group, 28% were A/A, 42% were A/G and 30% were G/G. A similar not significantly different distribution was found also in the recipient and in the donor group (recipients/donors: 14%/12% A/A, 42%/44% A/G, 44%/44% G/G). 16/43 of the recipients developed post-DLI GvHD; their allele distribution was not significantly different compared to the control group, as well as compared to the distribution of the rest 27 recipients who did not develop post-DLI GvHD. The distribution of the 16/43 matching donors whose graft led to GvHD development was significant different to the controls (6% A/A, 37% A/G and 57% G/G, p=0.05), whereas the distribution of the rest 27 donors did not differ to controls. Accordingly to response, 23/43 of the recipients responded to DLI and their C/T60 SNP distribution (4% A/A, 44% A/G, 52% G/G) differed significantly from the control group (p=0.02). The allele distribution of the matching 23/43 donors did not differ to the controls. The distribution of the rest 20 non-responders recipients and of their matching donors did not differ from the control group. Conclusion: Our data indicate that CTLA-4 C/T60 SNP of DLI donors correlates with the risk of GvHD development and CTLA-4 genotype of the recipients correlates with response to DLI. Clinical characteristics of chronic graft-versus-host disease following umbilical cord blood transplantation for adults K. Sugimoto* (1), H. Narimatsu (1) , T. Kawase (2) , H. Iida (3) Chronic graft-versus-host disease (GVHD) is a significant complication for long-term survivors following allogeneic hematopoietic stem cell transplantations; however, the clinical characteristics of chronic GVHD following cord blood transplantation (CBT) in adults have not been well described. To assess the clinical characteristics of chronic GVHD following CBT, we investigated a total of 77 patients who underwent CBT at 8 transplantation centers of the Nagoya Blood and Marrow Transplantation Group between March 2001 and November 2005. Of the 77 patients, 29 survived without graft failure or progression of underlying diseases for at least 100 days after transplantation. The median age of the 29 patients was 42 years (range, 18-67 years). Primary diseases included acute myeloid leukemia (n=12), acute lymphoblastic leukemia (n=6), chronic myeloid leukemia (n=5), malignant lymphoma (n=3) and others (n=3). The patients received myeloablative (n=14) and reduced-intensity (n=15) preparative regimens. The median follow-up of surviving patients was 35.3 months (range, 4.2-70.5 months). Seven patients developed chronic GVHD (extensive, n=4; limited, n=3) disease. The cumulative incidence of chronic GVHD 1 year after day 100 was 24% (95% confidence interval (CI), 11-41%). According to the grading proposed by the National Institutes of Health consensus development project (BBMT. 2005; 11 :945), 6 of these patients were categorized into mild, and 1 was categorized into moderate. The type of onset included quiescent onset (n=1), de novo onset (n=3) and progressive onset (n=3). The organs involved by chronic GVHD included the skin (n=6), oral cavity (n=4), liver (n=1) and gastrointestinal tract (n=1). In 3 patients, chronic GVHD resolved with supportive care, and the remaining 4 patients were successfully treated with additional immunosuppressive therapy. Overall survival rate of the 29 patients 3 years after day 100 was 53% (95% CI, 32-70%). Overall survival rates of patients with and without chronic GVHD 3 years after day100 were 83% (95% CI, 27-97%) and 44% (95% CI, 22-64%), respectively (P=0.076). Event-free survival rate of the 29 patients 3 years after day100 was 47% (95% CI, 28-65%). Event-free survival rates of the 29 patients with and without chronic GVHD 3 years after day 100 were 83% (95% CI, 27-97 %) and 36% (95% CI, 17-56%), respectively (P=0.047). These results suggest that chronic GVHD following CBT is mild and has a graft-versus-malignancy effect. A.L. Amir*, R.S. Hagedoorn, W.A.F. Marijt, R. Willemze, J.H.F. Falkenburg , M.H.M. Heemskerk. Leiden University Medical Center (Leiden, NL) Single locus HLA mismatched stem cell transplantation (SCT) and donor lymphocyte infusion (DLI) is applied in patients with hematological malignancies who may benefit from allogeneic transplantation but lack an HLA-matched donor. Based on the high frequency of allo-HLA reactive T-cells, and on the expression of HLA class I by almost all nucleated cells, one would expect all single HLA class I mismatched transplanted patients to develop severe GVHD. HLA mismatched DLI has a higher likelihood of inducing graft versus host disease (GVHD), however not all patients develop GVHD after DLI. We hypothesized therefore that the presentation of the HLA class I mismatched allele on cells of the patient is not sufficient to elicit an effective allo-immune response. We characterized the allo-immune response in a patient with acute myeloid leukemia who was treated with a T-cell depleted SCT from a sibling donor who was HLA identical except for HLA-A2. Six months after SCT, DLI was given for mixed chimerism. No clinical response and no GVHD developed. 12 months after SCT the AML relapsed with 9% blasts in bone marrow for which a second DLI was given. Five weeks after the DLI the patient died of grade IV GVHD. During the GVHD conversion to donor chimerism developed and in peripheral blood 90% of CD8 and 40% of CD4 donor T-cells were activated as determined by HLA-DR expression. To analyze the nature of the immune response, the activated CD8 and CD4 donor T-cells were single cell sorted, expanded and tested for alloreactivity. 94% of the CD8 T-cell clones were alloreactive and restricted to the allo-HLA-A2 molecule. 26% of the CD4 clones were alloreactive and recognized epitope 103-120 of the hypervariable region of HLA-A2 in the context of HLA-DR1. Both T cell responses were highly polyclonal. Prior to the first DLI, only HLA class I expressing T-cells and non-hematopoietic patient derived cells were present, which were isolated and shown to activate the CD8 clones but not the CD4 clones. Leukemic blasts isolated at the time of the second DLI, expressed HLA-DR, and activated the CD4 as well as the CD8 clones. We hypothesize that the HLA class II expression on hematopoietic cells of the patient at the time of the relapse was essential for the development of GVHD. These results indicate a role for patient leukemic blasts acting as host APCs in initiating the GVHD by activating both a CD4 and CD8 T-cell response in an HLA class I mismatched setting. Long term T cell reconstitution after Hematopoietic Stem cell Transplantation (HSCT) has been shown to be dependant of the patient thymic function and affected by Graft Versus Host Disease (GVHD). To confirm the role of acute GVHD on thymic function, we followed a cohort of 93 genoidentical HSCT patients that were grafted with Bone Marrow (56) or Peripheral Blood (PB) Stem Cell (37) mainly for hematological malignancies (81). The median age was 35 years (range and 68 of the patients had aGVHD of grade ≥1. PB samples were collected before graft and at 3, 6, 12 and 24 months. Numbers of CD3 and naïve T cells were measured by flow cytometry using antibodies against CD3, CD4, CD8, CD45RA and CD62L. Thymic output was measured by "signal joint T cell Receptor Excision Circles" (sjTREC) real time PCR quantification on all patients. We quantified also, in a selected group of 20 patients matched for age (mean 29.9 and 30.5 years for the 13 aGVHD and 7 no-aGVHD patients), the sjTREC (generated just before the T cell Receptor (TCR) alpha chain recombination) and beta TREC that are generated during the TCR beta chain recombination; sj/beta TREC ratio being a marker of thymocyte proliferation. For the 93 patients, aGVHD was linked to a decrease in the absolute number of naïve CD4 T Cells (but not CD8) at 6 and 12 months (p =.04 and .02). Absolute number of sjTREC was also lower at 6 months in aGVHD patients (median 3.8 vs 38.2 sjTREC/mL of blood, p= .014). If we looked at patients that were less than 25 year old, we found the same significant difference (median 13.5 vs 254.7 p= .005), suggesting an effect independent of age. Moreover, in the age-matched groups of patients, sjTREC/150000 PBMC or absolute number of sjTREC were significantly lower for aGVHD patients (114 vs 1327 sjTREC/150000 PBMC p=.035 and 7.8 vs 61.2 sjTREC/mL of blood p=.043, respectively). Mean betaTREC /150000 cells was also reduced for aGVHD patients at 6 months (12.8 vs 214.6, p=.02). Finally, there was no significant difference between the 2 groups for the sj/beta ratio. These data confirm that aGVHD patients have a lower thymic output in the first months after HSCT. This decrease in the thymic output could rather be explained by a decrease in the number of thymocyte precursors that start to differentiate and recombine their beta chain TCR (as measured by beta TRECs) than by a reduced thymocyte proliferation. Long-term follow-up of a phase II study of pentostatin in patients with steroid refractory acute intestinal graftversus-host disease S. Klein* (1), S. Mousset (2) , G. Bug (2) , B. Wassmann (2) , H. Martin (2) (1)Clinic Bayreuth (Bayreuth, DE); (2)Unicersity Clinic Frankfurt (Frankfurt, DE) Steroid refractory intestinal acute graft-versus-host disease (aGvHD) is a mayor complication after allogeneic hematopoietic cell transplantation. Survival rate of severe intestinal aGvHD is low. There is no standard therapy available. A promising strategy is the use of pentostatin, an inhibitor of adenosine deaminase inducing lymphocyte apoptosis. Here we report the 7 year follow-up of a phase II study on salvage therapy of aGvHD by pentostatin. We treated 18 patients with steroid refractory intestinal aGvHD stage III or IV with pentostatin (8 female/10 male). The mean age was 45 (range: 25-61 years). The underlying diseases were ALL (4), AML (10), MM(2), HD (1) and CML (1) . Eight patients were allografted with HLA-identical sibling donors, three with related donors with a single HLA class I mismatch, seven with matched unrelated donors. All patients were allografted with peripheral blood stem cells. As GvHD prophylaxis ciclosporine A in combination with mycophenolate mofetil or methotrexate were used. Eleven patients had aGvHD stage III, seven stage IV. All patients had a severe gastrointestinal-tract involvement (grade III or IV). After failure of steroid treatment (prednisolone >2 mg/kg for at least 3 days) pentostatin was applied as a salvage therapy (1 mg/m2 for three consecutive days). Eight patients received between one and three further courses of pentostatin every three to four weeks. Therapy was well tolerated. No severe neutropenia was observed. Except one case of hemolytic uremic syndrome (HUS) no impairment of renal function was observed. Eleven patients achieved complete, five partial remission of aGvHD. Time until a clinical improvement could be observed was 12-14 days. Two patients died before day 12 without improvement of GvHD symptoms. Seven patients (39%) are alive (960-2510 days post first cycle of pentostatin), two with extensive chronic GvHD. All one-year-survivors stayed alive. Eleven patients died (two due to relapse of leukemia, nine transplant related [1 x interstitial pneumonitis, 1 x pneumonia, 1 x chronic GvHD, 4 x aGvHD and multiple organ failure (MOF), 1 x parvo B19 infection and MOF, 1 x HUS and MOF]). In summary, in this phase II study pentostatin has been demonstrated to be a highly effective and well tolerated drug for salvage therapy of steroid refractory acute GvHD with intestinal involvement. Moreover, compared with earlier studies on steroid refractory aGvHD a high rate of long term survivors could be observed. We report on the occurrence of severe acute graft-versus-host disease (GVHD) 10 months after an allogenic sibling donor transplantation in a 62-year-old man. The GVHD was developed three weeks after the institution of glucosamine hydrochloride (Artrox®) treatment. Although no conclusive proof is available, the graft-versus-host reaction could have been triggered by glucosamine stimulation of alloreactive Tcells. A study in 7 healthy individuals confirmed a significant allostimulatory effect of Glucosamine hydrochloride treatment. To understand the immunobiolgical effects of Glucosamine in vivo we studied the alloimmunostimulatory effects in mixed lymhpocyte reactions (MLR) and cytokine production in a pilot study. Seven healthy individuals, 4 males and 3 females, with a median age of 49 (range 28-63) years were treated with Arthrox® 625 mg twice daily for 4 weeks. Blood samples was taken before the study and on days 14, 30 and 90. A significantly increase in Mixed Lymphocyte Reaction (MLR) and soluable IL2 receptors were seen after 2 and 4 weeks of treatment with Artrox®. The MLR 2 months after Glucosamine discontinuation (day 90) went back to the same level as compared before the study started. No effect was seen on Hemoglobin, Platelets, WBC´s, liver and renalfunction. Our interpretation is that Glucosamine compounds should be used with caution in patients after allogeneic hematopoietic stem cell transplantation due to the immunostimulatory effect in vivo by activation of primed T-cells and the risk of lifethretening graft-versus-host reaction. E. Distler*, S. Thomas, E. Schnürer, C. M. Britten, M. Schuler, C. Huber, K. Kolbe, R. G. Meyer, T. Wölfel, W. Herr Johannes Gutenberg-University Mainz (Mainz, DE) Diagnosis of graft-versus-host disease (GVHD) is mainly based on clinical features and on tissue biopsies. However, there are cases in which GVHD cannot be distinguished from opportunistic infections. This may pose a therapeutic dilemma of whether to modify immunosuppressive treatment or to administer donor lymphocyte infusion (DLI) for promoting antimicrobial immunity. We observed a 68-year-old patient with myelodysplastic syndrome who developed steroid-responsive acute GVHD IIo of skin and gut at day (d) 16 after T-cell depleted reducedintensity transplantation (Fig. 1) . From d90-230, the patient relapsed with massive diarrhea and recurrent gastrointestinal bleedings. Histopathology of gut biopsies showed a mixed picture with signs of GVHD and ulcerative inflammation. In stool screening, norovirus type 2 was detected from d99-d216, thereby confirming the longest infection with this virus ever reported. Because of severe lymphopenia, we considered DLI therapy to promote T cell reconstitution and norovirus clearance. To balance the risk for DLI-induced exacerbation of GVHD, CD8 T cells isolated from PBMC were analyzed for anti-recipient reactivity ex vivo by IFN-g ELISPOT assay. Due to the limited availability of recipient cells and a single HLA disparity between donor (B*3508) and patient (B*3503), we used HLA-deficient K562 cells transfected with mismatched (B*3503) or matched (A*0201) HLA alleles as antigenpresenting cells. Post-transplant CD8 T cells specifically recognized disparate HLA-B*3503 (Fig. 1) , but not shared HLA-A*0201. Mismatch-reactive CD8 T cells were detectable during the first episode of GVHD (71/10 5 ) and correlated closely with the intensity of diarrhea beyond d110. Maximum anti-HLA-B*3503 reactivity (439/10 5 ) occurred on d205. Considering this vigorous alloreactivity, scheduled DLI was omitted due to our concern it would boost GVHD rather than facilitating norovirus clearance. Patient's clinical condition improved spontaneously around d220. In conclusion, we introduce a T cell assay for the rapid detection of anti-HLA mismatch reactivity using K562-HLA transfectants as substitutes for patient cells. We validated the assay in two further HLA-incompatible donor-patient pairs and generated off-the-shelf K562 transfectants for more than 15 HLA alleles. Ex vivo alloreactivity toward mismatch HLA might be a surrogate marker to facilitate GVHD diagnosis and guide therapy in ambiguous clinical situations, such as the coincident viral infection described herein. Mycophenolic acid trough level monitoring in graftversus-host disease treated with Mycophenolate mofetil: correlation with albumin levels and clinical response P. Hiwarkar, S. Kulkarni, M. Dungarwalla, R. Saso, B. Shaw, S. Evans, J. Treleaven, G. Morgan, M. Ethell, M. Potter Royal Marsden Hospital (Sutton, UK) From January 2004 to November 2007, 32 patients (16 -60 yr) who underwent haematopoietic stem cell transplant (sibling donor: 15, unrelated donor: 17) for haematological malignancies (Ac. leukemia: 16, Ch. leukemia: 7, other: 9) were treated with Mycophenolate mofetil (MMF) for graft versus host disease (GvHD). Conditioning was TBI in 14 and only chemotherapy in 18 patients. GvHD prophylaxis was CyA with MTX (n=14) or without MTX (n=18). Campath was used for in vivo T-cell depletion in 11 patients. Nineteen patients had acute and 13 had chronic GvHD. The overall grade of acute GvHD was I (n=6), II (n=5), III (n=6), IV (n=2) and 7 had limited and 6 had extensive chronic GvHD. First line therapy for acute GvHD was steroids in all 19 patients and for chronic GvHD was CyA (n=1), MMF (n=2), and steroids (n=10). The indications for treating with MMF in patients with acute GvHD were steroid refractoriness (n=7), steroid dependence (n=8), intolerance to Cya (n=1) & breakthrough on Cya (n=3). In the chronic GvHD group, the indications were steroid refractoriness (n=6), steroid dependence (n=3), intolerance to Cya (n=2), breakthrough on tacrolimus (n=1) & recurrence after stopping MMF prophylaxis (n=1). Response was defined as resolution of symptoms of GvHD without addition of another immunosuppressive agent and steroids were spared. 21 (65%) patients achieved a good response whereas 11 (35%) were non-responders. 12 (63%) of 19 patients with acute GvHD and 9 (69%) of 13 patients with chronic GvHD achieved good response. In the steroid refractory or dependent group there were 24 patients, of which 16 (66%) responded to MMF. In all 21 patients who responded to MMF, mean Mycophenolic acid (MPA) levels were in therapeutic range. Six of 11 patients who did not respond to MMF had sub-therapeutic mean MPA levels (p=0.0005). On correlating mean MPA levels with mean of corresponding albumin levels, all 16 patients who had mean albumin ≥35 g/L had mean MPA levels in the therapeutic range, whereas 6 of 16 patients who had mean albumin < 35 g/L had sub-therapeutic mean MPA levels (p=0.02). Thus, suggesting that patients with low albumin may require higher doses of MMF. No grade 3-4 toxicities and no correlation between mean MPA levels and infectious complications were noted. In conclusion, MMF is efficacious in steroid refractory and dependent acute or chronic GvHD and therapeutic MPA trough levels correlate significantly with the albumin levels and clinical response. (1) Objective: Tyrosine kinase inhibitors (TKIs) like imatinib, nilotinib and dasatinib or donor lymphocyte infusions (DLIs) might be administered to CML/ALL patients with refractory disease or at relapse after allogeneic stem cell transplantation (allo-PBSCT). TKIs inhibit the proliferation of CML progenitor cells, but might also hamper CD8+ T cells mediating the graftversus-leukemia (GVL) effect and CD8+ T cells specific for cytomegalovirus (CMVpp65). Methods: Imatinib, nilotinib and dasatinib were added at different concentrations to proliferation assays of Tregs and CD8+ T cells. Mixed lymphocyte peptide cultures (MLPCs) were performed with peptides derived from influenza matrix protein (IMP), CMVpp65 as a viral antigen and the receptor for hyaluronic acid mediated motility (RHAMM-R3) as a leukemiaassociated antigen. CD8+ T cells from these MLPCs from healthy donors and patients with CML after allo-PBSCT were screened by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays. Tregs were measured after 3 days of culture with anti-CD3 and anti-CD28. The activity of T cells from the peripheral blood of patients under dasatinib and after withdrawal of dasatinib was tested. Western blots (WBs) for T cell receptor (TCR) related molecules and NFkB were performed. Results: The release of interferon gamma and granzyme B by CD8+ HLA-A2 /tetramer+effector T cells specific for each peptide as well as the proliferation and function of Tregs was inhibited by all TKIs in a dose-dependent fashion and correlating to the time of TKI exposure. The inhibitory effect was reversible by wash-out of the drug. In WBs, TKIs decreased the expression of ZAP70, Lck and Akt, as well as NF-B p65/p100/p105 and c-Rel. The potency of T cell inhibition was imatinib : nilotinib : dasatinib = 1:2:40 in in vitro T cell assays. Of note this immunosuppressive effect was also seen in vivo in patients off/on dasatinib administration. Conclusion: When administering TKIs, inhibition of both CD8+ cells and Tregs must be taken into consideration with respect to GVL and anti-viral T cell response. These effects are mediated by down-regulating the phosphorylation level of TCR and NF-B family members. Treatment of refractory chronic graft-versus-host disease with low-dose methotrexate M.L. Battista, F. Patriarca, A. Sperotto, F. Zaja, M. Medeot, M. Cerno, R. Fanin Division of Haematology (Udine, IT) Objectives: In this retrospective monocentre study, we report our experience with 9 patients receiving low-dose methotrexate (MTX) for the treatment of refractory chronic graft-versus-host disease (cGvHD) with the aim of evaluating clinical response rate and toxicity. Patients and methods: From February 2004 to October 2007, 9 consecutive patients with extensive cGvHD, refractory to at least 2 immunosuppressive therapies including prednisone and cyclosporine, received low-dose MTX (7,5 mg/mq i.v. weekly). Table 1 summarizes patients' main characteristics. The most frequent sites involved were: skin (9/9), with sclerodermic features (7/9), and oral mucosa (7/9) (table 2). The median cumulative MTX dose was 520 mg (range and the median duration of treatment was 15 months (range 2,5-24). Results: A complete resolution of cGvHD manifestations in each organ involved (complete response) was observed in 4 out of 9 patients (44%) and a regression of cGvHD signs >25% (partial response) was observed in another patient, with an overall response rate of 56%. The responses for each organ were: skin 5/9 (56%), sclerodermia 5/7 (71%), mouth 2/7 (28%), eyes 1/5 (20%), liver 3/4 (75%), lung 0/3, joints 3/3 (100%) (table 2). All the responses were sustained at the last evaluation with a median duration of response of 29 months (range 12-41); all the responding patients were able to decrease or discontinue concomitant immunosuppressive treatments. None of the patients experienced ≥ grade 2 WHO haematological toxicity. No significant change in immunoglobulin serum levels and lymphocytes subset counts was documented. We observed renal toxicity in 2 cases (one of them had a previous renal function impairment). Four out of 9 patients developed infectious events (only in one case severe), a patient experienced acute myocardial ischemia and another one had leukemia relapse and later died. Two patients interrupted the therapy because of the development of adverse events (infections or relapse). Conclusions: Our results indicate the potential activity of lowdose MTX for the treatment of refractory cGvHD with a quite safe toxicity profile. Furthermore they highlight a major efficacy of MTX in cGvHD involving skin (also with sclerodermic features), liver and joints. The efficacy of prophylaxis regimens for acute graftversus-host disease in patients undergoing bone marrow and peripheral blood stem cell transplantationsystematic review and meta-analysis R. Ram*, A. Gafter-Gvili, M. Yeshurun, M. Paul, P. Raanani, O. Shpilberg Beilinson Hospital (Petah Tikva, IL) Background: Currently, there is no consensus regarding the optimal graft versus host disease (GVHD) prophylaxis regimen among patients undergoing allogeneic bone marrow (BMT) or stem cell transplantation (alloSCT). Objectives: This study aims to evaluate the efficacy and safety of different prophylaxis regimens for acute GVHD. Methods: Systematic review and meta-analysis of randomized controlled trials of patients undergoing alloBMT/SCT that compare the following regimens: the addition of MTX to various regimens, the addition of corticosteroids (CS) to various regimens and a comparison between Cyclosporin (CsA)+MTX and tacrolimus (FK506)+MTX. Electronic search was conducted until 2007. Outcomes assessed were: allcause mortality, acute GVHD (aGVHD), chronic GVHD (cGVHD) and adverse events. Relative risks (RR) with 95% confidence intervals (CIs) were estimated and pooled. Results: Our search yielded 22 trials, 7 assessed the addition of MTX to CsA-based regimens, 8 compared between MTX and other regimens (most were CsA based), 5 evaluated the role of CS and 3 compared between FK506+MTX and CsA+ MTX. The addition of MTX to CsA/FK506, yielded a significant decrease in aGVHD (RR 0.49; 95% CI 0.38-0.65, 6 trials, Fig.1) , albeit with only a trend for reduced mortality at 100 days (RR 0.72; 5 trials) . There was a strong trend towards a reduction in cGVHD in the MTX arm (RR 0.84; 95% CI 0.70-1.02, 5 trials). The addition of CS to the same regimen without CS yielded no statistically significant differences in all outcomes. The comparison of FK506+MTX with CsA+MTX yielded a significant reduction in aGVHD in the FK506+MTX arm (RR 0.62; 95% CI 0.48-0.96, 3 trials, Fig.2 ). There was no difference in all-cause mortality at 100 days (RR 1.18; 95% CI 0. 89-1.57, 3 trials) or in cGVHD (RR 1.05; 95% CI 0.87-1.25, 3 trials). Conclusions: Addition of MTX to a CsA based regimen reduces acute GVHD, and thus, according to the common practice, is the first line preventive measure for acute GVHD. FK506 with MTX reduces acute GVHD compared with CsA with MTX and should be considered a superior alternative for the common CsA plus MTX regimen. Infliximab treatment for steroid-resistant GvHD M. López Duarte*, A. Insunza, A. Bermudez, L. Yañez San Segundo, S. Gonzalez de Villambrosia, M. Colorado, J. Nuñez, A. Iriondo H.U. Marqués de Valdecilla (Santander, ES) Introduction: Graft versus host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Almost 40% of patients show no response to a course of steroids. In this group there is no standard therapy established. Tumor necrosis factor alpha is an important cytokine involved in the pathophysiology of acute GVHD. Infliximab may offer possible benefit as treatment for this group. Patients and methods: We retrospective analyzed 15 patients with GVHD refractory to corticosteroids who were treated with infliximab between January 2004 and December 2007.The median age at transplant was 32 (range 5-55). Eight of the 15 were male.The conditioning regimen was myeloablative in 10 patients, donors were unrelated in 11 cases (73%) and eight patients received bone marrow as the source of stem cells.GVHD prophylaxis included cyclosporine A plus mycophenolate mofetil for transplants with reduced intensity regimen and methotrexate for the rest, with one exception. No antibiotic prophylaxis was used. Twelve patients have grade IV acute GVHD unresponsive to 1.5 -2 mg/kg/day methylprednisolone and three patients had extensive chronic GVHD unresponsive to conventional therapy.The main system involved was gastrointestinal in 7 cases. Results: No infusion-related side-effects were noted. Two patients were not analyzed because of insuficient number of doses (1) and cytomegalic disease. The median number of doses were 5 (range 2-9). Eleven of the 13 patients (84%) responded to infliximab: responses were complete (CR) in 3 and partial (PR) in 8. The median time between HSCT and infliximab therapy in 3 CR was 50 days (50-126) and 20 days in those without response.The median time between infliximab administration and response was 26 days.Twelve out of 13 patients developed one or more infectious episodes: 10 cases of septicemia, 2 cases of CMV reactivation, 2 cases of VHS-6 viremia,one case of VEB and 6 mycotic infections. Seven patients died, 6 because of infections. Six patients are alive, four whitout immunosupresion therapy and limited chronic graft versus host disease at a median time of 25 months. No disease recurrence has been observed. Conclusion: Infliximab shows an encouraging response rate.Responses are more frequent when the time between HSCT and infliximab administration is longer.The incidence of infections was is high, as expected, so that prophylactic strategies should be considered. J.L. Piñana, D. Valcarcel, R. Martino, E. Moreno, A. Sureda, J. Delgado, J. Briones, S. Brunet, J. Sierra Hospital De La Santa Creu I Sant Pau (Barcelona, ES) Background: Steroid-refractory acute graft versus host disease (aGVHD) is one of the main causes of non-relapse mortality after allogeneic hematopoietic stem cell transplantation. Anti-interleukin-2 receptor monoclonal antibody (anti-IL-2-Re) is a new treatment option with a different mechanism of action that may be useful as salvage treatment in this setting. Patients and Methods: We retrospectively analyzed the results in 59 adult patient who received Inolimomab as salvage therapy for steroid-refractory aGVHD between June 1999 and September 2006. The median follow-up for survivors was 580 days (range, 110-1336) . GVHD prophylaxis was based on cyclosporine A in 59 patients, 35 with a short course of methotrexate, 18 with mycophenolate mofetil. Inolimomab was administrated at a median of 19 days (3-101) after aGVHD onset. Forty-four patients received 1 course, twelve received 2, and three received 3 courses. Inolimomab was given intravenously at a dose of 11 mg/day for 3 days, then 5.5 mg/day for 7 days and 5.5 mg every other day for 5 doses. Results: aGVHD occurred at a median of 34 days (9-173) post-transplant. aGVHD severity was grade II in 3 patients, grade III in 30 and grade IV in 26 patients. Inolimomab was well tolerated, with only one drug-related infusional reaction in a patient who received 2 courses. Thirty-five (59%) patients responded to inolimomab; 20 (35%) patients showed a complete response and 15 (24%) a partial response. Patients with isolated hepatic aGVHD had a higher overall response rate than those with gastrointestinal or multiorgan aGVHD (100% vs 66% vs 48%, respectively; P=0,03). Neither prior use of ATG, nor number of prior lines used before Inolimomab had a significant influence on the response rate. Response to Inolimomab had a favorable impact on 1-year overall survival (OS) (58% vs 0%; P<0,001) by univariate analysis. Patients showing response at day 15 had a higher 1-year OS (53% vs S227 12% respectively, P<0,001). Of note, we observed higher absolute lymphocyte counts prior to therapy in responder patients (0.525 vs 0.266 x109/l, p=0.02). Infections were common: there were 18 cases of CMV infection, 12 severe infections due to other viruses, 16 invasive fungal infections and 12 severe bacterial infections. Conclusion: Inolimomab may be an effective salvage treatment for steroid-refractory aGVHD. Randomized clinical trials concerning its use are warranted. J.L.Piñana is supported by grants (expedient CM06/00139, ISCIII. Spain) J.L. Piñana, D. Valcarcel, R. Martino, P. Barba, A. Sureda, J. Delgado, J. Briones, S. Brunet, J. Sierra Hospital De La Santa Creu I Sant Pau (Barcelona, ES) J.L.Piñana is supported by grants from the Instituto de Salud Carlos III (expedient CM06/00139, Ministerio de Sanidad. Spain) Background: Graft-versus-host disease (GVHD) is a major obstacle to successful allogeneic stem cell transplantation (SCT). Cyclosporine A (CsA) in combination with methotrexate (MTX) is the most commonly used regimen for GVHD prophylaxis. Mycophenolate mofetil (MMF), together with CsA, has been recently introduced and is associated with earlier hematologic recovery and less mucositis. In this casematch study, we retrospectively analyzed the introduction of MMF as GVHD prophylaxis in comparison with a short course of MTX in the setting of reduced intensity conditioning allogeneic SCT (RIC-Allo). Patients and methods: We analyzed 59 RIC-Allo recipients of an HLA-identical sibling allograft from April 2000 to June 2006. The median follow-up was 473 days (range, 8-2139 days) for the entire cohort. Median age was 59 years (range, 43-72), and 68% were male. Diagnoses were acute leukemia/myelodysplastic syndrome (n=19/22), multiple myeloma (n=6), chronic myeloid leukemia (n=5) and non-Hodgkin lymphoma (NHL) (n=4). GVHD prophylaxis comprised CsA/MTX (MTX group) in 37 patients and CsA/MMF (MMF group) in 22 patients. Conditioning regimens were fludarabine plus busulfan (n= 42) or melfalan (n= 17). Results: The occurrence of grade II-IV mucositis was higher in the MTX group than in the MMF group (62% vs 28% p= 0.015). No significant differences were found between MTX and MMF groups in terms of time to neutrophil recovery (16 ± 3 days vs 15 ± 2days) (P=0.5) or one-year non-relapse mortality [14% (95% CI, 6-31%) vs 28% (13-60%); (P=0.2)]. Cumulative incidence of acute GVHD was 49% (35-68%) and 68% (51-91%) for the MTX and MMF groups, respectively (P= 0.6). Patients in the MTX group showed similar incidence of chronic GVHD compared to the MMF group: 55% (40-74%) vs 42% (23-75%) (P=0.2). No differences were found between MTX vs MMF groups in one-year overall survival [78%(64-92%) vs 53%(38-76%); P=0.1] or one-year relapse rate [37% (95%CI 21-64%) vs 19% (95%CI 10-37%), P=0.1]. Conclusion: We conclude that the CsA/MMF combination appears at least equivalent to the standard CsA/MTX regimen for GVHD prophylaxis in patients undergoing RIC-Allo. Furthermore, MMF showed less gastrointestinal toxicity as reflected by significantly less mucositis. Monitoring of circulating dendritic cell subpopulations predicts transplant complications after allogeneic haematopoietic cell transplantation S. Eder*, R. Weigl, K. Feldmann, M. Kouba, H. Greinix Medical University of Vienna (Vienna, AT) Introduction:Allogeneic hematopoietic cell transplantation (HCT) has an important curative potential in many hematological disorders. Recovery of dendritic cells (DC), B, T, and natural killer (NK) cells after HCT is important for allograft responses and thus, for treatment outcome. DC may play a role both in initiating graft-versus-host disease (GvHD) and in promoting immune reconstitution after HCT. We studied the clinical relevance of regeneration of immune cells including DC subpopulations on outcome of GvHD and severe infections in the early phase after HCT. Methods: We investigated 66 adult patients (34 male, 32 female) with a median age of 43 (range, 19 to 60) years undergoing myeloablative (n=37) or reduced-intensity conditioning (n=29) from HLA-identical siblings (n=29) or unrelated donors (n=37). The vast majority of patients received peripheral blood stem cells. Peripheral blood (PB) samples were drawn from patients at 1, 3, 6, and 12 months after HCT. Lineage marker-negative HLA-DR+CD11c+ DC (DC1) and lineage marker-negative HLA-DR+CD123+ DC (DC2) as well as monocytes, lymphoid subsets and CD3-CD56+ NK cells were enumerated by flow cytometry. Results were correlated with occurrence of severe infections, acute and chronic GvHD, relapse, transplant-related mortality (TRM) and overall survival. Results: The initial recovery of DC occurred simultaneously with myeloid engraftment. Low PB DC counts at +1 month were associated significantly with the development of severe infections, acute GvHD (median 10.65 DC/ul vs 21.92/ul in no complications), chronic GvHD (9.37/ul), and TRM (11.32/ul). PB DC2 were almost undetectable in patients with severe infections, acute and chronic GvHD and TRM. Whereas patients with acute GvHD responding to therapy had at +3 months PB DC and DC1 values comparable with patients without HCT complications, DC and DC1 remained significantly lower in patients not responding to steroids, chronic GvHD and TRM. PB DC2 were significantly lower at +3 months in all patients with HCT complications compared to ones with unremarkable courses. No correlation of DC counts with relapse was observed. At +12 months DC and DC1 were reconstituted equally in all patients except severe chronic GvHD. Conclusions: Assessment of DC subpopulations one month after HCT allows prediction of complications including chronic GvHD and TRM. Rise in DC subsets in the first year after HCT documents response to immunosuppressive therapy and immune reconstitution. A. Picardi*, R. Cerretti, L. Cudillo, A. Lanti, A. Di Veroli, A. Ferraro, M. Mirabile, G. De Angelis, L. Di Caprio, G. Adorno, W. Arcese University of Tor Vergata (Rome, IT) Chronic graft versus host disease (cGVHD) is often characterized by deep multiple variable-sized wounds that heavily impair the patients' quality of life. It is known the role of platelets (PLTs) gel in tissue regeneration of diabetic/surgical wounds through the realising of growth factors such as bFGF, PDGF and VEGF. We report 5 patients (pts) with skin lesions as manifestation of cGVHD who underwent PLTs gel as local therapy. Objective: The aim of this study was to verify the efficacy and the safety of PLTs gel in the treatment of cGVHD ulcers. Methods: We enrolled in this trial 5 pts with median age of 49 years (23-65), underwent allogeneic HSCT (4 related/1 unrelated) for their haematological malignancies, who developed ulcers related to extensive cGVHD. The pts showed skin lesions although the onset of conventional therapy. The patients' recruitment and the response rate were evaluated on the basis of the following wounds parameters: size (cm²), depth (mm), pain, microbiological assessment and granulation tissue forming. The depth of lesions was evaluated according to diabetic ulcers classification. Homologous haemocomponents were used to obtain PLTs gel with Vivostat System, respecting donor/patient AB0 compatibility. The final product was a gel aliquot of 8 ml with a PLTs concentration of 2 x 10 6 /microliter and the application was performed once a week. Results: All pts showed multiple skin lesions with median initial size of 5 cm² (4-15), involving dermis (grade1) and subcutaneous (grade2) in 2 and 3 cases, respectively. Ulcers duration prior PLTs gel use was from one month to 1 year old. Microbiological assessment resulted positive in 1 case so that PLTs gel was preceded by antibiotic therapy. Complete response was observed in 3 pts after a mean of 8 applications (7-9). The other 2 pts achieved a partial response: both stopped the gel treatment too early for low patient's compliance and for death, respectively. The pain disappeared in all cases after the 2nd gel application, while granulation tissue was observed after the first application in the 3 pts with grade 2 lesions. No side effects were documented. Conclusions: These preliminary data show the safety of the procedure in pts who develop skin lesions due to cGVHD. The efficacy of the treatment have to be confirmed by larger cohort of pts. However, these results lead to consider the use of local therapeutic options as useful support in the management of cGVHD. Donor response to G-CSF is the best predictive factor of extensive chronic graft-versus-host disease after nonmyeloablative allogeneic peripheral blood stem cell transplantation N. Dhedin*, T. Prébet, D. Réa, M. Tanguy, M. Kuentz, N. Piard, F. Norol, J.P. Jouet, J.A. Rubeil, R. Tabrizi, B. Rio, B. Lioure, P. Tiberghien, J.H. Bourrhis, A. Sirvent, P. Bordigoni, D. Blaise, M. Michallet, J.P We have previously reported that the incidence of acute graftversus-host disease (GVHD) after myeloablative peripheral blood stem cell transplantation correlates more strongly with the blood donor CD34+ cell count after G-CSF mobilization than with the dose of CD34+ cells infused to the recipient (Dhedin et al. Exp Hematol 2006) . The aim of this study was to confirm this finding in non myeloablative transplantation. We studied 92 patients transplanted with HLA-identical sibling donor after a conditioning regimen associating 2GY total body irradiation and fludarabine. The median blood donor CD34+ cell count on day 5 of G-CSF administration was 70/µl (range 9-210/µl). The median infused CD34+ and CD3+ cell doses per kilogram of recipient bodyweight were respectively 6.4 x 106 (range 1. 3-22.6) and 275 x 106 (range 88-839). The cumulative incidence rates of grade II-IV aGVHD was 34.4% (se=0.05). The cumulative incidence rates of extensive cGVHD at 24 months was 36.2% (se=0.06). Donor and recipient characteristics, the doses of infused CD34+ and CD3+ cells, and the donor CD34+ cell count after G-CSF mobilization were analyzed as potential risk factors for GVHD. As in several previous studies, the CD34+ cell dose tended to influence the incidence of extensive cGVHD, but the correlation was not statistically significant (p=0.14). In contrast, the blood donor mobilized CD34+ cell count, divided into quartiles, was the only factor significantly associated with extensive chronic GVHD: In univariate analysis, the incidence of extensive cGVHD was significantly higher in the quartile of patients (N=24) whose donors had the highest mobilized CD34+ cell counts (quartile n°4: >109 CD34+ cells/ml at day 5 of G-CSF mobilization), compared to the three other quartiles (cumulative incidence of extensive cGVHD in quartile n°4 was 58.9% compared to respectively 20.8, 37.5 and 25% in the third, second and first quartiles (p=0.03)). In multivariate analysis, the donor CD34+ cell count was the only variable associated with the risk of extensive cGVHD: HR associated with the highest quartile was 2.59 (95% CI [1.30-5.18 ], p=0.007).In conclusion, we confirm in non myeloablative transplants, that donor response to G-CSF is a better prognosis factor of GVHD, than the dose of CD34+ cell infused. "Outstanding mobilizers" seemed to have greater alloreactive potential. These findings have direct implications for transplant management,and especially for the modalities of GVHD prophylaxis. Long-term survival after inolimomab therapy for refractory steroid acute graft-versus-host disease S. Mercère-Girerd, C. Giraud, I. Jollet, I. Princet, F. Guilhot, M. Renaud CHU Poitiers (Poitiers, FR) Background: Steroid refractory acute Graft Versus Host Disease (aGVHD) remains an important issue after allogeneic Bone Marrow Transplantation (BMT). The monoclonal anti-CD25-antibody LEUCOTAC® (Inolimomab) has proven effective against serious aGVHD. Here we report the long term outcome of 32 patients who were treated by Inolimomab for steroid refractory aGVHD. Patients: During the last five years, 161 patients have been treated by allogeneic BMT in our center. The median age was 49 years (range: 16-66 years). The underlying diseases were ALL (19), AML (51), myelodysplasic syndroms (11), CLL/NHL (46), Hodgkin disease (3), multiple myeloma (18), aplasia (4) and myeloproliferative syndroms (9). 52 patients were allografted with HLA identical sibling donors, 60 with unrelated matched donors, 25 with mismatch unrelated donor and 24 with unrelated mismatched cord blood. 86 patients received a reduced conditioning regimen. 32 patients (20%) had a grade II-IV aGVHD refractory to steroid (9 cutaneous, 19 intestinal and 4 hepatic GVHD). All these patients received Inolimomab (0,3 mg/kg) daily during 10 to 28 days and then tapered. Median follow up was 18 months. Results: Therapy was well tolerated for the 32 patients. Among them, 16 achieved a complete remission and 13 had a partial remission of aGVHD. Failure was observed in three cases. Complete remission occurred more frequently for cutaneous aGVHD (78%) than for intestinal aGVHD (37%). TRM was 6%, 17% and 22% at 100 days, one and two years respectively. Ten patients died from day 79 to day 853 post transplantation : cause of death was refractory aGVH for three patients, late infections associated with refractory chronic GVHD for three patients and hemopathy relapse in four cases. EFS and overall survival at 2 years were 64% and 69% respectively. Interestingly, during the same period, overall survival was similar for these 32 patients as compared to the overall survival for the other patients without refractory steroid aGVHD. However, the occurrence of extensive chronic GVHD was particularly elevated after Inolimomab therapy (52%). Conclusion: Our report confirms that Inolimomab is an effective therapy for steroid refractory aGVHD with a high rate of long term survival. However, quality of life of the patients was frequently impaired by a chronic GVHD. M.Y. Shapira*, I.B. Resnick, P.D. Tsirigotis, M. Aker, P. Stefansky, B. Gesundeheidt , R. Or Hadassah -Hebrew University Medical Center (Jerusalem, IL) Alefacept (Amevive®) is an immunosuppressive dimeric fusion protein that is currently used for psoriasis control. We have recently showed its effect in acute steroid resistant/dependent and chronic extensive GVHD, in a protocol using similar timing as in psoriasis (e.g. once or twice weekly). In this study, we describe the use of induction of alefacept treatment (e.g. 7 consecutive days of IM 15mg alefacept) followed by bi-weekly maintenance (if needed) in combination with tacrolimus in acute steroid resistant/dependent GVHD. Patients: 14 patients were treated in this cohort (8 males, 6 females, median age 48.5 years (range 4-66)). Pretreatment GVHD grade ranged 2-4 (median 3) and involved the skin (14), gut (12) and liver (5). All patients received at least cyclosporine and steroids before the induction of alefacept and tacrolimus. The median time from transplantation to alefacept and from GVHD to alefacept was 41 and 14 days respectively (range 22-110d and 5-65d) and a median of 7.5 (range 5-20) injections were given per patient. Results: 11 out of the 14 patients showed response. The response was either marked (n=7) or moderate (n=4).In 9 of the patients, we were able to decrease the steroid dose significantly and in 4 of them it was completely stopped. Complications included diabetes and infections. All these events may be related to other drugs given simultaneously. Currently, 6/14 patients are alive all with improved GVHD. Eight patients died due to GVHD progression (4), infections (2), GI bleeding (1) and relapse of the basic disease (1) . Conclusion: alefacept is effective and safe for the treatment of acute steroid resistant/dependent GVHD. We feel that an induction treatment may be more effective, although dose and treatment's time intervals should be further explored. Acute graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic stem cell transplantation (SCT), especially when the graft is from unrelated donors or HLA-nonidentical family members. Because cytokines are important mediators, the modulation of different cytokines in the microenvironment may play an important role in determining the occurrence and severity of an immune response. Previous studies show that inflammatory cytokines such as tumor necrosis factor alfa (TNFa), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-18 (IL-18) are involved in the pathogenesis of aGVHD, and that the excess of these cytokines is associated with severity and mortality of aGVHD. We hypothesized that removal of these excessive cytokines from patients f blood at the onset of aGVHD might improve the treatment outcome. A novel absorbent CTR can effectively adsorb small-to middlesized proteins like cytokines and enterotoxins in vitro. In view of future exploitation of extracorporeal treatment using CTR column, we tested whether CTR could remove these inflammatory cytokines from blood. When the serum containing a mix of recombinant cytokines was incubated with a CTR adsorbent for 2 hrs, 55% of TNFa, 81% of IL-6, 83% of IL-8, and 22% of IL-18 were successfully removed. Next, we measured TNFa, soluble TNFa receptor 1 (TNFR1), IL-6, IL-8, and IL-18 levels in serum samples obtained from 5 patients (median age 38y, range 26-63y) who underwent myeloablative SCT in 4 and non-myeloablative SCT in 1. AGVHD developed in 2 with grade 3 and in 3 with grade 2. When cytokine levels in patients were expressed as a ratio to the mean cytokine level in control serum samples obtained from three healthy individuals, the mean ratios of TNFa, TNFR1, IL-6, IL-8, and IL-18 at the onset of aGVHD were 6.0 (range, 1.2-12.0), 6.5 (2.5-9.0), 274 (3.5-651), 48.3 (11.3-75.2) , and 6.7 (3.2-10.8 ). The CTR adsorption considerably reduced the concentrations of these cytokines except for IL-18. The adsorption rates of these cytokines were 64% for TNFa, 48% for TNFR1, 59% for IL-6, more than 94% for IL-8, and 0% for IL-18. The efficient removal of inflammatory cytokines suggests that extracorporeal blood purification with CTR column may be effective in the treatment of aGVHD. This treatment strategy may be promising because it essentially has no deleterious effects on immune functions of SCT recipients unlike other GVHD treatments. The dose of CD34+ cells negatively impacts the outcome of patients treated with genoidentical allo SCT prepared with a reduced-intensity conditioning D. Blaise*, L. Farnault, C. Faucher, S. Furst, J. El Cheikh, P. Ladaique, N. Vey, R. Bouabdallah, A.M. Stoppa, C. Lemarie, B. Calmels, M. Mohty, C. Chabannon Institut Paoli-Calmettes (Marseille, FR) The impact of graft composition on transplant outcome has been widely analyzed in the setting of myeloablative regimen for both BM and PBSC. However little is known for reducedintensity conditioning (RIC) prepared allo PBSCT. We report here such an analysis in a cohort of 100 patients with hematological malignancies (HM) treated with geno-identical SCT after the same RIC in a single center from 2000 until 2006. All grafts were PBSC from a match sibling donor. All grafts were monitored for CD34 (5. 6 (1.5-22) All patients received oral busulfan (8mg/m), thymoglobulin (2.5 mg/kg) and Fludarabine (150 to 180 mg/m) (FBT conditioning). All patients received post-graft CSA. Median age was 50 (18-64). Diagnoses included acute leukemia (39%), Myeloid (16%) or Lymphoid (45%) malignancies. 53 pts were in CR. All but one engrafted reaching full lymphoid donor chimerism prior to day 100 in 85% of the cases. 55 pts presented aGVHD (G1: 12; G2: 22; G3: 12; G4: 9) for a cumulative incidence (CI) of G2-4 aGVHD of 43% (33-53); 91 patients were evaluable for cGVHD with a 79% (71-87) CI (Lim= 20%; Ext: 80%). In a multivariate analysis cGVHD occurrence could be predicted by 2 independent factors: lower dose of CD34 (but no impact of CD3, CD4, CD8, CD19 or CD56) (odd ratio (OR): 0.79 (0.69-0.90)) and grade 2-4 aGVHD (OR: 1.16 (1.01-1.32) ). TRM CI was 14% at 12 months. Relapse CI was 15% (8-22) at a median of 169 days (30-769). Disease control was significatively associated with cGVHD occurrence (Relapse CI: cGVHD: 10% (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) ; no cGVHD: 42% (14-70): p=.01). 5 year overall survival (OS) and LFS probability estimates are 62 % (50-72) and 60% (48-72) with a plateau starting after 3.5 years. Outcome was similar for the patients above or under 50 and for the different diagnoses. In a Cox model analysis, LFS was independently positively affected by only 2 pre-transplant variables: a CR status at time of transplant (OR=0.45; p=.022) (LFS: CR: 67%; non CR: 52%; p=.03) and a lower dose of infused CD34+ cells (OR: 2.04; p=.039) (LFS: < 5.6x10 6 : 69%; > 5.6x10 6 : 50%: p=.04). These results suggest that after RIC, graft composition probably in relation with G-CSF mobilization might impact allogeneic effect and invite revisiting this subject in this context. Proportions of CD4+CD25++ cells were also higher 1-2 weeks after beginning of clinical manifestation (all patients except two were on steroids) as well as after completion of steroid therapy (3-4 weeks after first measurements), (ii) more frequently FoxP3+ lymphocytes percentage above 10% (9/31 vs 1/21, p=0.03) and (iii) higher levels of CD4+ and CD4+CD25++ cells co-expressing CD134 (5.7%±0.9 vs 2.4%±0.4, p=0.0006 for CD4+ cells and 0.1%±0.02 vs 0.03%±0.007, p=0.03 for CD4+CD25++ cells), as compared to those lacking aGvHD. CD4+CD25++ cells were positive for FoxP3 in 76.3%±2.0 while CD4+CD25-cells had FoxP3 in 12.9%±1.2 only. Higher percentages of CD4+CD25++ cells were associated with a higher risk of (i) post transplant sever complication including Herpes viruses reactivation, grade IV aGvHD or disabilitating extensive cGvHD, fatal septic complications, relapses, marrow failure (0.52%±0.07 vs 0.21%±0.05, p=0.04), (ii) lack of CD8+ cells specific for CMV and EBV peptides (positive result for CMV or EBV pentamer staining was seen only in 2 out of 12 measurements when CD4+CD25++ were above 0.5%, in contrast, when CD4+CD25++ cells were lower than 0.5% of lymphocytes 24 and 27 out of 74 cases were positive for CMV and EBV pentamer staining, respectively). In conclusion, CD4+CD25++ cells being frequently activated (CD134+) and in a majority furnished in suppressor cell machinery (FoxP3+) were higher in a proportion when aGvHD was clinically apparent, also a number of CD134+ cells and contribution of CD4+CD134+ to the pool of CD4+ cells was elevated in aGvHD cases. Therefore, activation of the immune system during alloreactivity is likely balanced by an increase in a proportion of Treg cells (CD4+CD25++FoxP3+) and this increase constituted a risk factor of (i) viral reactivation, (ii) fatal outcome of aGVHD, (iii) marrow failure and/or relapse. Supported by FP6 AlloStem project (LSHB-CT-2004-503319) and 2P05E 037 30 grant from the Polish Ministry of Science and Higher Education. Impact of high-resolution HLA typing on outcome after ATG-based unrelated stem cell transplantation N. Kröger*, T. Zabelina, T. Binder, F. Ayuk, U. Bacher, J. Petersen, H. Lellek, A. Muth, M. Hartwig, C. Wolschke, G. Amtsfeld, R. Erttmann, T. Eiermann, A.R. Zander University Hospital Hamburg (Hamburg, DE) Allogeneic stem cell transplantation (SCT) from unrelated donors is an acceptable transplant option for patients lacking a HLA-identical sibling. We evaluated the impact of high resolution HLA-typing of HLA-A,B,C,DRB1,DQB1 on outcome in 269 patients with a median age of 41 years (r, 1-68), who received unrelated stem cell transplantation after reduced (n=105) or standard myeloablative conditioning (n=164). The diagnosis were Acute leukemia (n=106), MDS (n=29), CML (n=39), lymphoid malignancies (CLL, Myeloma, NHL,HD) (n=60), myelofibrosis (n=14) and non malignant disorders (n=21). Stem cell source was BM (n=104) or PBSC (n=165) and diseases were classified as bad (n= 160) or good risk (n= 109). All patients received ATG-Fresenius® at a median dose of 60mg/kg (r, 20-120 mg/kg). Completely matched HLA (10/10) graft was transplanted in 110 (41%) of the patients, while transplantation with at least one mismatch was performed in 158 (59%) of patients. In those 158 patients one mismatch was seen in 91, 2 mismatch in 44, 3 mismatch in 15 and 4 mismatch in 8 patients. The HLA-mismatches were located in class I (n=134) or II (n=54). The incidence of acute graft-versus-host disease (GvHD) grade II-IV for the entire group was 36% and grade III/IV was 18%. The incidence of aGvHD grade II-IV did not differ significantly p=0.6) between the HLA-groups: 10/10: 34%, 9/10: 42%, 8/10: 44% and 7or 6/10: 36%. The same was true for non-relapse mortality: 10/10: 29%, 9/10: 32%, 8/10:43% and 7or 6/10: 22% (p=0.6) resulting in a similar event-free survival at 5 years: 10/10: 41%, 9/10: 41%, 8/10:37% and 7or 6/10: 42%. In a multivariate analysis the most significant factor for NRM was age (HR: 1.024; 95%CI: 1.010-1.037, p<0.001), for relapse bad risk disease (HR:2.966: 95% CI: 1.682-5.229, p<0.001) and for EFS: age (HR:1.013; 95% CI:1.003-1.022, p=0.007) and bad risk (HR: 1.946: 95% CI: 1.344-2.817, p<0.001) These results show that HLA mismatch allogeneic stem cell transplantation from unrelated donor with an ATG based conditioning can be performed without worsening the results in comparison to a fully (10/10) matched unrelated graft. (BU+Cph) was used in 39 pts, redused-intensity in 10 pts. The control group consist of 8 pts, undergoing autologous HCT after myeloablative conditioning and 17 healthy donors. Results: acute GVHD in 23 pts (47%) was diagnosed and s-HLA in 21 pts with aGVHD (91%) were revealed. In patients without aGVHD s-HLA were not revealed excluding 4 pts in whom in 19, 27, 41 and 84 days after HCT s-HLA were observed. In all of this pts cGVHD was diagnosed later. A correlation between severity of aGVHD and the level of s-HLA was estimated. It is interesting that in pts after myeloablative conditioning the time of appearance s-HLA and development of aGVHD coincided just as in pts after redused -intensity regimen the time of appearance s-HLA preceded of clinical manifestations of aGVHD (p< 0,05). Chronical GVHD in 30 pts (61%) was diagnosed, s-HLA in 97% of these pts were revealed. Therefore the presence of s-HLA associated with acute or chronical GVHD in the most of the patients. The appearance of s-HLA in some pts without aGVHD in 61,0 ± 8,12 d after HCT preceded of development of cGVHD in all cases in 149,0±14,8 days , p < 0,05 ). Conclusion: The appearance of s-HLA after HCT is specific for acute and cGVHD and can be used for diagnose , what is important particularly in pts with absence of clear clinical manifestations of diseases. Rituximab as salvage therapy for refractory chronic graftversus-host disease M. Mohty, N. Marchetti, J. El-Cheikh, C. Faucher, S. Fürst, D. Blaise Institut Paoli-Calmettes (Marseille, FR) The incidence of chronic graft-versus-host disease (cGVHD) is likely to continuously increase as a result of the increasing use of allo-SCT in older patients, PBSCs as stem cell source, and frequent DLIs, all of which are known to increase the risk of cGVHD. Standard primary treatment of cGVHD remains a combination of corticosteroids (CS) and calcineurin inhibitors. There is no standard therapy for those who fail to respond to CS, and CS-resistant GVHD is associated with high morbidity. The aim of this report was to analyze the outcome of patients treated with rituximab RTX as salvage therapy for refractory cGVHD. 15 consecutive patients experiencing severe or refractory GVHD received IV infusions of RTX (375 mg/m²/infusion) at weekly intervals for 4 weeks. Responding patients were allowed to receive one or 2 courses of maintenance therapy. Response to RTX was assessed one month after the last infusion. Patients' characteristics, GVHD features and outcome are summarized in the Table below. RTX was administered at a median time of 178 (range, 69-1136) days after allo-SCT, and patients received a median of 5 (range, 1-12) infusions. Most have received and failed at least 2 lines of immunosuppressive therapy prior to RTX. With a median follow-up of 118 days (range, 21-834) from first infusion of RTX, no major toxicities directly related to RTX were observed. Overall, 10 patients responded to RTX administration (66%; 95%CI, 42-90%) with 3 CRs. In those responding patients, the patient felt improvement as soon as one week after the first RTX infusion. In addition, RTX allowed a significant reduction of CS dosage (range, 0-83%). 4 patients did not respond and died from refractory GVHD, while one responding patient died of disease progression. With a median follow-up of 461 days from onset of cGVHD (range, 91-1192), the actuarial survival rate from the first RTX infusion was 60% at one year. We conclude that despite its limited size, this cohort demonstrates evidence of beneficial activity of RTX, mainly in the classical cutaneous, mucosal and liver cGVHD. Patients with involvement of other sites are not likely to benefit much from RTX. Results achieved with RTX in the cGVHD refractory setting, pave the way for further developments. As such, the addition of RTX to prednisone for the initial treatment of cGVHD is worth further investigations, both to increase the overall response rate, and to enable a more rapid CS taper while incurring less long-term toxicity. (2), Y. Hicheri (2) , C. Pautas (2) , R. Barouki (1) , G. Socie (3), M. Kuentz (2) , C. Cordonnier (2) ( Introduction : We have shown [1] that a calcineurin activity (CA)<28 pmol RII/mg/min was associated with the absence of acute GVHD after allogeneic stem cell transplant (SCT). We thus expected that CA monitoring would be more useful in adapting the cyclosporine (CsA) doses for GVHD prophylaxis than CsA blood levels. The aim of this prospective study was to evaluate the feasibility of adapting the CsA doses to the CA values either until GVHD occurs, or until day 100, whichever occurs first. A first clinical evaluation at day 35 was designed in the study, and we report here its results. Methods : CA and CsA levels were measured, as previously reported [1] , at least once a week from day d0-d15 and then twice a week from d16-d35. The CsA doses were adapted in order to maintain a CA<28 pmol/mg/min from transplant by 20% progressive changes of the CsA daily dose. Adaptation was stopped in case of CsA blood levels ≥600 ng/ml, creatinine clearance <40 ml/min, serum bilirubin >40 mmol/L, or severe clinical toxicity. Results : 39 patients were included and the frequency of severe GVHD occurence was similar to that reported in the litterature. However, 2 groups of patients were clearly identified when we looked at the frequency of CA determinations performed during the period of time d0-d20, i.e. from the day of transplant to the median time of GVHD occurence. The 2 groups of patients have similar ratio of HLAid /unrelated donor, and a median time of GVHD occurrence of 18 days. Severe GVHD occured for 10 out of the 21 patients monitored for CA every 3 to 5 days whereas it occured for 13 out of the 18 patients monitored for CA every 6 to 7 days. The predictivity of CA monitoring for the occurrence of GVHD was higher when it was performed every 3 to 5 days vs every 6 to 7 days (53% vs 20%, odds ratio of 4.57 with a 95%CI of 0.95-22.01). The frequency of severe GVHD occurence was reduced in the group of patients with CA monitoring every 3 to 5 days compared to the group of patients with CA monitoring every 6 to 7 days (47.5% vs 72%, odds ratio of 2.86 with a 95%CI of 0.76-10.75). Conclusion: These results establish that CsA adaptation is not only feasible on the basis of CA levels, but also that repeated CA determinations are required to avoid missing an increase of CA during an episode of acute GVHD. In conclusion, a regular and frequent CA monitoring appears as a promising functional management of GVHD prophylaxis. [1] Sanquer et al: Transplantation 2004; 77:854-8 . Treatment of acute and chronic GvHD by extracorporeal photophoresis H. J. Kolb*, J. Ullmann, J. Tischer, G. Ledderose University of Munich (Munich, DE) Acute and chronic graft-versus-host disease are the major obstacles of allogeneic stem cell transplantation. Unlike prevention treatment of GVHD has remained a difficult task particularly in patients not responding to the treatment with corticosteroids. Refractoriness to corticosteroids was defined as no improvement or progression after 3 days of treatment with 1 -2 mg/kg methylprednisolone every 8 hours. Concommittant immunosuppressive prophylaxis consisted of cyclosporin A only or in combination with and mykophenolate mofetil. Sixteen patients were treated for acute GVHD and 26 patients for chronic GVHD. At the start of extracorporeal photophoresis (ECP) 2 treatments were given per week until clinical response and one ECP per week thereafter. Complete response (CR) to ECP was defined as the possibility of discontinuation of immunosuppressive therapy including discontinuation of methylprednisolone. Partial response (PR) was defined as the discontinuation of immunsuppressive therapy and reduction of methyprednisolone treatment to less than 20 mg per day. Using these criteria 3 out of 15 patients with acute GVHD had a CR and 9 patients a PR. At the time of evaluation patients were alive between 96 and 655 days. Chronic GVHD responded to the treatment with ECP in 5 out of 26 patients with CR and in 12 out of 26 patients with PR. In addition 7 patients had stable disease without deterioration. Ther observation time after ECP was between 359 and 2517 days. At the time of evaluation onepatient had died of cerebral hemorrhage and one patient of progressive disease. Particularly encouraging was the finding that 2 patients with bronchiolitis obliterans improved lung function after treatment with ECP and rapamycin. We concluded from our observation that ECP is a valuable form of treatment of acute and chronic GVHD. A prospective randomized study using the same entry and response criteria should be performed. Background: The role of FoxP3+/CD4+/CD25+ regulatory Tcells (Tregs) in graft-versus-host disease (GVHD) is still unclear. In murine models, Tregs promote donor bone marrow engraftment and decrease the severity of GVHD after allogeneic haematopoietic stem cell transplantation (HSCT). In humans, an increased number of peripheral blood CD4+/CD25+ T cells is associated with chronic GVHD. In contrast, FOXP3 mRNA expression is reduced during acute and chronic GVHD. Because the evaluation of Tregs in peripheral blood may not reflect their presence in target organs, we evaluated the number of Tregs in skin specimens obtained from GVHD patients. Patients and Methods: Skin specimens were taken from biopsy of 22 allogeneic HSCT recipients with cutaneous rashes clinically suspicious for GVHD. 12 pts received an HSCT from identical sibling donor and 10 from an unrelated one. 8 were T-cell depleted HSCT. Stem cell source was peripheral blood in 15 pts, bone marrow in 6 pts and cord blood in 1 pt. Conditioning was myeloablative in 6 pts, reduced intensity (RIC) in 16 pts. 17 pts experienced aGVHD (grade I 6 pts, II 2 pts, >II 9 pts), 6 pts cGVHD (2 limited, 4 extensive). Tregs were analyzed by immunohistochemistry in serial sections from 23 paraffin-embedded skin GVHD specimens using anti-FoxP3, anti-CD4 and anti-CD25 antibodies. The number of FoxP3+/CD4+/CD25+ Tregs was quantified (mean number/HPF calculated in 5 HPF) and related to the number of CD3+ T lymphocytes. Results: We found that GVHD skin specimens were characterized by a CD3+ prevalent lymphocytic infiltrate. All FOXP3+ cells were CD3+ and co-expressed CD4 and CD25. In GVHD targeted skin specimens we found a low mean number of FOXP3+/CD4+/CD25+ Tregs in comparison with CD3+ cells (6.29±7.30/HPF vs 88.87±100/HPF, p<0.0001) with a mean percentage of Tregs of CD3+ T lymphocytes of 10.5%. The mean number of FOXP3+/CD4+/CD25+ Tregs was higher in myeloablative vs. RIC (11.17±10.29 vs. 4.57±5.31, p=0.05); and higher in GVHD grade I vs. >I (9.46±11.3 vs. 3.85±3.48, p=0.12), although such differences did not reach statistical significance. No difference was found between the number of Tregs in acute vs. chronic GVHD. Conclusion: The present finding of a low number of Tregs in GVHD skin specimens, may explain the deficient control by this lymphocytic subtype on GVHD. Such result should be confirmed in a large cohort to support strategies aimed to increase the number of Tregs following allogeneic HSCT. M. von Bonin, F. Stoelzel, M. Binder, A. Goedecke, K. Richter, N. Wuschek, G. Ehninger, T. Illmer, U. Platzbecker, M. Schaich, J. Schetelig, K. Holig, M. Schmitz, J. Babatz, M. Bornhauser TU Dresden (Dresden, DE) Acute GvHD remains the major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Patients with grade III-IV acute GvHD who do not respond to steroid treatment after 5-7 days have a disappointing outcome with all immunosuppressive salvage regimens tested so far. Mesenchymal stem cells (MSC) possess immunomodulatory properties in-vitro and have been successfully used in patients with steroid-refractory GvHD. Ten patients with haematological malignancies who suffered from grade III (n=3) and IV (n=7) acute GvHD received between 2 and 5 intravenous infusions of 0,555 -1,1 x10 6 /kg third-party MSC within individual compassionate use applications. MSC were expanded using lysates of thrombapheresis products as GMP-grade serum source. Quality criteria included sterility, clonogenicity (CFU-F), osteogeneic/adipogenic differentiation and suppression of PHA-induced CD4 T cell proliferation. All patients had grade 2-4 hepatic (n= 7) and/or gastrointestinal (n=9) and/or skin (n=3) GvHD by the time of MSC infusion. Previous and concomitant treatment for acute GvHD included methylprednislone (2-5 mg/kg, n=10) , infliximab (n=8), budesonide (n=10), pentostatin (n=5), alemtuzumab (n=1) and MTX (n=1). Seven out of ten patients responded to MSC application within 28 days after the first infusion. Five out of seven patients with initial liver involvement responded to therapy whereas only 4 out of 9 patients with dominating gut GvHD experienced clinical improvement. Extracorporeal photopheresis was used as maintenance therapy in 4 patients. One patient received two courses of pentostatin salvage therapy. After median follow-up of 191 days (range 60 to 326), five patients are alive without relapse of the underlying malignancy. Reasons for death were liver failure (n=3), diffuse alveolar hemorrhage (n=1) and transplant associated microangiopathy (n=1) all probably related to GvHD except for one due to liver toxicity. No surviving patient is off immunosuppression, so far. In summary, our experience supports the potential efficacy of MSC in the treatment of steroid refractory acute GvHD. Predominant reponses of hepatic GvHD might point towards a preferential homing of MSC in some cases. Further controlled studies will hopefully define the appropriate timing and dosing of MSC infusions and potential response predictors in patients receiving MSC as salvage therapy for acute GvHD. Autoantibodies are detected in patients with chronic GvHD as well as in the absence of GvHD after allogeneic HSCT I. Hilgendorf *, B. Mueller-Hilke, R. Claus, J. Casper, C. Junghanss, M. Leithaeuser, M. Freund, D. Wolff University of Rostock (Rostock, DE) Background: Chronic graft-versus-host disease (cGVHD) and autoimmune disorders have many clinical and laboratory features in common. In this study, we analysed the frequency of autoantibodies in 31 patients after allogeneic haematopoietic stem cell transplantation (alloHSCT). Methods: During routine follow-up visits peripheral blood samples from alloHSCT recipients were tested for: antinuclear antibody (ANA), anti-neutrophil cytoplasmatic antibody (ANCA), antimitochondrial antibody (AMA), anti-smoothmuscle antibody (ASMA) and double stranded DNA (dsDNA). Furthermore the counts of B-and T-lymphocyte subpopulations including naïve CD4+ CD45RA+CD31 recent thymic emigrants and CD4+CD25+FOXP3+ regulatory T-cells were determined by flowcytometric analysis. Chronic GVHD was evaluated using the criteria and guidelines of the National Institute of Health. Results: 20 patients with and 10 patients without history of cGVHD were analysed. The median age was 49 years (20-67), the median time after transplantation was 832 days (124 -2914). In patients with cGVHD ANA was positive in 35% (7/20), ASMA in 20% (4/20) and AMA in 5% (1/20) . ANA was positive in 36% (4/11) and ASMA in 27% (3/11) of patients without cGVHD. AMA and dsDNA were negative in all patients without cGVHD and ANCA was negative in all tested patients. More than one antibody occurred in 10% (3/31) of patients. ANA was found in 47% (7/15) of mVUD and 31% (4/13) of mVRD transplant recipients, whereas ASMA was positive in 33% (5/15) of mVUD and 15% (2/13) of mVRD transplant recipients. No antibodies were detected in mmVUD (3/31) transplant recipients. The count of CD4+CD45RA+CD31+ Tcells was 17.5x10 6 /l (0.57-194) in patients with and 20.5x10 6 /l (2.2-237) in patients without cGVHD. The median count of CD4+CD25+FOXP3+ regulatory T-cells was 4.8x10 6 /l(0.87-33.9 )in patients with and 5.29x10E6/l(0.68-28.5)in patients without history of cGVHD. Conclusion: In this small cohort no difference was detectable with regard of the occurrence of autoantibodies as well as the counts of naive thymic precursors, regulatory T-cells and Bcells in the peripheral blood in patients with or without cGVHD and autoantibodies are not a distinctive feature of chronic GVHD. Since peripheral lymphocyte counts are influenced by the time point after transplantation as well as immunusuppression a prospective evaluation of the association of lymphocyte subsets with the GVHD status is crucial. (2), F. Aljassim (2) , M. Brune (1) (1)Sahlgrenska University Hospital (Gothenburg, SE); (2) Queen Silvia Children's Hospital (Gothenburg, SE) Background and Methods: Obliterative bronchiolitis (OB) with chronic airway obstruction is a potential fatal complication after allo-SCT. It is considered to be the Graft vs Host Disease (GvHD) of the peripheral airways and is tentatively defined as a FEV1 <80% of predicted value. We studied multiple-breath inert gas washout (MBW) in addition to spirometry for the assessment of lung clearance index (LCI) and ventilation inhomogeneity in conducting airway zone (Scond) and more peripheral airways (Sacin) using SF6 as an inert marker gas ( Am J Respir Crit Care Med 1998; 157: 1573) . Aim: To use MBW for the assessment and characterization of airway obstruction in pts with and without OB, as defined above. Subjects (1), lymphoma (7), myeloma (2) . Mean follow-up post allo-SCT was 3.8 (1.3-7.3) years and mean age was 49.5 (20-72) years. At the point of investigation, 29 pts were on corticosteroids due to ongoing or recent GvHD Results: 12 patients (20%) fulfilled FEV1 criteria for OB. They showed significantly greater LCI and particularly elevated (Sacin), as assessed by MBW, see Table ( all FEV1 are given as % of predicted value). We also found 6 patients (10%) who had slight elevated LCI, but FEV1 >80%. Conclusion: Airway obstruction as assessed by MBW after allo-SCT affects the most peripheral airways, as shown by elevated Sacin, and to a lesser extent the conducting airways. Using MBW instead of spirometry, for characterization of airway obstruction seemingly gives more information of what airway level is affected. Perhaps can MBW detect early changes in the peripheral airway, as suggested by the finding of elevated LCI in pts with normal spirometry. Sirolimus combined with tacrolimus as graft-versus-host disease prophylaxis after allogeneic stem cell transplantation J. Mattsson* (1), P. Svenberg (1) We enrolled 24 patients in a phase II study in order to test the efficacy of sirolimus and tacrolimus in both children and adults after SCT. The results were compared with control patients receiving cyclosporine (CsA) based GVHD prophylaxis. The controls were matched for diagnosis, age, donor, stem cell source and conditioning therapy. Diagnoses in both groups included SAA (n=7), inherited disorders (n=13), Fanconi anemia (n=3) and ALL (n=1). Median age for patients receiving sirolimus and tacrolimus was 11 (1.5-63) years compared to 9 (0-39) years for patients given CsA (ns). Sibling donors were used in 7 and 8 transplants and 13 patients received stem cells from matched unrelated donors in both groups. TBI combined with cyclophosphamide (Cy) were given to 4 and 5 patients in each group. Busulfan combined with Cy were administered to 5 and 7 patients in each group. Reduced intensity conditioning was given to 15 patients in the sirolimus group compared to 12 in the controls. Twelve patients in the control group received CsA combined with MTX as GVHD prophylaxis. In 5 patients receiving cord blood, CsA was combined with prednisolone. Bone marrow was the main stem cell source in each group. Sirolimus was discontinued median 3.25 (1.5-11) months after SCT. Tacrolimus was discontinued 14 (3-29) months compared to 17 (9-39) months for patients receiving CsA. One patient in the sirolimus group was diagnosed with VOD after SCT and none in the controls. Moderate to severe mucositis was more common in patients receiving sirolimus, 16 patients vs 4. There was a tendency for faster engraftment in the CsA group 16 (11-54) vs. 21 (11-37) days (p=0.06). Bacteraemia was diagnosed in 5 (21%) patients receiving sirolimus compared to 11 (46%) in the controls (p=0.12). Rejection occurred in 4 patients given sirolimus compared to 2 patients in the CsA group (ns). The cumulative incidence of acute GVHD was 22% in the sirolimus group vs. 17% in the controls (ns). No patient developed acute GVHD grades III-IV. The cumulative incidence of chronic was 29% and 31% (ns). The 3-year overall survival was 100% in patients treated with sirolimus+tacrolimus compared to 84% in patients receiving CsA based regimen. Conclusion: Sirolimus combined with tacrolimus as GVHD prophylaxis is safe, associated with low incidence of moderate to severe acute GVHD and excellent survival. We have therefore now started a prospective randomized study comparing sirolimus+tacrolimus with CsA+MTX. A. Müller*, K. Kentouche, J. Dörnbrack, R. Häfer, F. Zintl, J.F. Beck Friedrich Schiller University Jena (Jena, DE) Detection of inflammatory cytokine producing cells by the ELISPOT assay is a sensitive but time consuming method. Current flow cytometry assays for cytokine detection do not represent the in vivo situation. We developed a 3 dimensional ELISPOT-like assay for the determination of cytokine producing cells. Microbeads were covalently coated with catch antibodies. The beads were cultivated together with the patients cells and after incubation separated from the cells. Cytokines bound on the beads were detected by a second direct fluorescence dye marked antibody. Positive events were measured by flow cytometry. As a negative control the beads were cultured and processed without cells. As a positive control served a known standard concentration. We further confirmed the result using two different cell concentrations. For verification of the assay we used MNC from 5 healthy donors treated with/ without staphylococcus enterotoxin B (SEB). The number of cytokine producing cells are shown in Tab.1: Stimulation of MNC from 4 healthy donors using LPS resulted in a much higher number of cytokine producing cells Tab.2: First results of testing MNC from patients suffering from extended cGvHD during extra corporeal phototherapy points to a very early detection of viral (IFN-a) or bacterial (IL-6) infections before clinical onset. Moreover the frequency of TNF-a and IL-1 á producing cells decreased in patients with a positive therapy course. Summary: The 3-dimensional ELISPOT assay may be a useful method for guiding immunotherapy in patients suffering from severe complications after stem cell transplantation. Donor/recipient matching for short tandem repeat D3S1358 predicts severe acute GvHD in a chimerism mediated fashion after HLA-identical allogeneic stem cell transplantation C. Manzano*, D. Barroso, P. Balsalobre, D. Serrano, A. Gómez-Pineda, J.L. Díez-Martín, I. Buño Hosp. G.U. Gregorio Marañon (Madrid, ES) Background: Short tandem repeats (STR) are routinely used to distinguish donor (D) and recipient (R) DNA and therefore analyse chimerism after allogeneic stem cell transplantation (SCT). However, the influence of particular STR alleles in D and R on the outcome of SCT has been scarcely studied. Objective: To evaluate the association between D/R STR matching and the dynamics of chimerism and the development of complications post-SCT (rejection, relapse, graft versus host disease -GVHD-and exitus). Patients and methods: 32 patients that received SCT from an HLA-identical related donor were analysed. Multiplex PCR was performed to amplify 11 STR (SGM AMPflSTR Plus, Applied Biosystems) loci from D and R genomic DNA (QIAamp Blood Minikit, Qiagen) obtained from peripheral blood samples before SCT. PCR products were revealed by capillary electrophoresis and analysed using the Genotyper software (Applied Biosystems). Results were analyzed using Fisher's exact test due to the reduced sample size. Results: An association with statistical significance was observed between D/R full allele (2 out of 2) matching for STR D3S1358 and the development of severe (grades III-IV) acute GVHD (62,5% in fully matched D/R pairs vs 16,7% in partially matched/mismatched D/R pairs; p=0,023, Table 1 ). Furthermore, these patients had a significantly lower incidence of mixed chimerism (MC) in peripheral blood during the first month post-SCT (0% vs 43,5%; p=0,032; Table 1 ). Discussion: STR D3S1358 locus is localized within the LARS2 gene which encodes for an enzyme that catalyzes the charging of tRNAleu with leucine, an essential step in protein synthesis. Although the impact of STR D3S1358 on LARS2 gene expression or protein function is unknown, other polymorphisms (SNP A3243G) in this gene have been associated with susceptibility to the development of Type 2 diabetes (Diabetes 2005; 54:1892) . In spite of this lack of knowledge, our results suggest that D/R identity for STR D3S1358 would promote a higher alloreactivity of donor cells, which would favour the establishment of complete chimerism and subsequently increasing the risk of acute GVHD. If these results were confirmed, LARS2 would represent a novel chimerism mediated GVHD susceptibility gene. We report our results using extracorporeal photopheresis (ECP) for the treatment of chronic graft-versus-host disease (cGvHD). Between 2002 and 2007, 30 patients with steroidrefractory or -intolerant cGvHD received ECP for a median duration of 9.6 (range 3.5-58.8) months. HSCT types were 18 HLA-matched related donor (MRD), 11 HLA-matched unrelated donor (MUD) and 1 HLA-mismatched unrelated cord blood donor. Standard GvHD prophylaxis included cyclosporine-based (7) and tacrolimus-based (23) regimens. The median follow-up time after ECP initiation was 17.5 (5.3-71.8) months. Among 26 patients with adequate follow-up (>6 months post ECP initiation), 9 (35%) and 17 (65%) cases had moderate and severe cGvHD by CIBMTR criteria. The sustained overall and complete response (OR and CR) rates were 62% (16/26) and 35% (9/26) with the maximum response occurring at the median time of 1.3 (0.2-3) months from ECP initiation. OR and CR rates by CIBMTR severity grading were 67% (6/9) and 56% (5/9) in moderate group, and 59% (10/17) and 24% (4/17) in severe group. Steroid-sparing (steroid discontinuation or requiring adrenal doses only) was achieved in 58% (15/26) of this cohort and 81% (13/16) of the responders, and occurred a median 4.13 (0.9-29.7) months after ECP. Among the 16 responders, five cases (31%) had recurrent cGvHD with a median cGvHD progression free survival of 20.4 (95% confidence interval (CI),18.7-22.1) months. Overall, four patients required re-initiation of steroid with a median steroid-free period of 45 (95%CI, 0-90.4) months. Factor associated with OR was the Johns Hopkins prognostic scores at ECP initiation of ≥2.5 (P=0.045), while that with CR was early initiation of ECP within 12 months of cGvHD diagnosis (P=0.045). The overall *mean survival time after ECP initiation was 53.9 (95%CI, 40-68) months. Type of donor was associated with survival post ECP initiation (P=0.0468). The patients with HLA-MRD survived longer than those with HLA-MUD (*mean survival times (95%CI), 59. 3 (44.8-73 .7) and 29.7 (19.7-39.8) months). However, OR and CR to ECP were not associated with prolonged post ECP initiation survival. In summary, our results suggest that ECP is an effective second-line treatment option for cGvHD especially with early initiation. HLA-MUD was associated with poor survival rates in patients receiving ECP for cGvHD. Whether response to ECP would be translated to higher survival requires a larger study. *Median survival times were not reached. Methods: A decision analytic model was developed based on a double blind randomized trial that compared posaconazole with fluconazole antifungal prophylaxis in recipients of allogeneic HSCT with GVHD who were receiving immunosuppressive therapy (Ullmann et al, 2007) . Following initiation of prophylaxis, clinical events are modeled with chance nodes reflecting probabilities of IFIs (9% for fluconazole prophylaxis), IFI related death and death from other causes. It is assumed that patients surviving the prophylactic period will have a life expectancy that reflects that of the underlying condition. This allows translation of the trial outcomes to a lifetime horizon. Data on life expectancy, quality of life, medical resource consumption and costs were obtained from the literature. Model outcomes include incremental cost per life year gained and incremental cost per QALYs gained. Results: The total cost (prophylaxis + treatment of breakthrough IFI) with posaconazole amounted to €9,428 (95% uncertainty interval €7,743 -€11,388), which is €4,566 (€2,460 -€6,854) more than the cost with fluconazole. Posaconazole prophylaxis resulted in 0.17 (0.02 -0.36) QALYs gained compared to fluconazole prophylaxis, corresponding with an incremental cost effectiveness ratio (ICER) of €26,225 per QALY gained. Results from a probabilistic sensitivity analysis indicate that there is 79% probability that posaconazole is cost-effective at a threshold of €50,000 per QALY. A scenario analysis demonstrated that the probability of being cost-effective improved significantly at an increased background risk of contracting an IFI: ICER of posaconazole versus fluconazole was €13,462 per QALY gained at a background IFI risk of 15%. Conclusion: Given the underlying data and assumptions, and taking into consideration the severity of the disease and the financial investments made for HSCT, our economic evaluation demonstrated that posaconazole prophylaxis is likely to be cost-effective at a threshold of €50,000 per QALY compared to fluconazole in recipients of HSCT developing GVHD in the Netherlands. Reference: Ullmann AJ et al. N Engl J Med 2007; 356(4) :335-347. The analysis of chronic GvHD after cord blood transplantation in comparison with bone marrow transplantation S. Seo, N. Uchida, H. Yamamoto, N. Matsuno, K. Ishiwata, N. Tsuji, S. Takagi, D. Kato, K. Masuoka, A. Wake, S. Taniguchi Toranomon Hospital (Tokyo, JP) Backgrounds: Umbilical cord blood can be an alternative stem cell source for the patients with hematological malignancies requiring allogeneic stem cell transplantation. However, little is known about graft versus leukemia/lymphoma (GVL) effect in cord blood transplantation (CBT). Here, we analyzed chronic GVHD (cGVHD) in CBT compared with that in BMT and evaluated the relevance between cGVHD and GVL. Patients/methods: We retrospectively studied 162 patients who had been free from disease progression for more than 100 days after either unrelated BMT (n=75) or CBT (n=87) at Toranomon Hospital from January 2002 to December 2006. Median age of the patients was 52 years old (BMT vs CBT: 49 vs 53). Underlying diseases were acute leukemia (n=88), myelodysplastic syndrome (n=17), lymphoma (n=39) and others (n=18). Conditioning regimens were mainly composed of Fludarabine 125-180 mg/m² with several combinations of Melphalan 80-140 mg/m², Busulfan 8-16mg/kg and/or total body irradiation (4-8 Gy). Results: The Median observation period after the transplantation was 612 days (range, 109-1944) . The cumulative incidence of cGVHD was 84% in BMT and 62% in CBT (p=0.09). The severity of cGVHD was analyzed based on its type; limited or extensive. In CBT, the percentage of the former type was 34% (vs 25% in BMT) and the latter was 23% (vs 48% in BMT). High-risk disease (p=0.03) and preceding acute GVHD(p=0.03) are related to the occurrence of cGVHD. RICBT tended to increase cGVHD compared to CBT using BU/CY or CY/TBI regimen. Multivariative analysis showed that cGVHD increased over-all survival (p<0.01) and suppressed recurrence of the disease (p<0.01). During observation period, no patients were died of cGVHD. Discussion: We demonstrated that cGVHD in CBT is tolerable compared with that in BMT and that the occurrence of cGVHD could result in good prognosis. Our analysis also suggested that CBT could have GVL effect as well as BMT. M. Sartor*, A. Yeung, S. Wong, D. Gottlieb Westmead Hospital (Sydney, AU) Naturally occurring regulatory T cells (Treg) are actively involved in the control of peripheral immunity. A number of animal studies have demonstrated the critical role of these cells in the outcome of allogeneic hematopoietic stem cell transplantation (HCT). In these models, Treg can exert a potent suppressive effect and prevent graft versus host disease (GvHD) but an imbalance between Treg and conventional T cells during immune reconstitution may impair regulatory mechanisms and contribute to the development of GvHD. We therefore measured Tregs in the blood of normal donors as well as 12 patients receiving an allogeneic HCT. Blood samples from patients were obtained twice weekly following transplantation to day100. Treg cells were characterised by the co-expression of CD4+CD25+ and CD127 Lo. Our data show that Tregs reconstitute promptly after HCT. Longitudinal analysis over the first 6 weeks shows that in allogeneic transplant recipients Tregs as a percentage of the lymphoid compartment in peripheral blood equals and sometimes exceeds what is observed in normal donors. Thereafter, the percentage of Tregs falls into or below the normal range. The overall recovery of CD4 + cells within the lymphoid population was significantly lower (median 27.8% range 8.5 -44.4%) than that observed in normal individuals (median 40% range 31.8 -61.4%)(p=.001). The percentage of Tregs within the lymphoid population in patients that developed aGvHD grade I (n=6) and grade II (n=1) was significantly lower (p=0.0009) than the percentage of Tregs in patients that did not develop aGvHD (n=5). One patient relapsed; this patient's donor stem cell product contained a greater than two fold increase in the percentage of Tregs within the lymphoid population compared to other donor stem cell products within this study (5.3% vs median 2.5% range 1.7 -4.3%). These data demonstrate that in comparison with other lymphoid cells, Tregs reconstitute rapidly post transplant. Subsequently, their growth lags behind other lymphoid cells. Our data suggest that lack of expansion of Tregs may be associated with development of GvHD. Early onset of acute graft-versus-host disease indicating a worse prognosis in terms of chronic graft-versus-host disease compared to late onset: multicentre retrospective study S.K. Sohn (1) Background: Acute graft-versus-host disease (GVHD) is an important risk factor for predicting chronic GVHD. The transplant outcome can be influenced by the onset time of acute GVHD in patients who received allogeneic stem cell transplantation (SCT). Method: The medical records of 301 patients with hematological diseases who received allogeneic transplantation in three SCT centers were retrospectively reviewed. Results: Median onset time of acute GVHD was 21 days (range 1-112). One hundred two (33.9%) patients developed acute GVHD within D+21 after allogeneic SCT (D+21 group). The cumulative incidence of chronic GVHD and extensive chronic GVHD were 58.2% and 41.9%, respectively. The chronic GVHD and extensive chronic GVHD were observed in 70.5%, 57.6% (p=0.002) and 51.3%, 38.2% (p=0.033) in D+21 group, respectively. On multivariate analysis, grades II-IV acute GVHD devveloped before D+21 (HR 2.422, p<0.001) and CD3+ cell count at transplants (HR 1.638, p=0.029) were identified as independent variables predicting chronic GVHD. But overall survival rate was not different between D+21 group (p=0.670). Conclusion: Acute GVHD of early onset (within D+21) and CD3+ cell count at transplants were regarded as a worse prognostic indicator in terms of chronic GVHD. Introduction A variety of different Anti T Lymphocyte globulins (ATG) is used for GvHD prophylaxis in allogeneic stem cell transplantation. Different mechanisms of cell death mediated by ATG including complement mediated lysis and antibody dependent cell mediated cytotoxicity have been reported. We investigated the signal transduction dependent cell death pathways by surface epitope crosslinking of rabbit rATG (Fresenius®) in the human lymphoblastic cell line Jurkat, that is used as immunogen for rATG generation. Material and Results Jurkat cells were incubated with increasing doses of rATG and cell death was assessed by intracellular staining for active caspase 3, controls were incubated with FAS activating antibody CH11. While FAS induced a strong caspase 3 activity, rATG was unable to activate caspase 3. In contrast, when phosphadityl serine exposure was measured by annexin staining or mitochondrial apoptosis by loss of DIOC6 uptake rATG induced high rates of cell death. We further investigated the influence of the caspase inhibitor zVAD, FAS inhibitory antibody ZB4, serine protease inhibitors DCI, TLCK and TPCK as well as the reducing agent NAC on rATG induced cell death. We found that as a single substance only the serine protease inhibitor DCI significantly abrogated rATG induced cell death. In combination with the trypsin inhibitor TLCK this effect could be synergistically enhanced. Conclusion We found that rATG induces high rates of cell death in Jurkat cells by cell surface epitope crosslinking/signal transduction pathways. In contrast to established pathways this type of cell death is not caspase dependent, but involves the activity of serine proteases. Candidate serine proteases and their transduction pathways are only subject to speculation since no consistent data exist to date. One previous study implicated lysosomal cysteine proteases in rATG induced cell death, we hypothesize that lysosomal serine proteases might be involved in this alternate transduction pathway. Extracorporeal photopheresis in chronic graft-versushost disease: update of a single-centre experience K. Hölig*, K. Zimmer, M. Blechschmidt, U. Platzbecker, A. Kiani, G. Ehninger, M. Bornhäuser University Hospital Dresden (Dresden, DE) Background: Extracorporeal photopheresis (ECP) is widely accepted as 2nd line treatment of chronic graft versus host disease (cGvHD). We present our experience with ECPtreatment between 1/2000 and 12/2007. Patients and Methods: We treated 26 patients (16 males, 10 females) with extensive cGvHD after allogenic blood stem cell transplantation. The main manifestation of cGvHD was skin with different degree of mucosal involvement. Other involved organs were liver (12 patients), gut (4 patients) and lung (2 patients) . ECP treatments were generally performed on 2 consecutive days (=1 cycle) every week during the 1st month and every 2nd week for the following 2-3 months. Thereafter treatment intervals were tapered according to individual response. ECP procedures were performed with the UVAR-XTS device (Therakos, Exton, PA, USA). Peripheral venous access was obtained in 19 patients, 1 pediatric patient was treated via a central line (Hickman Catheter) and 6 patients were treated via Port systems (Vortex™ TR Port, Rita Medical Systems, Atlanta, Ga, USA). Additional immunosuppression consisted of calcineurin inhibitors, other immunosuppressants and corticosteroids. Results: 26 patients underwent a median of 9,5 (4 -32) ECP cycles. ECP treatment has been completed in 19 patients. The procedures were well tolerated without severe treatmentrelated side effects. Technical problems were related to difficult venous access, low hematocrit and/or hyperbilirubinemia. Implementation of Vortex™-Port systems facilitated ECP-treatment in 6 patients. Clinical improvement could be observed in 18 patients (69%). Skin symptoms improved remarkably in 10 and moderately in 7 patients. In 3 patients no change of cGvHD-symptoms could be observed, 2 patients died from infection, 1 patient died from liver failure due to refractory cGvHD. 3 patients suffered exacerbation of skin (2) and mucosal (1) GvHD. Overall survival is 23/26 (88%). The steroid dosage could be reduced remarkably in all responders. Conclusion: Our data are in accordance with the literature confirming ECP as a feasible, safe and effective therapeutic modality in cGVHD patients not responding to conventional treatment. Best response rates are achieved in skin involvement. Mucosal and especially ocular lesions seem to be very resistant to treatment. The application of Port-Systems is a valuable opportunity for patients with very difficult venous access. Introduction: Graft versus Host Disease (GvHD) remains the most important complication after allogeneic haematopoietic stem cell transplantation (HSCT). It is recognized that Daclizumab (Dac) is effective in the treatment of steroidrefractory acute GvHD (aGvHD) in adults, but an increased risk of infections has been reported. We describe 15 children (pts) treated with Dac for refractory aGvHD after allogeneic HSCT Materials and methods: between November 02 and October 07, 15 children (median age at HSCT: 8.6 years; 2.4-16.4) received Dac for steroid-refractory or-dependent aGvHD after a median of 43 days from HSCT (15 days-3 months). Four and 6 out of 15 pts had grade 2 and 3 aGvHD, and the remaining 5 patients were treated for grade 4 aGvHD. Dac was given intravenously at the dosage of 1 mg/Kg on days 1, 4, 8, 15 and 22 . Clinical response and occurrence of infectious events were evaluated 30 days after the first dose of Dac. We defined complete response (CR) the absence of aGvHD, partial response (PR) the decrease of at least one grade in the overall aGvHD score Results: overall, 6 out of 15 pts (40%) obtained CR, 8 pts (53%) PR,1 failed to respond. All cutaneous involvement reached CR; 50% of gastrointestinal involvement obtained CR, 34% a PR and 16% failed; hepatic aGvHD failed to respond in 54% of cases, PR was obtained in 37% and CR in 9%. Additional treatment included extracorporeal photophoresis (ECP) in11 pts. As for opportunistic infections, 6 pts developed a CMV reactivation at a median of 22 days from Dac;1 pt had an EBV reactivation (6 days after Dac); and 1 Gram positive septicemia (54 days after Dac administration. Outcome: 7 pts died after a median of 8.9 months from Dac:3 disease recurrences,1 enterococcus septicemia,1 aGvHD, 1 cGvHD, 1 secondary malignancy. Among the 8 surviving pts, 6 are still under immunosuppression (IS),4 of them without any sign of cGvHD and 2 with persistent cGvHD, 2 alive off IS without cGvHD Conclusions: in our experience Dac was active in steroidrefractory or dependent aGvHD, in particular for cutaneous and intestinal involvement and in combination with ECP. Its early administration seemed useful to control the flare of aGvHD waiting for the immunoregulatory action of ECP. Careful early diagnosis of opportunistic infection (especially viral) is strongly recommended. In conclusion, Dac can represent an effective therapeutic opportunity for treatment of acute GvHD also in paediatric patients The aim of this study was to investigate the relation between graft vs. host disease and the degree and type of lineagespecific chimerism in peripheral blood cell subsets. Patients and methods: 33 patients (AML-18, ALL-6, CML-1, MPS-2, MDS-1, HD-1, NHL-2, AA-2 patients), 15 conditioned with reduced-intensity and 18 with conventional myeloablative regimens were included in the study. The median patients' age was 33 years (range 18-67), male/female ratio 16/17 and sex disparity 26/33. Lineage-specific chimerism evaluation was performed in peripheral blood mononuclear cells (PBMCs) and separated (Whole Blood MicroBeads and auto-MACS Separator) T lymphocytes (CD3+), monocytes (CD14+) and granulocytes (CD15+). Chimerism status (% of donor chimerism and type according to the EBMT 2004 criteria: donor chimerism and transient, stable and progressive mixed chimerism) was determined on day 30, 60, 120 after SCT and at the final analysis point (range 161-700 days) on the basis of STR/VNTR polymorphism analysis with the help of PCR and capillary electrophoresis. Statistics was performed using the Fisher's exact test and a multiple logistic regression. Results: In all patients engraftment was confirmed. Acute GvHD was observed in 58% (II-IV grade in 15 cases), while chronic GvHD in 48% of patients (limited -2, extensive -14 cases). On day 30 after SCT in RIC patients the average value of donor chimerism in PBMCs and other studied cell subsets was higher than in cohort of MA patients. This was also observed on day 60, 120 after SCT and at the final point of analysis in any cell subsets (with PBMCs exemption). On day 30 after SCT in both group of the patients the degree of donor chimerism in PBMCs, T lymphocytes CD3+ cells, monocytes and granulocytes did not correlate with aGvHD occurrence. The incidence of aGvHD was associated, however, with changes of kinetics of hematopoietic chimerism in CD3+ cells (p=0,031) and in PBMCs (p=0,004). This relationship was not confirmed in the multivariate analysis, in which only CD34+ cell number (above 4x10 6 kg) in the graft had an impact on aGvHD incidence (OR 38; CI 2, p=0, 009) . Moreover, chimerism types with higher degree of donor cells in T lymphocytes CD3+ was associated with aGvHD occurrence (p=0,016). A similar relation was not found in terms of aGvHD grade and cGvHD extent and type of chimerism in T lymphocyte CD3+. A major challenge in the field of hematopoietic stem cell transplantation (HSCT) is to prevent the alloreactivity of donor T-cells which leads to acute graft-versus-host disease (GVHD) while preserving a graft-versus-tumor (GVT) effect. Selective depletion using anti-CD25 immunotoxin (IT) can eliminate harmful alloreactive T-cells while preserving other donor Tcells with antileukemic/antitumor reactivity. Allodepleted donor cells were then repeatedly stimulated using purified leukemia/tumor cells from the same cancer patient. Leukemia/tumor-reactive donor T-cells were purified by immunomagnetic separation on the basis of INF-g production. 23 MLRs (12 with acute myeloid leukemia cells, 3 with acute lymphocytic leukemia cells and 8 with renal carcinoma cells) were performed. Selective depletion of alloreactive donor T-cells with anti-CD25 IT led to more than 2log depletion (99,2 -100%, median 99,7%). Graft-versus-leukemia (GVL) effect of donor T-cells was well preserved (4,7% of donor T cells were GVLreactive) while the graft-versus-host (GVH) reactivation of donor cells was negligible even after 3 rounds of repeated stimulation with patient's non-leukemic PBMC. In the case of renal cell carcinoma, GVT effect was less dominant (1,8% of donor T cells were GVT-reactive clones) and GVH reactivation of selectively allodepleted donor T cells was negligible even after 3 rouds of repeated stimulation with patient´s PBMC. In conclusion, it is possible to selectively deplete donor alloreactive T-cells with anti-CD25 IT. This strategy eliminates alloreactivity but preserves tumor antigen reactivity in patients with leukemia as well as in patients with renal cell carcinoma. Supported by the Ministry of Education of the Czech Republic, NPVII-2B06058 Changes in airway responsiveness to methacholine following haematopoietic stem cell transplantation G. Barisione*, A. Bacigalupo, E. Crimi, M.T. van Lint, T. Lamparelli, V. Brusasco, A. Ibatici, S. Bregante, A.M. Raiola, C. Di Grazia, D. Occhini, F. Gualandi, A. Dominietto S.Martino's Hospital (Genoa, IT) Background: Patients undergoing allogeneic haematopoietic stem cell transplantation (AHSCT) are susceptible to develop pulmonary complications. Among them, are the bronchiolitis obliterans (BO) and the bronchiolitis obliterans organising pneumonia (BOOP). The former results in a purely obstructive abnormality, whereas the latter results in a restrictive abnormality with reduction of DL,co. Airway hyperresponsiveness to methacholine chloride (MCh) was repeatedly reported in lung transplant recipients and suggested to be a risk factor for development of BO. Airway responsiveness after AHSCT was only occasionally reported. Aim of the study: This study aimed at investigating changes in lung function at 3 and 12 months (mos) after AHSCT and their impact on airway responsiveness to MCh. Patients. Twenty-three pts were studied. A standard incremental-dose MCh challenge was obtained in 17 pts (Study 1) 1 week before, 3 (n=16) and 12 mos (n=13) after AHSCT. The remaining 6 pts (Study 2) were challenged before and 3 mos after AHSCT using a modified MCh protocol in which deep breaths were prohibited and changes in airway caliber were inferred from changes in partial expiratory flow (V'part). Occurrence of air trapping was inferred from measurement of RV and from changes in the linear regression of all values of FVC vs. FEV1 measured during the challenge. Results: In both studies, AHSCT was followed by a reduction in TLC and DLco. Study 1: After both 3 and 12 mos, AHSCT was associated with the development of a mild restrictive lung function abnormality, without any signs of BOOP. There were no significant changes in airway responsiveness to MCh, as assessed by PD20, FEV1 or slope, but FVC decreased less after AHSCT, suggesting less air trapping for a given level of induced bronchoconstriction. Study 2: At 3 mos after AHSCT, the reduction of V'part at low lung volume induced by MCh was significantly more reversed by DIs after than before AHSCT. The increased relaxant effect of DIs was significantly correlated (r =0.88; P = 0.02) with the reduction in TLC. Conclusions: Pts undergoing AHSCT may develop sub-clinical restrictive abnormalities, which are associated with reduced tendency to develop air trapping upon induced bronchoconstriction. The effect of repeated DIs in reversing MCh-induced bronchoconstriction is enhanced after AHSCT and is presumably due to the reduction in TLC. We speculate these effects may be the results of an increased distending force acting on airway walls. Background: Chronic graft versus host disease (cGVHD) is the major late complication after allogeneic stem cell transplantation. Standard therapy is steroid and Cyclosporine-A (CyA); however, immune suppression (ISS) related infections or unresponsiveness to ISS, are major mortality causes. Extracorporeal photopheresis (ECP) has shown activity in treatment of cGVHD, but its use has been limited to first-line-unresponsive cGVHD. Aim of the study. This is a single center pilot study testing feasibility of a programme of photopheresis in association with standard therapy as first line treatment in high risk cGVHD. High risk was defined as the presence of parameters predicting high cGVHD-related mortality. Secondary objectives were response and complications incidence. Patients: Among 9 pts fitting enrolling criteria, 2 refused due to logistic problem or low compliance with the procedure, 7 were enrolled. Median age was 40. Donor was HLA identical sibling in 6 cases and MUD in 1. All cases presented with extensive/moderate-severe cGVHD; Akpek score was > 0 in 3/7 pts. Treatment plan. Pts started with Prednison (PDN) 1 mg/kg and CyA at cGVHD diagnosis; ECP was started with a frequency of 4 application/month in the first 3 months and 2/month for the subsequent 9 months; PDN and CyA were slowly reduced until suspension, or otherwise modulated. Study duration was 1 year. Pts were ruled out the study in case of ECP suspension; requirement of other ISS drugs in case of GVHD progression unresponsive to standard therapy, or severe infections. Response was evaluated with standard criteria, as progression, partial response (PR), very good PR (vgPR) or complete response (CR). Results: Adherence to protocol was: 5/7 pts at 3 months, 4/7 at 6 and 9 months, 3/7 at 12 mm; exit from the study was due to infectious complications (2), ECP suspension due to venous access related thrombosis (1) and clear cGVHD progression (1) . In evaluable pts, response (CR+very good PR / ≤PR) per trimester was 4/5, 2/4 and 3/4 at I, II and III respectively; at the IV trimester, 1 very good PR, 2 PR and 1 progression were observed. Complications were evaluated in 4 pts and are reported on the table. At 1 year, 2/7 pts died (1 TRM, 1 relapse). Conclusion: ECP in association with standard therapy is feasible; complications incidence seems to be similar to those observed in patients not treated with ECP; a larger group of patients is needed to evaluate response in this setting. The early diagnosis can be done through biopsies of oral mucosa with or without apparent injury clinic. The association between the clinical and histopathological tables of GVHD, especially through the use of recent consensus established in these areas, can bring new benefits to physicians. Objective: This study aims to apply and compare the two classifications histological to GVHD, Horn (1995) and Consensus (2006) in specimens obtained from clinical oral lesions suggestive of GVHD; correlate them with clinical classification according Akpek (2001) and with the survival of the patients. Methods: For this study were analyzed histological samples of oral mucosa of 10 patients with clinical diagnosis of GVHD, which have areas of epithelium, and salivary gland. The histopathological aspects were observed by the author of this work and his advisor, so blinded by applying the criteria for Horn and the Consensus. We consistency between the two classifications obtained and histological correlation with the clinical classification of GVHD (Akpek, 2001) and the overall survival. Results: The histological classification according Horn showed that in most cases there was presence of GVHD with low degrees of severity; Already classification of the Consensus showed that there were in full presence of GVHD of moderate degree. Clinically (Akpek) ranged from grades 2 and 3, whereas the patients with grade 3 was no death in 75% of them. Conclusion: The data found most studies suggest the need for the diagnosis of GVHD, so that you can correlate with more accurate data clinical, histological and the survival of these patients. Background:Monitoring the specific immune response against CMV could optimize the timing for anti-viral therapy administration in order to avoid related toxicity and to reduce CMV related mortality and morbidity after alloHSCT. Aims: to test a new ELISA based method (QuantiFERON®-CMV) for functional immune-monitoring (IM) after allogeneic stem cell transplantation (alloSCT). Methods:31 whole blood specimen from 13 patients (pts)undergoing alloHSCT were analysed in a 2 phases experiment. In the first one, QuantiFERON®-CMV (together with a positive and a negative control) was performed on whole blood of 10 patients at any time from transplant,in order to test the reliability and the performance. In the second phase, 3 CMV-seropositive (CMV+) pts (at the time of the analysis) were enrolled in a prospective monitoring study. The patient #1 (pt1) has been transplanted from a CMV+ sibling donor; the pt2 received a haplo-transplant from a CMV+ sister (CD19/CD3 depletion) with strong reactivation of CMV and marrow involvement after 2 months from alloHSCT; the pt3 has been transplanted from a CMV+ sibling donor after a micro-conditioning.All the pts were monitored every 2-4 weeks since engraftment.The IFN-g response in the CMV Ag tube was considered positive if significantly above the Nil IFN-g IU/mL value.The Mitogen-stimulated plasma sample was used as an IFN-g positive control for each specimen tested.GVHD extension was arbitrary defined by the number of involved sites. Results:Several months after transplant the production of specific IFN-g against CMV was still low.A statistically significant correlation between aspecific IFN-g and chronic GVHD (cGVHD) extension was found (R=0.68).IM of pt1 identifies an aspecific production of IFN-g that anticipates the onset of cGVHD,increases over time and dropped after the treatment of the cGVHD.The immune-monitoring of the haplotransplanted pt provides evidence of an impaired T-function either specific nor aspecific.The pt is currently treated with CMV specific CTL from the donor (Pt2).The only pt conditioned with a non myeloablative transplant achieve an optimal specific response shortly after transplant and this response remains over time (Pt3). Conclusions:Notwithstanding the low number of cases, the preliminary data suggest that QuantiFERON®-CMV could represent a reliable test for immune-monitoring for pts undergoing alloHSCT. This test could identify pts with specific anti-CMV immune response, which looks to anticipate the onset of cGVHD. However, larger studies are needed. A. Lanti (1) , N. Ramundo (2) Chronic graft-versus-host disease (GVHD) is a major limitation of successful allogeneic hematopoietic stem cell transplantation (HSCT). The current standard strategies for therapy include immunosuppressive drugs like: cyclosporine, mycophenolate mofetil, tacrolimus. Extracorporeal photopheresis (ECP) is a relatively new therapeutic option adopted to control the clinical manifestations of cGVHD resistant to conventional therapy. In our study we evaluate 14 (10 males, 4 females) patients affected by cGVHD, immunosoppressive therapy resistant or with controindication to the therapy, treated with ECP. The ECP treatment was started, on average, 16 months after the cGHVD diagnosis and 22 months after the transplant. The protocol consisted of 2 ECP per week in the first 2 weeks of the treatment, 1 ECP per week between the 3rd and the 14th week, and 2 ECP per month from the 4th month onward. 85% of the patients with skin lesions and 60% of the ones with hepatic problems showed a slight or remarkable improvement of the clinical signs. 65% patients ECP allowed progressive reduction or discontinuation of the concomitant pharmacological immunosuppressive therapy. Through the adoption of the Pearson correlation coefficient, we evaluated the degree of association between the number of cells irradiated with ECP per patient and the degree of improvement of the hepatic and cutaneous cGHVD. The correlation coefficient showed a good grade of association between the number of cells treated with ECP and the improvement of the serum concentration of ALT and GGT, (r=0.70). Unlike, the grade of association with the cGVHD cutaneous was less remarkable, r=(0.55). Our study has highlighted: • the ECP in the cGVHD treatment is responsible for a cutaneous and hepatic improvement in a high percentage of patients; • All the patients respondent to the treatment with ECP can reduce the immunosoppressive therapy; • An important predicting factor of the response to the treatment is the number of cells collected and irradiated during the ECP. Treatment of steroid-refractory acute graft-versus-host disease with ATG (Fresenius) J. Kamelander, M. Navratil*, Z. Koristek, M. Doubek, M. Krejci, Y. Brychtova, Z. Racil, D. Mendelova, M. Weinreb, J. Mayer University Hospital Brno (Brno, CZ) Introduction: Several studies tested Anti-thymocyte globulin (ATG) for the treatment of steroid-refractory Graft-versus-host disease (SR GvHD) with, unfortunately, usually very poor long term results. Here, we retrospectively analyze our experience with ATG (Fresenius), because different brands of ATG/ALG differ in their anti-T-cell potency and virtually no data exist concerning this particular drug in the setting of SR GvHD. Patients and Methods: Upon the first experience, ATG is now given early in the course of SR GvHD, predominately not in patients with isolated liver GvHD, and at the dose of 10 mg/kg/day for 4 doses every other day (treatment course). However, some first patients received this salvage therapy quite late and were not given 4 doses. Results: In 16 pts, 26 treatment courses were given. The number of involved organs before the first ATG course weregut: n=12; liver: n=9; skin: n=5. Median ATG doses in the first course was 2 (1) (2) (3) (4) , and in the next courses, usually 1 or 2 doses were given. Other immunosuppressive therapy was continued and in the case of response, steroids were tapered. Eight patients received ATG during the first SR GvHD attack. Other 8 patients, however, received ATG later in the course of SR GvHD for a new attack or disease worsening after prior improvement upon another therapy than ATG. In general, ATG was administered at a median of 4 days (1-30) after the start of the GvHD attack considered as SR. Regarding the first ATG treatment course, the best responding organ involvement was skin (80% response rate, RR) and gut (75% RR), liver involvement responded in only 56%. There were 2 striking patterns of response to ATG treatment: 1) very quick signs of improvement (median: 3 d; 1-10 d) and 2) tendency to relapse (median: 13 d; 3-40 d). After a relapse, retreatment with ATG was possible and successful. CMV and BK virus reactivation occurred in 8 pts (50%) and 2 pts (13%), respectively. Ten pts (63%) died, usually from GvHD progression, various infections, or both. Several interesting case-reports will be presented. Conclusion: Based on this experience, ATG (Fresenius) at the dose of 10 mg/kg/d every other day for 4 doses is now our standard treatment of gut and/or skin SR GvHD, with starting this salvage therapy early upon failure of the first-line steroids. Vigorous anti-infective measures are mandatory. Retreatment of GvHD relapse with lower total dose of ATG is possible and feasible. The effects of Thymoglobulin in a non-myeloablative conditioning regimen that included fludarabine and busulfan (Flu-Bu) on immune reconstitution are not well characterized. This study examined immune reconstitution after Thymoglobulin and the associations between immune reconstitution and GVHD. Methods: 23 patients received a non-myeloablative conditioning regimen with Thymoglobulin (1.5 mg/kg Day -6, -5, -4, -3) and Flu-Bu. Lymphocytes, memory cells, NK, and DC cells were measured by flow cytometry at baseline, engraftment, and months 2, 3, 6, 9, and 12. Results: This cohort consisted of 8 AML, 7 MM, 3 MDS, 2 NHL, 1HD, 1 ALL, and 1 DLBL patients. The mean age was 52±14 years and 61% were male. 87% had high risk disease. All patients received peripheral blood stem cell transplant (61% related and 39% unrelated). 22(96%) patients had successful engraftment at a median of 20 days posttransplant. 12(52%) patients developed acute GVHD [Grade 1 (17%), 2 (58%), 3 (17%), 4 (8%)] at 51±37 days posttransplant. Patient survival rates at 30 days, 100 days, and last follow-up (median 186 days) were 95.7%, 87.0%, and 43.5%, respectively. Disease relapsed/progressed in 10 (44%) patients at 109±112 days posttransplant. Post-engraftment, 8 patients developed opportunistic infection with CMV being the most common. T and B lymphocytes, memory cells, DC, and NK cells recovered promptly after engraftment and remained stable during follow-up. The relationship between the immune composition and outcomes were examined. Surprisingly, at baseline, patients without acute GVHD had significantly higher CD3, CD4, CD16CD56, CD45RACD4+, CD45ROCD4+, CD25CD3, and CD25CD4 cell counts than those with acute GVHD. However, post-engraftment and follow-up, none of these cells were different between groups, except for NK cells which remained higher in patients without GVHD. T and B lymphocytes, memory cells, DC, and NK cells appeared to be associated with patient survival or disease relapse. Conclusions: In this high risk cohort, a non-myeloablative conditioning regimen with Thymoglobulin, Flu-Bu appeared to be safe, did not adversely affect immune reconstitution, and was associated with acceptable outcomes. Higher baseline pre-transplant immune cells lowered risk of GVHD suggesting role of recipient cells in immune tolerance. Patients who developed acute GVHD also had significantly lower NK cells after engraftment, suggesting a role of NK cells in protection against GVHD. Background: We and others previously reported that rituximab is associated with delayed-onset neutropenia (DON) in patients with B-cell non-Hodgkin lymphoma (B-NHL). However, its clinical significance after autologous stem cell transplantation (ASCT) has not been well described in a large series. We conducted a retrospective cohort study to examine the impact of DON on clinical outcome. Patients and Methods: Subjects were consecutive 109 patients with B-NHL receiving ASCT between June 1991 and January 2007 in our institute. We defined DON as absolute neutrophil count < 1.0 x 109/L at any point after 30 days post ASCT without apparent causes of neutropenia. Documented infectious events were reviewed between 30 days and 1.5 years after ASCT with and without the period of DON, and risk factors associated with infectious events were evaluated including DON, rituximab using, sex, age, bulky lesion, bone marrow involvement, CD34 selected ASCT, number of prior chemotherapy, number of prior local radiation therapy and total body irradiation as a part of conditioning regimens. Results: Forty-nine patients had diffuse large B-cell lymphoma, 35 follicular lymphoma, 21 mantle cell lymphoma and four other B-NHL. Fifty-two percent of patients received rituximab in combination with chemotherapy before ASCT. The median number of prior treatment regimens was two. Seventeen percent of patients received CD34 selected ASCT. DON was observed in 50% of 109 patients. The median value of nadir neutrophil count during DON was 0.446 x 109/L (range; 0.009-0.968) at a median of 96 days (range; 32-686) after ASCT. Twenty-four events of infection were documented in the period of DON, and 95 events in the period without DON. DON was an independent risk factor of total infectious events (p<0.001), irrespective of the period with or without DON. The frequency of varicella zoster virus (VZV) infection was 69 % and 31% with and without DON group, respectively. The development of DON (p=0.02) and CD34 selected transplantation (p=0.007) were associated independently with an increased occurrence of VZV infection. With a median follow-up of 3.7 years, estimated 3-year overall survival was 83% and 68% with and without DON group, respectively (p=0.09). Conclusion: DON was associated significantly with the occurrence of VZV and total infectious events in all period after ASCT, which did not affect survival. Careful follow-up is needed after ASCT. A. Huynh*, M. Roussel, L. Ysebaert, C. Recher, F. Huguet, C. Nouvel, G. Laurent, M. Attal CHU Purpan (Toulouse, FR) Introduction: Mantle cell lymphoma (MCL) is an agressive non Hodgkin lymphoma with median overall survival (OS) in most series of 3-4 years. Although frontline therapies with autologous stem cell transplantation (ASCT) induce high rates of complete remission (CR), relapse is usual.RCHOPalternating RDHAP followed by ASCT recently showed promising results in terms of response rates and OS. Bortezomib (VEL) has demonstrated clinical efficacy in relapsed and refractory MCL, a synergistic effect with Cytarabine and a lack of sustained haematological toxicity when combined to high dose Melphalan (in multiple myeloma). The combination of VEL to BEAM regimen could be a logical approach to improve the OS in MCL. The purpose of this monocentric prospective study was to evaluate the feasibility and safety profile of this VEL-BEAM association. Methods: Between 05/06 and 08/07, 8 consecutive patients with MCL were enrolled to receive intensification with VEL-BEAM followed by ASCT. VEL (1,3 mg/m²) was delivered on days -6, -3, + 1, + 4 and BEAM (300 mg/m² BCNU, 4x200 mg/m² Etoposide, 4x200 mg/m² Cytarabine and 140 mg/m² Melphalan) on day -7 to -2. Peripheral blood stem cells (median 8,7x10 6 CD34/kg) were infused on day 0. We secondary conducted a retrospective comparison with 7 frontline MCL patients who received "classical" BEAM followed by ASCT. Results: Main characteristics of the patients in both groups are resumed in table 1. All patients except 1 were in CR after induction therapy with 3 RCHOP/ alternating RDHAP chemotherapy. VEL did not increase the haematological toxicity observed with BEAM (see table 2 ). Median duration of neutropenia (< 0.5 x 109/l) and thrombocytopenia (< 50 x 109/l) was 9 and 10,5 days respectively. No grade 3/4 extra-haematological toxicity was observed. One patient was transferred to USIC for septic choc with pneumoniae during neutropenic period. At time of reporting, 1 patient died in the VEL-BEAM group at 3,5 months (mo) of intracerebral hemorragia and 2 patients relapsed in the classical BEAM group at 20 and 25 mo post ASCT. Median follow-up (5,3 mo) is currently too limited to comment on whether the conditioning regimen with VEL-BEAM will translate into an improved progression free survival and therefore OS. Conclusions: These preliminary results suggest that VEL (1,3 mg/m 2 ) and BEAM is a feasible and safe conditioning regimen. Further studies are needed to evaluate the efficacy and therefore the improvement of survival. Allogeneic peripheral blood stem cell transplantion for treatment of high-risk relapse of aggressive lymphoma. Interim analysis of the DSHNHL R3 study B. Glass*, J. Hasenkamp, P. Dreger, M. Gramatzki, J. Schubert, G. Wulf, M. Nickelsen, L. Trümper, N. Schmitz on behalf of the DSHNHL High dose therapy (HDT) followed by autologous stem cell support has poor outcome in patients with primary progressive lymphoma or relapse after primary HDT due to high relapse. Allogeneic SCT may help these patients by exerting an GVL effect. Its use however is hampered by high incidence of severe GVHD and treatment related mortality (TRM) in this population. Rituximab has been claimed to solve this problem. We initiated a randomized phase II study using intermediate conditioning (Fludarabine 125 mg/m², Busulfan 12 mg/kg and cyclophosphamide 120 mg/kg) followed by GVHD prophylaxis with short term mycophenolat mofetil plus tacrolimus. Patients were randomized to receive two times four doses of rituximab (375 mg/ m²) post transplant starting day +28 and day +175 or no further GVHD prophylaxis. From January 2005 to August 2007 sixty patients with aggressive NHL were enrolled. Thirty one pts had DLBCL, 9 patients follicular lymphoma grade 3, 8 pts blastic mantle cell lymphoma, one patient agg. marginal zone lymphoma and 11 patients peripheral T cell lymphoma. The median number of prior treatment regimens was 3 (range 1 to 6) . 43 (72%) pts received at least one cycle of high-dose therapy and autologous SCT prior to alloSCT; 58% of patients had chemorefractory disease and 52 % had progressive disease with high or high intermediate age adjusted IPI immediately prior to conditioning. Allo-PBPC were obtained from HLA-identical siblings in 16 pts, from fully matched unrelated donors in 32 pts and from 1 locus mismatched unrelated donors in 12 pts . Engraftment of leukocytes was rapid (median 10 days, range 8-27) and all patients achieved complete (> 95%) donor type chimerism after alloSCT. Median observation time is 8 months (range 1-35 months). 32 pts died, in 20 patients death was attributed to treatment related causes. After one year, estimated overall survival is 47%, failure free survival is 43%, TRM is 37%, relapse rate is 33% and incidence of GVHD > grade 1 is 57%. There was a trend to lower relapse rates in patients with GVHD > grade 1 (25% vs 44%, p=0.14). Intermediate intensity conditioning followed by allogeneic SCT is a valuable treatment option in patients with high-risk relapse of aggressive NHL. The basic incidence of GVHD and TRM is high. Due to the short observation time, a definitive conclusion regarding the impact of post transplant Rituximab cannot be drawn presently. At time of presentation a first analysis of this topic can be given. Allogeneic stem cell transplantation (HSCT) is increasingly considered an option in refractory /relapsing lymphoma. Almost all patients with B-cell lymphoma are treated with monoclonal antibodies, such as the anti-CD20 antibody rituximab (R) prior to HSCT. R remains in the circulation of patients up to 6 months after the last administration. Patients receiving HSCT short term after R might therefore be at risk of impaired B-cell reconstitution, due to in vivo depletion of Bcells/ B-cell precursors as a result of circulating R. We studied B-cell immunereconstitution of 12 patients (6 male, 6 female) with lymphoma (5 follicular, 3 diffuse large B-cell, 2 mantle cell, 2 marginal cell lymphoma) receiving R 1 to 9 months (median 3 months) before HSCT by analyzing B-cell counts and levels of immunoglobulins (Ig) before HSCT, and 1, 3, 6, 12, and 24 months after HSCT. Controls were 12 consecutive patients (8 male, 4 female) with myeloid neoplasias receiving HSCT with no R pretreatment. Lymphoma patients were conditioned with BEAM, fludarabine and 2Gy TBI, control patients with leukemia received cyclophosphamide and 12Gy TBI. All patients were alive at 24 months of follow up. Patients with lymphoma showed significantly reduced B-cell levels at time of HSCT (median 0*10e6/l vs. 23*10e6/l for controls, p=0.002). B-cell reconstitution was delayed in patients with R pretreatment compared to controls (median absolute B-cell count at 6 months (0.5 vs 23 * 10e6/l, p=0.04) and at 12 months (15 vs 282 * 10e6/l, p=0.004). B-cell counts reached values comparable to controls 24 months after HSCT. CD4, CD8 and NK-cell reconstitution was not different from controls ( Figure 1 ). In parallel, Ig (IgA, IgG and IgM) levels were significantly lower in these patients (Figure 2 ). At 24 months post HSCT, Ig levels reached normal values. One patient experienced severe sepsis with U. urealyticum, an infection typically associated with severe B-cell deficiency. Other infections could not be related directly to B-cell deficiency.In conclusion, B-cell reconstitution is significantly delayed and Ig levels are significantly reduced up to two years post HSCT in lymphoma patients with a history of R treatment. Pretreatment with R appears to have clinical consequences beyond the immediate early post-transplant period. Autologous stem cell transplantation in elderly patients (≥ 60 years) with diffuse large B-cell lymphoma: an analysis based on EBMT registry E. Jantunen*, C. Canals, A. Rambaldi, G. Ossenkopple, B. Allione, D. Blaise, E. Conde, H. Tilly, G. Cook, C. Craddock, A. Gallamini, N.H. Russell, C. Gisselbrecht, P. Dreger, M. Autologous stem cell transplantation (ASCT) is a treatment option in patients with relapsed chemosensitive diffuse large B-cell lymphoma (DLBCL) but its role as a part of first line therapy is currently unknown. Limited experience on feasibility and efficacy of ASCT in elderly patients with DLBCL is available. Patients and transplant characteristics: We have analysed 2612 patients aged 18-75 years treated with ASCT 2000 ASCT -2005 and reported to the EBMT registry. 463 patients (18 %) were ≥ 60 years at the time of ASCT (311 pts 60-64 yrs, 139 pts 65-69 yrs, and 13 pts 70-74 yrs). When compared to 2149 patients < 60 yrs, the elderly patients had less commonly B-symptoms at diagnosis (43 % vs. 49 %, p=0.04) and less often bulky disease (19 % vs. 30 %, p<0.001). Elderly patients had received more commonly at least two treatment lines (76 % vs. 57 %, p< 0.001), were less commonly in the first complete remission at the time of transplant (23 % vs. 30 %, p=0.005) and received their transplants later after diagnosis (median time 14 months vs. 7.5 months, p<0.001). Results: Non-relapse mortality (NRM) was higher in elderly patients at 100 days (4.4 % vs. 2.8 %), at one year (8.7 % vs. 4.7 %) and at three years (10.8 % vs. 6.5 %) (p=0.002). The relative risk of NRM in patients 60 years or older was 1.6 (CI 1.1-2.3, p=0.01) when compared to younger patients. With a median follow-up of 12 months for the surviving patients for the elderly group and 15 months for the younger group, risk of relapse was 38 % and 32 %, respectively (p=0.006). Progression-free survival was 51 % and 62 % at three years (p<0.001). Overall survival was 60 % vs. 70 % at 3 years (p<0.001). Conclusions: ASCT is feasible in selected elderly patients with DLBCL although early and late NRM is somewhat higher than in younger patients. Both progression-free and overall survival is promising taking into account the generally poorer outcome of elderly patients with DLBCL. Prognosis for tumour stage Mycosis fungoides (MF) and Sézary syndrome (SS) is poor with median survival of approximately 5 years. Treatment of advanced stage is largely palliative, and allogeneic stem cell transplantation (SCT) with the possibility of inducing graft versus lymphoma effect (GvL) could result in long-term complete clinical and molecular remission. We report on a pilot study on the role of reduced intensity conditioned (RIC) HLA matched sibling SCT on survival in advanced stage MF/SS and the role donor lymphocyte infusions (DLI) for post-transplant relapse. Eight patients underwent HLA matched sibling allogeneic SCT, SS (n=3), tumour stage MF (n=3), and the erythrodermic variant of MF (n=2). All had advanced stage disease (> stage IIIA) at time of SCT. All received a reduced intensity regime using Campath-1H, Fludarabine with Cyclophosphamide (n=6) or Melphalan (n=2). Disease status pre and post-transplant was monitored clinically, radiologically and molecularly by PCR for TCR gene rearrangement. On post-SCT relapse, in the absence of significant GVHD an escalating dose regime of DLI with a (starting dose of 10x106/kg CD3+ve cells) was commenced. Seven patients entered clinical complete remission (CR), five also experiencing molecular CR (1 too early to determine). One patient died in CR of transplant related mortality (TRM) (invasive aspergillosis) and 1 had autologous reconstitution dying early of disease progression (TRM 12.5%). Four patients experienced an early relapse with cutaneous disease and the re-emergence of a T-cell clone (median time to relapse 82.2 days (range: 28 -100d)). Two patients developed acute GVHD on withdrawal of immunosuppression entering molecular CR. Three patients received DLI for relapse all developing acute skin GVHD, with concurrent regression of the relapsed disease. Two patients have not relapsed, one developed grade III skin GVHD. Five remain alive (median 669 d, range: 74 -5287) in CR. The minimum DLI dose to induce GVHD and CR2 was 10x106/kg. RIC-SCT results in clinical and molecular remissions in the majority of patients, and is appropriate therapy. Most patients experience early relapse. Reduction of immunosuppression and donor lymphocyte infusions induce GVHD followed by disease regression. We believe this is clear evidence of a graft versus cutaneous T-cell lymphoma effect. Whether patients with less advanced disease should receive SCT avoiding heavy pre-SCT therapy to reduce TRM is undetermined P826 BEAM-alemtuzumab followed by allogeneic SCT is a feasible and highly efficient treatment strategy in relapsed or refractory T-NHL A. Günther, H. Schade, A. Humpe, B. Gahn, A. Claviez, A. Schrauder, M. Gramatzki, R. Repp University of Kiel (Kiel, DE) For patients with relapsed or refractory T-cell lymphomas the prognosis is grim and no standard treatment strategy exists. In addition, mature T/NK-cell-lymphomas are an infrequent and heterogeneous group of lymphoid tumors. Ten patients (age: 11 to 59 y) with relapsed T-cell Non-Hodgkin's lymphoma (NHL) were treated with allogeneic stem cell transplantation (SCT) after 1 to 2 cycles of CLAEG (cladribine, cytarabine, etoposide/etopophos and G-CSF) induction therapy immediately followed by conditioning with BEAM (carmustine, cytarabine, etoposid/etopophos, melphalan) combined with alemtuzumab (Campath-1H). The following subtypes were included: 5 peripheral T-cell lymphomas (not otherwise specified), 2 angioimmunoblastic, 1 ALK negative anaplastic large cell, 1 extranodal NK/nasal type and 1 enteropathy-type T-cell lymphoma. All patients had at least 2 different treatment regimens previously. After induction chemotherapy with CLAEG, one patient achieved a CR, 6 patients a PR and 3 had SD. Conditioning with BEAM combined with alemtuzumab was well tolerated. Seven patients received peripheral blood stem cells (PBSC) from HLA-identical unrelated donors and 2 from matched sibling donors, 1 patient received bone marrow from an HLA one-allele mismatched unrelated donor. Engraftment was rapid and the treatment well tolerated. There were 4 cases of grade IV mucositis, 2 cases of sepsis in neutropenia, and 1 case of grade IV liver toxicity. One patient died from sepsis on d + 45. Prophylaxis for graft-versus-host disease (GvHD) consisted of cyclosporine A and MTX or mycophenolate mofetil. No grade III or IV acute GvHD was seen and only 3 patients presented with grade II GvHD. Two patients experienced limited chronic GvHD. Asymptomatic CMV reactivation was found in 4 cases and all were treated successfully. Late infections occurred in four patients, including CMV infection, viral enteritis, aspergillus pneumonia and streptococcal sepsis. All patients achieved a CR after allogeneic transplantation. Currently, 3 patients have relapsed: two of them died due to disease-related complications, one is receiving DLI. Another patient died in CR on day + 233 due to aspiration pneumonia caused by nonmalignant bowel stenosis. All other patients remained in CR for up to 26+ months. Allogeneic SCT with this strategy shows a surprisingly good outcome in a group of T-cell NHL patients with a very poor prognosis and seems to be a promising therapeutic option for these patients. Improved relapse free and overall survival is predicted by a negative pre-transplant FDG-PET scan following salvage chemotherapy for relapsed diffuse large B-cell lymphoma treated with autologous stem cell transplantation M. Dickinson (1), R. Hoyt (2) Introduction: FDG-PET imaging is a powerful predictor of both positive and negative outcome of initial therapy for DLBCL. Existing data on the utility of PET to predict outcome of ASCT have included a range of histological subtypes, limiting its direct applicability to the setting of DLBCL. Methods: Data from 43 consecutive patients from 2001-2007 who had PET scans immediately prior to undergoing ASCT for treatment of first relapse of de novo DLBCL were analysed. We compared the clinical characteristics, event free survival (EFS) and overall survival (OS) of those with residual FDGavid disease (n=21) against those without FDG-avid disease (n=22). Results: The median age was 53(18-71). Patient characteristics are shown in the table. At 28 months median follow up, EFS for PET positive patients was 38(±12)% vs 81(±8.5%) for PET negative patients (p=0.01). OS was 37(±17%) vs 83(±9%) (p=0.03) respectively. Conclusion: Excellent EFS and OS can be anticipated from ASCT when undertaken in those who have achieved a PET negative remission following salvage chemotherapy. Conversely, failure to achieve a PET negative remission after salvage chemotherapy in relapsed DLBCL predicts for a poorer, but not irretrievable outcome after ASCT, suggesting that a proportion of PET positive patients can be successfully salvaged by either ASCT of post-ASCT salvage therapies. The role of alloSCT in the treatment of pts with NHL is still controversial, but seems to be the only curative therapeutic option for pts in advanced disease stages. Treosulfan (Treo) is a newly-introduced drug for conditioning. Currently, no data are available, which allow an assessment of the tolerability and efficacy of Treo-based compared to standard preparative regimens in disease-specific transplant settings. We report 45 pts with NHL with mature B-cell neoplasm (n=33) of high grade (n=19), and low grade malignancy (n=14), and mature T-cell neoplasm (n=11) of high grade (n=8), low grade (n=3), and unspecified dignity (n=1). The Treo-based conditioning consisted of Treo (42g/m²) and fludarabine (150 mg/m²) (n=21) or Cy (120mg/kg) (n=4). Conventional preparative regimen was based on TBI either in combination with an alkylanting agent (n=13) or with fludarabine (n=7). Both cohorts were well comparable in terms of patient and donor characteristics. Median age was 41.5 years (18-62). All pts had received intensive and multiple courses of chemotherapy and 56% of them had undergone prior autograft. Eleven pts (24%) were transplanted in advanced CR, whereas 33 (73%) were not in CR. The remission status of one pt. was undefined. Donors were matched related (42%), or matched unrelated (58%). All patients engrafted. Median follow up was 5 (range 2 -8) years. For pts transplanted for low grade and high grade NHL, NRM at 100 days, 1 and 3 years after transplant was 12 vs. 22%, 18 vs. 30% and 31 vs 33%. NRM after Treo was 24%, 29% and 37% compared to 15%, 25% and 30% after standard conditioning (ns). No significant difference of NRM between the two regimens was detectable in the 33 pts not in CR. The RR increased with the grade of malignancy (low vs. high grade) and the disease stage (CR vs. advanced stages), but was not influenced by the conditioning regimen. The RR for pts with high grade NHL at 1 and 3 years was 20% and 29% after Treo-compared to 35% and 40% after TBI-based conditioning (ns). OS in pts at 1 and 3 years was 60% and 47% after Treo-compared to 55% and 50% after TBI-based conditioning (ns). This retrospective long-term follow-up analysis supports that Treo-based conditioning leads to equivalent results compared to a myeloablative TBI-based preparative regimen in pts grafted for advanced and unfavourable NHL. Appropriate patients should be considered for alloSCT when standard treatments are no longer capable of achieving prolonged remissions. To give an overview of allogeneic blood stem cell transplantations (SCT) performed for malignant lymphomas in Germany from 1998 to 2006 and to facilitate further clinical studies in the field the data sets of the DRST/EBMT activity survey and the Minimal Essential Data A (MED-A) project were evaluated. According to the EBMT classification system the term of malignant lymphoma was confined to Hodgkin`s Disease (HD) and all non-Hodgkin Lymphomas (NHL) with the exception of plasma cell disorders and lymphatic leukemias. Evaluation of Activity Survey Data: The total number of documented first transplants was n=947 for NHL and n=120 for HD. While the frequency of SCT for NHL increased form n=38 in 1998 to n=181 in 2006, the SCT frequency for HD remained more or less constant over the years with a maximum of n=20 in 2001. Since 1998 peripheral blood was increasingly preferred over marrow as stem cell source, and the most prevalent type of donor was "matched unrelated" since 2002. Evaluation of MED-A data: The total number of documented transplants (first, re-and additional SCT) was n=907 for NHL and n=150 for HD. The mean patient age was significantly higher (p< 0.001) in the B-cell NHL group (n=602,45 ±13 yrs) as compared to the T cell NHL (n=207, 36±15 yrs) or the HD (n=150, 32±11 yrs) cohorts. Precise histologic entities according to the WHO-classification, which was introduced in 2001, were reported for n=591 patients. The leading diagnoses were diffuse large cell, follicular cell and mantel cell B-NHL, with frequencies of 26%, 21% and 16%, respectively, followed by anaplastic large T-cell NHL with 7%. The disease stage at transplant was accurately defined in n=810 cases. While the ratio of early/advanced stages was balanced for T-NHL, the majority of SCT were performed for refractory/progressive disease or relapse in the B-NHL and HD cohorts. In 289/1095 of the cases (26%) a preceeding autologous transplantation was reported. Use or non-use of total body irradiation (TBI) as part of the conditioning regimen was documented in 1038 cases. TBI was more frequently used in T-and B-NHL (43%) as compared to HD (32%), p=0.02. Reduced intensity conditioning protocols were used in 40% of the HD and in 37% of the NHL patients (n.s.). The overall survival 5 years post transplant for the T-and B-NHL and the HD cohorts were 34 %, 32 % and 13%, respectively. Thus, the DRST data base has become a valuable tool for designing studies in the field of SCT for NHL and HD. Very late relapse (>5 years) after autologous stem cells transplantation for aggressive lymphomas: true recurrence or secondary lymphoma? C. Pilatrino* ASO S.Luigi di Orbassano (Orbassano, IT) Very late relapse of B cell aggressive lymphomas occur infrequently after conventional chemotherapy representing less then 10% of all relapsed patients. Furthermore late relapse after autologous stem cells transplantation (ASCT) are very unusual confirming that the eradication of the disease or the early relapse due to growth of chemoresistant clones are the more frequent clinical outcomes observed. On the other hand, the long interval between ABMT and relapse observed in these cases may suggest that, albeit in some cases, the recurrent lymphoma could be actually represent a secondary neoplasm. In a survey of patients who underwent ABMT for aggressive B cell Lymphoma (Diffuse Large Cell Lymphoma-DLCL and Mantle cell Lymphoma-MCL) in our centre in the last twelve years, we identified four patients (3 DLCL,1 MCL) who relapsed more than 5 years from ABMT . In all patients we were able to amplify DNA of the FR2-JH region from pathological samples at diagnosis and relapse according to Stilgenbauer et al. (Leukemia 16: 993, 2002) . The rearranged region was cloned and sequenced: in three patients the VH-DH-JH region was identical at diagnosis and relapse with an overlapping pattern of point mutations; on the contrary one patient showed a completely different VH-DH-JH repertoire at relapse with higher mutational status (13,8% versus 3,3% at diagnosis) compared to the germline sequence indicating that old and new lymphoma, albeit histologically identical, were actually not clonal related. Clinically these patients exhibited at relapse an unusual pattern of tissue involvement with predominant extra lymphatic sites but with scanty symptoms and limited extension of the disease. All relapsed patients were highly responsive to conventional chemotherapy and underwent a second complete remission; furthermore one patient was successfully transplanted with autologous PBSC and now is in CCR 26 months after ABMT. In conclusion our data indicate that very late "relapse" of aggressive lymphoma after ABMT may be actually a secondary lymphoma in some cases; on the other hand in patients with "true relapse" the disease was less aggressive than at diagnosis, exhibited an unusual tissue homing and showed a prompt and complete response to chemotherapy. All these data are in agreement with the hypothesis that these very late relapsed lymphomas may evolve from a "dormant" clone which persisted for several years despite the previous myeloablative treatment. High-dose sequential followed by autologous hematopoietic stem cell transplantation for Hodgkin's disease (hd): a Brazilian experience B. Duarte, I. Valente, A. Vigorito, F. Aranha, G. Oliveira, E. Miranda, I. Lorand-Metze, K. Pagnano, C. De Souza* State University of Campinas (Campinas, BR) From May 1998 to November 2006, 77 Hodgkin's disease (HD) patients who used the regimen of HD cyclophosphamide (CY) 7 or 4g/m², HD methotretaxe 8g/m² and HD etoposide 2g/m² followed by HD Therapy and autologous hematopoietic stem cell transplantation (AHSCT) were analyzed. Their median age was 25.8 (8.8-71.5) years, 46 males and 31 females. At diagnosis 50 (65%) were stage III or IV disease, 10 (13%) had bone marrow involvement, 29 (37.7%) bulky disease and 55 (71.4%) B symptoms. Besides that, all patients were submitted to a mean of 2 (1-4) chemotherapies and their status were 3 (3.9%) complete remission (CR), 17 (22.1%) partial remission (PR), 57 (74%) progression disease (PD) or in non-specified relapse. Concerning CY dose 30 (39%) received 4 g/m² and 47 (61%) received 7 g/m2. After the HDCY 16 (20.8%) were in CR, 22 (28.6%) PR, 28 (36.3%) remained in DP and 11 (14.3%) died most in PD. After a median follow-up of 3.97 (1.13-55.9) months, 53 (68.8%) patients were submitted to AHSCT and their present status is 30 alive [18 CR, 2PR and 10 DP]; 23 dead [2 CR, 14PD and 7 related to the procedure]. Overall survival for transplanted patients was 55% in 8 years. Currently we have 33/77 (43%) alive patients, 19 (57.5%) CR, 3 (9%) PR, 11 (33.5%) PD. Overall Survival (OS) for whole group was 32%, Disease Free Survival (DFS) 64% and Progression Free Survival (PFS) 40%. Patients who were in DP or relapse prior to the HDCY (57) compared to their status after that had a significant improvement (P=0,001), their OS was 40% to CR-PR group (24) versus 16% PD group (33). In general, mortality was 44 (57%), their cause was 19 PD (43.2%), 8 (18.2%) related to HDCY, 2 (4.5%) related to HDMTX, 7 (16%) related to AHSCT, 6 (13.6%) infections, 1 (2.3%) chronic GVHD after a reduced intensity conditioning regimen transplant and 1 (2.2%) AML. Besides that, 3 patients developed MDS and 1 developed AML. In conclusion, although it had happened a significant number of toxicity related deaths. The sequence is feasible, mainly for sensitive patients and presents an acceptable response. The authors emphasize the high frequency of poor prognosis patients and occurrence of MDS/AML in 4 (5.2%) patients. Brazilian experience using high-dose sequential followed by autologous haematopoietic stem cell transplantation for non-Hodgkin lymphoma B. Duarte, I. Valente, A. Vigorito, F. Aranha, G. Oliveira, E. The purpose of this study was the evaluation of the use of high-dose (HD) cyclophosphamide (CY) 7 or 4g/m2 and HD etoposide (VP-16) 2g/m 2 followed by HD Therapy and autologous haematopoietic stem cell transplantation (AHSCT) in pts affected by aggressive non-Hodgkin lymphoma (NHL). From Jan 98 to Nov 06, 106 pts, 66 males, 40 females, median age 47 (8-66) years were undergone into this regimen. The diagnosis was 83 (78.3%) DLBCL, 13 (12.3%) with T-cell Lymphoma and 10 (9.4%) Mantle Cell. At diagnosis, 88 (83%) were stage III or IV, 34 (32.1%) presented bone marrow involvement, 65 (61.3%) bulky disease, 67 (63.2%) B symptoms and 45 (42.5%) were high or high intermediate risk patients according to IPI. Prior to HDCY all pts had been submitted to a mean of 2 (1-3) therapy lines and 6 (5.7%) were in complete remission (CR), 38 (35.8%) partial remission (PR), 62 (58.5%) progression disease (PD) or relapse. Concerning CY dose 42 (39.6%) pts received 4 g/m 2 and 64 (60.4%) 7 g/m 2 , respectively. After HDS, 13 (31%) pts who had used 4 g/m2 and 30 (47%) who had used 7 g/m 2 were in CR (P= 0.03). After the HDCY 43 (40.6%) were in CR, 33 (31.1%) PR, 21 (19.8%) remained in PD and 9 (8.5%) died. After a median follow-up of 4.1 (1.5-57) months, 80 pts (75.5%) were submitted to AHSCT and their present status is 44 alive [33 CR, 4 PR, 7 PD]; 36 dead [4 CR, 1 PR, 2 relapse, 17 PD and 12 had death related to procedure]. Their OS was 45% in 8 years. Currently we have 50/106 (47%) alive pts, 35 (70%) CR, 6 (12%) PR and 9 (18%) PD. OS was 37%, DFS was 49%, PFS was 42%, EFS was 28%. OS by diagnosis was 42% DLBCL, 40% T-cell in 8 years whereas 20% Mantle Cell in 6 years (P=NS). OS by B symptoms patients was 22% versus 58% (P= 0.002) and EFS was 23% versus 37% (P= 0.03). Pts who were in PD or relapse prior to the HDCY (62) compared to their status after that had a significant improvement (P< 0.001), their OS was 38% to CR-PR group (38) versus 0% PD group (24). In general, mortality was 56 (53%), their cause was 23 (41.1%) PD, 7 (12.5%) related to HDCY, 12 (21.4%) related to AHSCT, 13 (23.2%) infections and 1 (1.8%) GVHD. Besides that 1 (0.9%) patient developed MDS and is alive. B symptoms at diagnosis have appeared as a predictor factor in survival, as confirmed by literature. Our study suggests HDS is an efficient treatment to improve status and to reduce tumoral burden. Although it presented high toxicity related mortality, we consider the treatment to be feasible, especially considering the patients' poor prognosis. were infused.Patients received a mean of 5.5 (range 0-22) irradiated and filtered packed red blood cells;platelet support was by apheresis (median 3.0, range 0-8) or randomly obtained platelet concentrates (median 4,5 range 0-32).4 pts had mucositis WHO grade 3-4;7 pts WHO grade 2;1 pt WHO grade 1;the remaining 28 pts did not show any sign of it.15 pts had fever (8 FUO,7 microbiologically documented).All pts engrafted at median day +11(range 6-19) PMN>100/mmc and +21(range 12-39) for PLT>30.000/mmc,respectively.The mean WBC count at nadir was 0.1 x 106/DL(range 10-700).4 pts developed a WHO grade 1 cutaneous aGvHD;WHO grade 4 liver aGVHD was seen in 2 pts.After a median follow up of 35,6 months(range 2-83),28 pts are still alive (20 CR, 5 PR, 3 in relapse), 4 pts experienced chronic GvHD (3 cutaneos WHO grade 1-2,1 pneumonial WHO grade 1).8 transplant related deaths were documented:2 liver aGVHD;1 cerebral vasculitis at 18 months to the transplant;1 acute respiratory distress syndrome at 2 months from transplant;2 acute bacterial pneumoniae;1 acute thrombotic thrombocytopenic purpura at 18 months from transplant;1 interstizial pnemoniae at 20 months to the transplant.3 death for disease recurrence at 10,17 and 36 months post-transplant,1 for Lymphoma non EBV related at 11 months from transplant.Chimerism study showed a full donor situation in 19 cases,mixed chimerism in the others.In conclusion, NST is feasible and considerable for high risk lymphoma patients. Introduction: Mantle cell lymphoma (MCL) belongs among high-risk lymphoid malignancies, with unfavorable prognosis and median overall survival of 3 to 4 years. Intensified first line treatment with anti-CD20 immunotherapy and high-dose chemotherapy followed by autologous peripheral stem cell transplantation (APSCT) probably leads to improved treatment response, however, the lymphoma remains incurable by means of chemotherapy. Goal: To verify effect of first line APSCT in newly diagnosed MCL. Methods: prospective, single center observational and comparative study. The treatment response was assessed by National Cancer Institute (NCI) criteria. Status of the disease was evaluated prior to and post APSCT. Event Free Survival (EFS) and Overall Survival (OS) of APSCT patients was compared to those treated only with induction chemotherapy. Patients: 48 patients with newly diagnosed MCL (sec. REAL and WHO classification) and treated with induction chemotherapy (CHOP-like regimen with or without rituximab anti-CD20 in 38/48, VAD+chlorambucil in 10/48) in 1996-2005. Results: 19/48 (39%) patients with median age of 54 (37-71) years, in first complete (CR) or partial remission (PR) after induction therapy were indicated to high-dose chemotherapy BEAM with APSCT. Prior to APSCT, 9/19 (47%) patients were in CR, 1/19 (6%) in CRu and 9/19 (47%) in PR. Post APSCT, 18/19 (94%) patients were in CR a and one patient (6%) remained in PR. 3-years PFS a OS in APSCT patients were significantly longer then in non-APSCT: 51% vs. 15%, p= 0,0003, resp. 87% vs. 42%, p= 0,0169. Median age and IPI index of 3-5 were significantly higher in non-APSCT patients: 69 (44-77) vs. 54 (37-71), p= 0,0002, resp. 65% vs. 27%, p= 0,017. Conclusion: The data shows that APSCT (BEAM) leads to better treatment response with higher number of complete remissions reached (from 47% to 94%), improved probability of 3-years PFS and OS (51% a 81%) compared to non-APSCT cohort. Larger studies would be needed to verify lower age and IPI index impact on results in APSCT cohort. MCL patients, however, still relapse and despite intensified first-line treatment with APSCT the prognosis remains poor. Allogeneic transplantation must be considered, especially in younger patients. The need of new effective regimen for high-dose chemotherapy followed by autologous stem cell transplantation (ASCT) in aggressive B-cell non-Hodgkin s lymphoma (NHL) patients and promising results observed so far in trials with 90Y-Ibritumomab tiuxetan containing regimens in ASCT strongly warrants the investigation of 90Y-Ibritumomab tiuxetan combined busulfan/ cyclophosphamide/etoposide (Z-BuCyE) high-dose chemotherapy with ASCT for relapsed, refractory, or high-risk B-cell NHL. Treatment consisted of two doses of Rituximab (250 mg/m², IV, day -21, -14) and a single dose of 90Y-Ibritumomab (0.4 mCi/kg, IV, day -14). All patients received conditioning regimen: busulfan (3.2 mg/kg, IV, day -7, -6, -5), etoposide (200 mg/m², IV, day -5, -4), and cytoxan (50 mg/kg, IV, day -3, -2) followed by ASCT (day 0). Nineteen patients were entered the trial with 13 patients actually evaluable. The median age was 46 years (range: 25-60), and 6 (46%) patients were male. Histology was diffuse large B-cell (n=10), follicular (n=1), Burkitt (n=1), and mantle cell lymphoma (n=1). The overall response rate was 76.9% (10/13): continued complete response (CR), 38.5% (5/13); induced CR, 23.1% (3/13); continued or induced partial response, 15.4% (2/13). Three patients (23.1%) had progressed after transplantation and two of these patients died of progression. Median followup duration was 6.0 months. Median progression-free survival and median overall survival has not yet been reached. Toxicity was principally non-hematologic. Grade 2 toxicity included mucositis (53.8%), nausea (61.5%), vomiting (15.4%), diarrhea (23.1%), and elevation of liver enzyme (7.7%). Grade 3 toxicity included mucositis (15.4%), nausea (23.1%), and diarrhea (23.1%). There was no grade 4 toxicity. Infection occurred in ten patients, bleeding in one patient, and there was no treatment related mortality. This preliminary analysis shows that the combination of Z-BuCyE and ASCT has excellent efficacy and is well-tolerated treatments for relapsed, refractory or high-risk B-cell NHL. FDG-PET early durino first-line chemotherapy of Hodgkin's lymphoma (HL) has a high positive and negative predictive value. The predictive value of functional imaging (FI) in refractory or relapsed HL after salvage treatment and prior to high-dose (HD) chemotherapy and autologous stem cell transplantation (ASCT) is less well estabilished. A restrospective analysis was undertaken of the clinical characteristics, FI results, event free survival (EFS) and overall survival (OS) data for 66 consecutive patients (pts) with refractory or relapsed HL treated with ASCT between 1997 and 2007. The median follow-up was 52 months (range 0-125 months). Following salvage therapy, and prior to ASCT, 63 out 66 evaluable pts (96%) had response assessment with a conventional CT scan, 53 pts also had FI with a Gallium scan (18), FDG-PET scan (31) or both (4). Of the 53 pts with FI prior to ASCT, 21 (40%) were in metabolic complete remission (FI-negative) and 32 pts (60%) had residual disease (FI-positive). The median EFS and OS for the whole cohort were 29 and 52 months, respectively. Median EFS was 31 and 13 months (p=0.096), and median OS was 60 and 39 months (p=0.35) for those with negative and positive FI, respectively. Significantly more pts with negative FI achieved CR with conventional imaging compared to those with positive FI (9/19 = 47% vs 2/32 = 6% p0.001). Male pts were disproportionately represented in the cohort with positive FI (20/32) compared to the pts with negative FI (7/12, p=0.038). There was no association between FI-positivity and the clinical features of age, short duration (< 12 months) of initial response, B symptoms or extranodal disease at the time of relapse, or the type of ASCT conditioning used. We didi not demonstrate a strong predictive value of FI outcome prior to ASCT for relapsed or refractory HL despite a trend for improved EFS for pts with negative FI. We did demonstrate that males had a higher rate of FI positivity suggesting this is also an adverse risk factor for failure of salvage therapy in relapsed/refractory HL. Although CT scanning remains the gold standard for the staging and follow-up of malignant lymphomas, fluorodeoxyglucose PET has a potential role in accurately staging disease and in predicting response to therapy. Compared to most types of lymphomas, PET-CT has excellent sensitivity for even small tumour burden in Hodgkin's disease (HD). A retrospective analysis was performed on all patients who underwent stem cell transplantation (SCT) between Mar 2005 and Feb 2007 for progressive or relapsed HD at a single centre. Post-transplant PET-CT evaluation was planned in all cases. During the treatment period, 24/27 patients underwent autologous SCT (ASCT), and additional 3/27 patients who failed previous ASCT underwent allogeneic SCT. The male/female ratio was 1,7/1. The median age at the time of SCT was 32 (range 19-60) years. The indication of ASCT was primary progressive HD, early relapse and late relapse in 13, 6 and 8 patients, respectively. The median follow-up of this study was 733 post-transplant days. 25/27 patients are alive, while 2 patients died on D14 and D27, respectively, before the first post-transplant staging. 1/25 patient relapsed as documented by Gallium-scans. PET-CT scans were performed in all of the remaining 24 patients at various time points (median D95 /range D67-467/). The first post-transplant PET-CT scan documented complete metabolic remission in 15/24 patients. All of them are in complete remission at the time of evaluation after a median follow-up of 740 (range 567-998) days. The 15 PET-CT negative cases included 3 chemoresistant patients. In 3/9 PET-positive patients rebiopsy or further scans documented progression or relapse. In 2/9 patients follow-up scans raised the possibility of false PET-positivity. Both of them remain free of progression after 720 and 893 days, respectively. In the remaining 4/9 PET-positive patients rebiopsy and/or additional PET-CT scans were planned to clarify disease status. One of them, an allo-transplanted patient, turned PET-negative in 138 days after discontinuing immunosuppression. In the other patients, evaluation is pending. The follow-up time of this retrospective study is relatively short, but a negative PET-CT scan after SCT seems to be a very good predictor for long term progression-free survival in refractory or relapsed HD. However, in the case of a positive post-transplant PET-CT scan, further investigation is warranted to distinguish false positive tests from impending progression. Peripheral T-cell lymphomas (PTCLs) are relatively common in Korea and known to have poor prognosis. There have been few reports comparing the outcome following autologous stem cell transplantation (ASCT) between PTCLs and diffuse large B-cell lymphoma (DLBCL). It was aimed to analyze the survival after ASCT in PTCLs and DLBCL. We reviewed ASCT registry of Asan Medical Center, Seoul, Korea. PTCLunspecified (n=20) and angioimmunoblastic T-cell lymphoma (n=3) were included as PTCLs for current analysis and pathology has been reviewed according to the WHO classification by a pathologist (JH). We have found 23 patients with PTCLs and 54 DLBCLs. There was no significant difference of patient characteristics regarding age and salvage chemotherapy regimens between the patients with PTCLs and DLBCL; however, there were more male patients in PTCLs (82.6%) than in DLBCLs (57.4%) (P=0.034). The regimens of high-dose therapy, number of infused CD34+ cells, time of and status at ASCT, and each category and score of International Prognostic Index (IPI) showed no difference between 2 groups. Overall survival (OS) at 2 years was 46% (95% CI, 33-59%) for DLBCL patients and 41% (95% CI, 21-61%) for PTCLs with no statistical difference (P=0.769). DLBCL patients showed 2 year event-free survival (EFS) of 40% (95% CI, 27-53%) with no difference to 2 year EFS of PTCLs (43%; 95% CI, 23-63%) (P=0.0833). On univariate analysis with pooled data of both groups, upfront ASCT, complete response status (CR) at ASCT, LDH/performance/stage categories of IPI, and low/lowintermediate IPI/aaIPI were significant prognostic factors for both OS and EFS. Subset analyses showed that PTCLs had better OS (P=0.0013) and EFS (P=0.0037) than DLBCL in the subgroup with high/high-intermediate aaIPI. Multivariate analysis showed that CR status and low or low-intermediate aaIPI were favorable for both OS (RR 2.97, 95% CI: 1.23-7.19; RR 3.76, 95% CI: 1.74-8.14, respectively) and EFS (RR 2.60, 95% CI: 1.17-5.81; RR 2.75, 95% CI: 1.30-5.84, respectively). Pathologic discrimination of PTCLs and DLBCL had no impact on OS (RR 0.56, 95% CI: 0.27-1.18) or EFS (RR 0.62, 95% CI: 0.30-1.30) with multivariate analysis. PTCLs do not appear to have inferior outcome following ASCT to DLBCL. Well known prognosticators such as disease status, clinical situation and IPI score at ASCT have impact on survival following ASCT regardless of T-or B-cell lymphoma phenotype per se. Indication of a strong graft-versus-lymphoma effect in patients with primary cutaneous T-cell lymphoma after allogeneic stem cell transplantation M. Nickelsen* (1), S. Schoenland (2) , U. Hegenbart (2) , P. Dreger (2) , M. Gramatzki (3) , N. Schmitz (1) ( We here describe 5 patients (pts) with an aggressive course of cutaneous T-cell lymphoma (cTCL) who received allogeneic stem cell transplantation (SCT) as the remaining curative treatment option. Patients: Histological subtypes of the 5 pts (4 male, 1 female) were PTCLu, Mykosis fungoides, gamma/delta TCL, blastic NK-cell and cytotoxic TCL. All pts had primary cutaneous manifestations of the TCL, two had additional nodal involvement (lymph nodes, spleen). Time from diagnosis to SCT varied from 5 months to 15 years. All pts had received at least 2 chemotherapeutic regimens (2-6) before conditioning and were refractory to at least 1 regimen. All pts reached PR before SCT with high-dose methotrexate-containing regimens with (3) or without (2) Peg-Asparaginase. Median FU after SCT is 18 (9-31) months. Results: Between 05/05 and 12/06 the pts underwent SCT after dose-reduced conditioning (1 x BEAM, 3 x FC, 1 x FBC). Donors were matched sibling (1), MUD (2) and mismatched unrelated donors (8/10, 2). Graft versus Host Disease (GvHD) prophylaxis consisted of CSA combined with short course MTX or MMF. 3 pts with unrelated donors received ATG (1) or Alemtuzumab (2) . There were no grade 3/4 toxicities immediately after SCT. All pts became full chimaeras (>95%) after SCT (day +60) and developed at least limited cGvHD of the skin. Two pts had extensive GvHD (liver, lung). 1 pt progressed within two months after SCT. 4/5 pts reached CR, two of these ongoing (+365, +630). There was one proven relapse (+390) 2 months after loss of complete chimaerism (60% donor) following immunosuppressive treatment of extensive GvHD of the liver. The pt received local radiotherapy and two DLI´s and is in cCR (+930) with limited cGvHD of the skin. Another pt had suspected relapse (+420). After tapering of CSA despite of cGvHD of the skin he is in cCR (+720). The pt with early progressive disease discontinued immunosuppressive treatment, too. She developed limited GvHD of the skin, mucosae and liver and reached ongoing stable disease (SD) (+270). Due to GvHD it was not possible, so far to stop immunosuppression or to administer DLI. Conclusion: We here report a relatively large series of 5 pts with aggressive cTCL. In all pts disease status improved after SCT (4 CR, 1 SD). Concordant to 27 published cases of SCT in cTCL, the correlation between disease status, immunosuppressive treatment, DLI and development of GvHD observed in our 5 pts strongly suggests an impressive GvLeffect in cTCL. The efficacy of sequential high-dose chemotherapy with autologous stem cell transplantation in relapsed and refractory lymphomas. Seventeen-year experience of a single centre G. Koumakis, N. Tsoukalas*, D. Tryfonopoulos, S. Demiri, K. Papadimitriou, G. Lipas, M. Vassilomanolakis, V. Barbounis, S. Droufakou, I. Filis, C. Panopoulo, M. Moraki, A. Efremidis "Saint Savvas" Anticancer Hospital (Athens, GR) Background: Traditionally high-dose (HD) chemotherapy of refractory lymphomas (Hodgkin and non-Hodgkin) consists of a single cycle of chemotherapy. In 1990 Gianni, et al. proposed sequential infusion with high doses of effective regimens as a conditioning and simultaneously therapeutic part of a megatherapy program which showed high response rates in refractory Hodgkin lymphomas. The aim of this study was to investigate the applicability of the sequential high dose therapy program in patients with refractory lymphomas as well as to estimate the therapeutic profit compared to single cycle megatherapy and transplantation. Materials and methods: Fifty one patients (median age 38 years, range 16-60) (23 females/28 males) who suffered from Hodgkin (21) and non Hodgkin lymphomas (30) were enrolled. All patients had received conventional chemotherapy ± radiotherapy and presented primary refractory disease or relapse within the first 12 months since the first treatment. Peripheral blood stem cells (PBSC's) were mobilized with HD-CTX 6gr/m 2 and growth factor successfully in all patients. Upon hematologic recovery they received sequentially HD-VP16 1400mg/m 2 , HD-MTX 8gr/m 2 , HD-VCR 1,4mg/m 2 and HD-Cisplatin 120mg/m 2 . Finally they received high dose chemotherapy with BEAM (BCNU 300mg/m 2 D1, Etoposide 200mg/m 2 D2-5, Aracytine 200mg/m 2 D2-5 and Melphalan 140mg/m 2 D6). After 72 hours from the end of chemotherapy PBSC's were reinfused. Results: Overall response rate was 86,27% [ 28 (54,90%) complete remission (CR) and 16 (31,37%) partial remission (PR) ] while seven patients (13,73%) presented deterioration (PD). Particularly from patients with Hodgkin disease 11 (52,38%) presented CR, 6 (28,57%) PR and 4 (19,05%) PD, while from patients with non Hodgkin lymphoma 17 (56.67%) presented CR, 10 (33.33%) PR and 3 (10%) PD. Toxicity was manageable. The mean overall survival (OS) was 112,72 months (SE=14,03) and the mean time to progression (TTP) was 111 months (SE=14,51). Conclusion: Sequential high dose chemotherapy followed by autologous stem cell transplantation is effective in patients with lymphomas refractory to conventional therapies and probably is better than classical programs with single cycle megatherapy and transplantation. Introduction: Autologous stem cell transplantation (ASCT) is the standard of care for relapsed or refractory Hodgkin Lymphoma (HL). Reduced intensity conditioning allogeneic SCT (RIC-Allo) has become a popular option for patients (pts) who have failed or are ineligible for ASCT. We wished to review the Canadian experience of RIC-Allo for HL in preparation for an innovative national multicentre Phase II trial. Methods: 24 pts with relapsed (REL) or primary refractory (REF) HL underwent RIC-allo at 8 Canadian centers between June 99 and Nov 06. Conditioning regimens consisted of: FLU-MEL:6, FLU-BU-P:7, FLU-BU:1, FLU-BU-TBI 2 Gy:1, FLU-CY:8 and FLU-TBI 2Gy:1. ATG was given in 11 cases; no alemtuzumab was used. Calcineurin inhibitor-based GVHD prophylaxis was used in all cases (CsA: 18, FK506: 6) with short course MTX (8) or MMF (11). Second line chemotherapy and ASCT was considered 1 regimen for calculation of prior therapy. REF HL was defined as progressive disease on or within 3 months of primary therapy. Results: The median age at diagnosis was 20 years (range 10 -39). Histologic subtypes were: nodular sclerosing (21), mixed cellularity (1) and lymphocyte predominant (2) . The median time from diagnosis of HL to ASCT was 12 months (range 8 -165). 23/24 pts had undergone prior ASCT (21 with REL HL, 3 with REF HL). Median number of chemotherapy regimens prior to RIC-allo was 3 (range 1-6). 22/24 had received prior radiotherapy. The median age at the time of RIC-allo was 25 years (range 18 -44). Median time from ASCT to RIC-allo was 16 months (range 2-92). Response to treatment immediately prior to RIC-allo was: CR/CRu: 3, PR: 11, SD: 4, PD: 1 and unknown: 3. Graft source was matched sibling (16) and unrelated donor (8; 2 with 1 HLA Ag mismatch). 1 pt developed graft failure and was subsequently re-transplanted. With a median follow-up post RIC-allo of 11.5 months (range 1 -92) for the entire cohort and 27 months among survivors, the 2-year overall survival was 40%, 2-year progression-free survival was 19% and the 2-year treatmentrelated mortality (TRM) was 27%. Conclusions: This Canadian review of RIC-allo in HL shows significant variation in type of preparative regimen and GVHD prophylaxis employed at individual centers. Even in this relatively young cohort, both TRM and relapse were significant and confirm the findings of other groups. Pts undergoing RICallo for HL should be enrolled on prospective studies that evaluate novel transplant strategies. Background: The safety and efficacy of non-myeloablative conditioning followed by allogeneic stem cell transplantation (NMSCT) is still under investigation in patients with resistant/refractory Hodgkin's Lymphoma. Patients (pts) and methods: from September 2003 to September 2007 we treated 11 pts with NMSCT, 10/11 with identical sibling peripheral blood stem cell progenitors (PBSCP), 1/11 with matched unrelated donor PBSCP. 9/11 were in disease progression (PD), 2/11 were in > 2nd complete remission (CR). All pts had previously received high dose chemotherapy with autologous PBSCP. Median age was 29 years (range 20-45), 8 (male) and 3 (female). 10/11 pts with sibling donor received a fludarabine-based conditioning regimen, 1 pt with a MUD received Treosulfan, Fludarabine, ATG and Rituximab 500 mg as EBV prophylaxis. Graft-vs-host disease (GVHD) prophylaxis was Cyclosporine A (CyA) and Mycophenolate Mofetil (MMF) for pts with sibling donor, CyA only for the patient receiving MUD. Results: all patients engrafted and full donor chimerism was achieved in all patients. Grade II-IV acute GVHD developed in 7 of the 11 pts (64%), chronic GVHD in 6 of the 11 pts (55%). After a median follow-up of 6 months (range 4-48), 3/11 pts died (27%) (1 GVHD, 1 sepsis, 1 Thrombocytopenic Thrombotic Purpura/Acute Renal Insufficiency). In the 11 evaluable patients, 1/11 achieved a PR, 6 a CR, 2 a SD and 2 progressed after transplant. 7/7 responding patients had GVHD, 8/11 pts are still alive (73%). Median progression free survival at 1 year is 50%, with a median survival of 1.05 years (95%CI 0.3-1.18). Conclusions: non myeloablative allogeneic transplantation (NMSCT) with Fludarabine-based reduced intensity conditioning regimen is a feasible salvage strategy. It has an acceptable transplant related mortality in such a poor prognosis group of pts with Hodgkin's lymphoma relapsed and refractory after autologous Peripheral Blood Stem Cell Transplant. We observed a high rate of acute and chronic GVHD possibly due to the fact that all pts received several lines of chemotherapy. Furthermore acute and chronic GVHD seems to be associated with graft-vs-lymphoma effect. A prolonged complete remissions were achieved in few pts. and most of them received subsequent chemotherapy after PD. The disease status at transplant is the main factor affecting the prognosis. Further investigations, considering also patients in PR or CR could be useful to confirm the clinical relevance of NMSCT. TBI was used in <2% of patients. Five year overall survival (OS) of all patients was 60%. Treatment related mortality was 2.3%. In univariate analysis, the following factors were significant predictors for OS: CD34+ cell dose/kg, histology, conditioning regimen, and mononuclear cell (MNC) dose/kg. OS (5 yr) significantly differed by histology: MM -67%, HL -62%, DLBCL/ST -50%, MCL -46% (p=0.0001) and by conditioning regimen: Melphalan 200 -69%, BEAM -68%, VIP -60%, BuCyV/TBI -48% (p=0.0000). Of interest, increasing MNC dose adversely influenced OS with 16% increment of mortality risk per 1x108/kg increase in MNC dose (p=0.0011). In multivariate analysis, higher risk of death was related to older age (continous variable), male sex, higher MNC dose, histology: DLBCL vs. other, and conditioning regimen: BuCyV or TBI vs. other. Patient survival was also worse in the period 1997-2002 (56%) than in 2003-2007 (70%) . Conclusion: these data demonstrate accepatable safety and substantial efficacy of autologous HCT in high-risk lymphoma patients (recurrent or hard-to-treat). However, more active conditioning regimens and better quality graft preparations are clearly needed. Adverse effect of higher MNC dose found in our study needs a further investigation. Fotemustine, etoposide, aracyitin and melphalan. Variation of a "well tested" contitioning regimen for lymphoma treatment with autologous haematopoietic stem cell transplantation R. Scalone* (1), A. Pinto (2) , G. Marcacci (2) , N. Cascavilla (3) , P. Scalzulli (3) Sollievo della Sofferenza (San Giovanni Rotondo, IT); (4)Ospedale "Vito Fazzi" (Lecce, IT); (5)Presidio Ospedaliero "Umberto I" (Salerno, IT) Patients and Methods: We replaced the carmustine of BEAM with fotemustine, with the primary end-point to evaluate the feasibility of fotemustine, etoposide, aracyitin and melphalan (FEAM). 33 patients were conditioned with FEAM. 22 were male, 11 female. Median age was 51 years (range, 20-70). 5 had HL, 28 had NHL. 9 patients were stage II (5 bulky), 8 stage III, 16 stage IV. Six patients had a bone marrow involvement, 3 a central nervous system (CNS) involvement. At the time of transplant, 9 patients were in complete remission (CR), 1 in very good partial remission (VGPR), 19 in partial remission (PR), 4 had progressive disease (PD), 14 patients were at first line of therapy, 19 at second or third line. Patients received fotemustine 150 mg/m² on days -7,-6, etoposide 200 mg/m² and aracytin 400 mg/m² on day -5,-4,-3,-2, and melphalan 140 mg/m² on day -1. The median CD34+ cells infused was 4.6x10 6 /Kg (range:1-21.8). Engraftment: The median time to neutrophil (N> 0.5x10 6 /L) and platelet (PLT >20x10 9 /L) engraftment was 11 (range, 9-17) and 12 days (range, 6-105) respectively. 16 patients (48%) received trasfusions of red blood cell units, with a median of 2 units (range, 1-8). All patients received platelet trasfusions with a median of 2 units (range, 1-10). Toxicity: No chemotherapy-induced nausea and vomiting (CINV) was observed in 6 patients (18%), 18 patients (55%) had CINV grade I-II, 9 patients (27%) grade III, no grade IV was observed. No mucositis was observed in 4 patients (12%), 17 patients (52%) had mucositis grade I-II, 10 patients (30%) grade III, 2 patients grade IV. No diarrhea was observed in 24 patients (73%), 7 patients had diarrhea grade I-II, 2 patients grade III, no grade IV was observed. No epatic toxicity was observed in 30 pts (91%), 1 patient had epatic toxicity grade I and 2 patients grade III, no grade II or IV was reported. No patient had renal toxicity. Fever >38,5°C was documented in 25 patients (76%) with a median duration of 3 days (range 1-15). Outcome: Three patients died, 2 for PD at day +110 and +75 from transplantation respectively, 1 for bacterial meningitis at day +45 from transplantation. TRM at 100 days was 3%. At a median follow-up of 4 months (range, 1-6), 17 patients were valuable, 4 patients maintened CR, 10 patients achieved CR (77%), two of these had CNS involvement. Conclusions. Our study demonstrated feasibility of FEAM. A longer follow-up is needed to valuate the efficacy of this conditioning regimen. Vinorelbine and low dose cyclophosphamide: a safe and effective new regimen for peripheral stem cell mobilisation in lymphoma patients G. Marcacci*, G. Corazzelli, F. Frigeri, F. Russo, M. Arcamone, G. Capobianco, C. Becchimanzi, A. Pinto National Cancer Institute, Fond. Pascale (Naples, IT) Objectives: High dose Cyclophosphamide (Hd-CTX) at doses ranging from 7 to 4 gr/sq plus Granulocyte-Colony Stimulating Factor (G-CSF) is commonly used to mobilize peripheral blood stem cells (PBSC) in Multiple Myeloma (MM) and Lymphoma. Such treatment is however associated with substantial haematologic toxicity and can require hospitalization for febrile neutropenia and hematological support therapy. A combination of low dose CTX (1.5 gr/sq) plus Vinorelbine (25 mg/sq)(VRL) and G-CSF 10 microg/kg/daily was recently demonstrated as a safe and highly efficacious outpatient procedure for mobilizing PBSC in newly diagnosed MM patients (pts) (Annunziata M et al, Ann Hematol 2006) . In this study, we evaluated safety and mobilizing efficacy of this less toxic outpatient combination (VRL plus CTX) plus G-CSF in 23 previously pretreated pts with non Hodgkin's (NHL) and Hodgkin's Lymphomas (HL). Methods: Twentythree pts (M/F=12/11), median age 44 yrs (range 27-61), with relapsed and refracory lymphoma (NHL/HL=21/2, DLBCL=11, FCL=9, Burkitt-like=1), stage at relapse (advanced=17, early=6), with a median of 1 line of previous chemotherapy (range [1] [2] were accrued after informed consent. Mobilizing chemotherapy consisted of VRL (25 mg/sq iv day 1) and CTX (1.5 gr/sq iv day 2) plus G-CSF 10 microg/kg/daily from day 4 until apheresis procedures. All pts received mobilizing chemotherapy in an outpatient setting and without any hospitalization. Results: Twenty pts (87%) successfully mobilized PBSC (median CD34 peak 86.21/microl, range 17.74-160.2/microl; median CD34 in the harvest 4.1 x 10 6 /Kg, range 1.1-8.41) while only 3 pts (13%) were considered as mobilization failures (median CD34 peak 8.2/microl, range 7.5-9.1/microl). Apheresis was performed at day of CD34 peak (median +11, range 9-12), with a median of 1 apheresis/patient (range [1] [2] . The median number of subcutaneous G-CSF doses administrated was 8 (range 7-9). None of the pts, both mobilizers and failures, required hospitalization for febrile neutropenia, hematologic support therapy or other therapyrelated or unrelated complications. Eleven pts proceeded to high dose therapy and stem cell transplantation with normal engraftment times and without cytopenia-related deaths (TRM=0%). Conclusion: Our results indicate that the combination of VRL, low dose CTX and G-CSF represents a safe and effective mobilizing strategy for previously treated patients with NHL and HL. High dose chemotherapy ( HDS) followed by Autologous Stem Cell Transplantation ( ASCT) is the treatment of choice for relapsed lymphomas ( HD and NHL). In our institution, from November 1995 to December 2007, 131 patients affected by HD or NHL underwent ASCT. Initially, we used BEAM as conditioning regimen (112 patient). Considering the excellent to this regimen and the incidence of a non negligible number of post ASCT relapse, we decided to intensify the conditioning regimen, in order to try to achieve a more eradicating activity using drugs never used during chemotherapy, thus we modified BEAM schedule by replacing etoposide and cytarabine, mostly used in the standard schedules of conditioning, with thiothepa (BTM). Doses and drugs administration timing were as follow: BCNU 300 mg/mq on day -6 THIOTEPA 150 mg/mq from day -5 to day -3 MELPHALAN 140 mg/mq on day -2 On day +1, after reinfusion, a single dose of Pegfilgrastrim is given. We compared 19 patients treated by BTM with 19 consecutive patients previously treated by BEAM after 2004 ( when the dose of pegfilgrastim was introduced in the schedule). In both groups, the median time to neutrophil engraftment (> 1000/mm3) was the same ( 9 days). The total amount of RBC transfusion was 0,5 U for the BTM group and 0,25 U for the BEAM group. The median time to platelet engrafment ( > 20.000/ mm3) ( 12 days) and the total amount of platelet transfusion ( 2 U ) were identical in the groups. The incidence of mucositis and the number of episodes of febrile neutropenia and septicemya did not differ between the two groups. Median OS and DFS was 21,6 and 18 months for the BEAM group; only 6 and 3 months for BTM group ( very short followup until now) Relapse rate was 43.75 for BEAM group and 10.5 for BTM group. Regimen-related toxicities were similar, except that BEAM group was associated with more gastrointestinal mucositis (median time of diarrhea 1 day). F. Pester*, A. Klink, S. Scholl, K. Schilling, L. Muegge, K. Höffken, H. Sayer Friedrich-Schiller University (Jena, DE) It has been shown that patients (pts) with chemosensitive relapsed Hodgkin-(HD) and Non-Hodgkin's lymphoma (NHL) were more likely to remain disease-free when consolidate high-dose therapy (HDT) with stem cell support was utilized. The standard regimen before autologous peripheral blood stem cell transplantation (PBSCT) is the combination of carmustine, etopophos, cytarabine and melphalan (BEAM). Pulmonary toxicity with fibrosis and progressiv reduced diffusion capacity is a specific complication of carmustine within this regimen. To reduce this pulmonary toxicity we compared the efficiency of replacing carmustine through the alcylating drug thiotepa in a non-randomised phase II study. Pts received 200 mg/m² thiotepa on day -6 instead of carmustine followed by 2 x 150 mg/m² etopophos (day -6 to -3), 2 x 200 mg/m² cytarabine (day -6 to -3), and 140 mg/m² melphalan (day -2) (TEAM), only if pulmonary function test or cardiac functions test showed significant impairment. Pts without impaired function test received the established BEAM protocoll. Between 06/1998 and 11/2007 20 female and 39 male pts (n=59) with relapsed NHL (n=45) or HD (n=14) were treated with BEAM (n=31) or TEAM (n =28) HDT followed by PBSCT. All pts had received at least two salvage chemotherapy courses prior to HDT. The median age was 46 years (range 19 -67) in both groups. After a median follow up of 36 months (range 4 -113) the relapse rate was similar in both groups: 32 % (n=10) in BEAM and 39 % (n=11) in TEAM. After reaching a complete remission (CR) prior PBSCT the overall survival was comparable (100 %, 12/12 pts for BEAM and 80 %, 4/5 pts for TEAM). Nevertheless the overall survival is significant different between the BEAM and TEAM group (84 % vs. 54 %), mainly due to the fact that only 21 % of the TEAM patients were in CR before transplant compared to 39 % of the BEAM pts. One of the TEAM patient developed and died by a secondary leukaemia some weeks after HDT. Another TEAM patient died by pneumonitis after consolidating radiation and one patient died with sepsis during the HDT prior 5 cycles of Dexa-BEAM salvage chemotherapy. The HDT with TEAM seems to have the same effectiveness as the BEAM-protocoll in the treatment of relapsed lymphoma for patients who reached a CR prior PBSCT. In pts not reaching adequate disease response TEAM shows no benefit in view of overall survival. Optimal remission status prior PBSCT is the main factor for favourable outcome in advanced lymphoma. Ifosphamide, etoposide and epirubicin as a salvage treatment and mobilising regimen for patients with refractory and relapsed non-Hodgkin lymphoma and Hodgkin's disease in single-centre experience A. Czyz, D. Dytfeld, L. Gil, A. Nowicki, M. Kozlowska, J. Parulska, K. Sawinski, M. Komarnicki University of Medical Sciences (Poznan, PL) Ifosphamide, etoposide and epirubicin (IVE) chemotherapy has been shown to be a salvage treatment option and effective peripheral blood stem cell (PBSC) mobilising regimen for patients with lymphoma. We report chemotherapy related toxicity, mobilisation details and treatment outcome of 29 patients, median age 34 years (range 19-60) with Hodgkin disease (n=18) and non-Hodgkin lymphoma (n=11) treated with 1-2 cycles of IVE (ifosphamide 9g/m², etoposide 600mg/m², epirubicin 50mg/m²) between January 2006 and December 2007 in our center. The indication for IVE was primary refractory disease (n=11), relapse (n=10) and failure to achieve CR with two or more previous chemotherapy lines (n=8). 27 of the 29 patients were treated with the intent to proceed to high-dose therapy supported with autologous hematopoietic stem cell transplantation (autoHSCT). Results: The toxicities of a total 41 cycles of IVE were analyzed. The neutrophil count decreased < 0,5G/L in all cycles. Infectious complications occured in 23/41 (56%) cycles represented by neutropenic febrile (n=21), bacterial endocarditis (n=1) and severe herpes zoster infection (n=1). 8 of the 41 (19%) cycles were complicated by transient encephalopathy (somnolency-4, meningismus-2, drowsiness-2) involving 8 patients. Two cycles were complicated by acute renal failure in 2 patients treated earlier with DHAP. 27 of the 29 patients were treated with the intent to mobilise PBSC and collect minimum 3x10 6 /kg CD34+ cells. The median total number of mobilised CD34+ cells was 5,12x10 6 /kg (range 0,77-11,8) collected with a median of 3 (range 1-5) leukapheresis per patient. 5 of the 27 (18%) patients failed a mobilisation attempt, 2 of them because of chemotherapy related toxicity. 21 of the 29 (72%) patients achieved a major response (CR -11, PR-10) after IVE. OS and PFS for the whole group were 86% (95% CI 72-100) and 29% (95% CI 17-56) respectively at 2 years estimated with the Kaplan-Meier method. Twenty patients who proceeded to autoHSCT are all alive with estimated PFS 41% (95% CI 14-68) at 2 years. Conclusions: IVE is an effective mobilising regimen in heavily pre-treated patients and has a satisfactory response rate in refractory and relapsed lymphoma with acceptable toxicity. The patients who are successfully mobilised with IVE and proceed to autoHSCT can achieve a sustained remission. High-risk Hodgkin's lymphoma: outcome after intensified chemotherapy, BEAM or CVB. A single-centre analysis of 60 patients H. Diedrich*, H. Kamal, D. Forstmeyer, E. Dammann, E. Weissinger, M. Stadler, M. Eder, A. Ganser Hannover Medical School (Hannover, DE) Introduction: Hodgkin´s lymphoma (HD) can nowadays be cured in about 75% of patients (pts) by using multimodality therapy with chemo-and radiotherapy. However, for pts with bad response to conventional treatment, relapse or resistant disease the therapy of choice is an intensive conditioning regimen followed by autologous or allogeneic stem cell transplantation (SCT). We here report the outcome of 60 pts with HD after autologous and allogeneic SCT. Patients and methods: From December 1986 to May 2007 56 pts with HD with either insufficient or delayed response to standard therapy were treated with myeloablative chemotherapy and auto-SCT (3 pts with tandem SCT). Median age was 32 years (range: 17-58) with 35 and 21 females and males, respectively. At time of SCT 13 pts were in CR, 22 pts in 1st or subsequent PR, and 21 pts had less than PR upon standard therapy. Time from last conventional treatment to SCT was median 47 days (range 16 -160). BEAM and CVB was used in 33 and 20 pts, respectively, whereas 3 pts received individualized high-dose therapies. Stem cell source was bone marrow (BM) in 17 pts and peripheral blood (PBSC) in 38 pts with one pt receiving BM plus PBSC. Four pts were allografted for relapse after auto-SCT upon reduced intensity conditioning. Results: With a median follow-up of 31 months (range: 0.3-228) 34 pts (61%) treated with auto-SCT are alive whereas 22 pts (39%) died, 7 of them within 100 days after SCT. Death was mostly due to progressive disease (n=10) or infectious complications (n=6). Time to engraftment (neutrophils >500/µl) was 11 days (range . Overall survival at 12 months after auto-SCT is 71 % and reaches a long lasting plateau at 55,5% level, ± 0,73% after 65 months. In addition, four pts received allogeneic SCT for relapsed HD after prior autol-SCT. Fludarabin and Melphalan were used for conditioning for allogeneic SCT from MMRD (n= 1) and MUD (n= 3). GvHD prophylaxis consisted of ciclosporine ± MMF or methotrexate. Two pts. are alive at +31,9 months with newly relapsed HD and +32,1 months in persistent CR, whereas 2 pts died after 9 and 13.2 months due to progressive disease and infectious complications. Conclusions: These data demonstrate that high-dose chemotherapy followed by autologous SCT can result in longterm survival for more than half of pts with relapsed or high-risk HD. In addition, allogeneic SCT may be considered for patients with relapse or progressive disease after autologous SCT. Mobilized peripheral blood stem cells (PBSC) are widely employed in the management of patients with high-risk non-Hodgkin's lymphoma (NHL). The DHAP regimen has thus been integrated into various treatment plans tailored for NHL patients as a second line therapy and salvage chemotherapy for PR patients, or as an intensification and mobilizing regimen in CR HiRisk. Treatment with rituximab is widely used for B cell NHL. However, its effects on peripheral blood stem cell mobilization are not completely known. In our study we retrospectively evaluated 62 NHL patients (30 with follicular and 32 with diffuse large B cells) mobilized with the DHAP regimen, 34 of whom received rituximab while 28 did not, to evaluate whether there were any differences in the number of CD34+ cells (x106/kg) or engraftment kinetics. Patients mean age was 42 years (range 17-65); 48 patients (77.4%) had stage III-IV disease; 41 patients (66.1%) had bone marrow involvement; systemic B symptoms were present in 35 patients (56.5%). At the time of PBSC mobilization, 35 patients (56.5%) were classified as responsive (complete remission, partial remission or sensitive relapse) and 27 (43.5%) as not responsive (refractory relapse or refractory to therapy). Median CD34+ cells collected were 6.2 x10 6 /kg in patients receiving rituximab vs 8.3 x10 6 /kg CD34+ cells (p=n.s.) in the non rituximab-treated group. Failure to mobilize, defined as failure to reach a circulating CD34+ cell count of 10/mcl, occurred in 3 patients (8.8%) in the rituximab group and 4 (14.3%) in the non rituximab group. All patients were transplanted using the myeloablative chemotherapy conditioning regimen (BEAM); G-CSF was administered subcutaneously from day +3 at a dose of 5 microg/kg body weight/day. Comparison of the two groups showed no statistically significant difference between median days to absolute neutrophil >0.5 x10 9 /L and platelet >20 x10 9 /L counts after autologous stem cell transplantation, and no differences in the incidence and severity of infections, days of fever or duration of antibiotic treatment between groups. In conclusion, the use of rituximab in association with DHAP does not affect the ability to collect an adequate number of PBSC for autologous stem cell transplantation in NHL. Further studies are warranted in larger populations to determine the impact of rituximab on stem cell collection, engraftment and survival. Autologous stem cell transplantation (ASCT) is considered a standard of care in patients with chemosensitive relapsed diffuse large B-cell lymphoma (DLBCL). We have evaluated the outcome of these patients. Patients and methods: In 1996-2006, 68 patients with relapsed DLBCL received ASCT in five centres. There were 44 males and 24 females with a median age of 54 years (24-68) at ASCT. Only eight patients (12 %) had received rituximab during the first-line therapy. The median duration of the first remission (CR1) was 13 months . Ageadjusted IPI (AA-IPI) at relapse was 0-1 in 36 patients (53 %) and 2-3 in 29 patients. Twenty-eight patients (43 %) received rituximab during relapse treatment. The most common highdose regimen was BEAM (36 patients, 53 %) followed by BEAC (29 patients, 43 %). Results: Two patients (3 %) died due to the early complications and were not evaluable for response. Further, two patients did not respond. Sixty four patients (94 %) achieved a complete or partial remission. With a median follow-up of 26 months from ASCT, relapse or progression has been observed in 30 patients (44 %)-The median time to relapse or progression was 7 months (2-51 months) from ASCT. Progression-free survival at 2 years was 59 % and overall survival 61 %, respectively. In multivariate analysis of factors predicting progression-free survival, only rituximab in relapse treatment was significant (p=0.02). Moreover, rituximab in relapse treatment (p=0.006) and treatment response after high-dose therapy (< 0.001) predicted overall survival. Duration of the first remission, AA-IPI at relapse, remission status at ASCT and high-dose regimen used were not predictive for outcome. Conclusions: ASCT is feasible treatment in patients with relapsed DLBCL with a low treatment-related mortality. Rituximab-therapy during relapse treatment predicted improved outcome. Due to high-risk of early relapse additional treatments including radioimmunotherapy and allogeneic stem cell transplantation are worth considering in future trials. First experience with treosulfan-based preparative regimen in advanced poor-risk CLL T. Gromke*, R. Trenschel, N.K. Steckel, M. Ditschkowski, L. Kordelas, D.W. Beelen University Hospital of Essen (Essen, DE) Purpose: Allogeneic SCT is a potential curative treatment option for chronic lymphocytic leukaemia (CLL). Here we report the first results of allogeneic SCT after a treosulfanbased preparative regimen in 7 patients (pts) with very advanced and prognostically unfavourable CLL. Methods: Between 01/2005 and 10/2007, 7 pts (male/female: 4/3, median age 57 [range 45 -63] years) underwent alloSCT with an unrelated donor (3 matched and 4 mismatched). Median disease duration prior to transplant was 6 (range 2 -11) years. The indication for alloSCT was incomplete response or failure of fludarabine -containing combination chemotherapy (n=5) alone or together with an adverse karyotype (n=2). Four of these patients received a second alloSCT following graft failure after reduced intensity conditioning with fludarabine and cyclophosphamide ± TBI (2Gy) applied before the first alloSCT. All pts were previously treated with more than 3 different chemotherapeutic regimens and one patient had relapsed after autologous SCT following myeloablative radiochemotherapy. The preparative regimen consisted of treosulfan 42g/m² in combination with fludarabine 150mg/m². NCI complete remission (CR) criteria, flow cytometry, cytogenetics and chimerism data, as available, were used to assess response to therapy. Results: All 7 pts engrafted with a median neutrophil engraftment time (ANC>500) of 14 days (range 13 -18). Five pts developed acute GVHD (grade I: n=2, grade II: n=2, and grade III: n=1) after a median time of 14 days after transplant. In 7/7 patients CR was documented on day +100 by marrow morphology and flow cytometry, and all pts demonstrated complete hematopoietic chimerism. So far, after a median follow-up time of 11 months (range: 7 -36), no relapse and no fatal adverse event has occurred and 6/7 pts remain in CR. Conclusion: In this pilot study, we observed excellent tolerability and high efficacy of treosulfan-based conditioning for alloSCT in patients with extremely advanced CLL. These first results support that alloSCT with this treosulfan-based conditioning regimen warrants further evaluation in CLL pts. Outcome of haematopoietic stem cell transplantation for patients with Hodgkin's disease at a cancer centre, Jordan: a young program in a developing country experience F. Abdel-Rahman*, H. El Taani, O. Abu Hlalah, R. Rihani, A. Ahmad, F. Badaineh, A. Badeeb, A.H. Al-Zabin, A. Addasi, T. Nseerat, M. Sarhan King Hussein Cancer Center (Amman, JO) Purpose: to evaluate the outcome of patients with Hodgkin disease who underwent hematopoietic stem cell transplantation at the young KHCC BMT program Patients and methods: Over the past 4 years,41 patients had autologus stem cell transplant(ASCT),and two patients had allogenic stem cell transplant without prior autologous transplant.Among the ASCT recipients there were 35 adults (85%),and 6 children (15%). The median age was 29 and 15 years respectively. There were 9 (22%) patients in complete remission (CR) and 32(78%)with chemotherapy responsive disease.Prior to transplant, one patient (2%) received only one chemotherapy line, 22(54%) received two lines, 11 (27%) received three lines and 7 had four(17%) or more lines. All patients received BEAM as pre transplant conditioning except one patient received CBV conditioning. The median CD34 positive cell dose was 5.3x10*6/kg (range, 1.5-12x10*6). Results: the main end points of the study are overall response rate (ORR) at day 100, Event-Free Survival (EFS) and overall survival (OS). The median follow up time was 19.2 months (range, 0. 2-45.7months ).The median time for the WBC engraftment was 10 days,and for platelets was10 days. The ORR at day 100 was 75% (35%CR and 65%PR).The median survival and EFS for the whole group were 40.6 and 20 months, respectively. A total of seven patients underwent reduced-intensity conditioning (RIC) allogeneic stem cell transplantation from a matched sibling one as tandem, 4 after relapse following ASCT and 2 received allogeneic without prior ASCT. Six patients were conditioned with Fludarabin and 200 cGy total body irradiation and one patient received Fludarabine with Cytoxan. The median follow up time for this group was 21 months. The median survival and EFS were 38 months and 38 months, respectively. Four of the 7 patients relapsed; one of the relapsed patients is still alive getting donor lymphocytes infusion. Three patients died, two secondary to disease relapse, and one died with lung graft versus host disease. Conclusion: Despite its young age, the BMT program is able to perform autologous and allogeneic stem cell transplantation for Hodgkin Disease at a reasonably acceptable mortality and with an outcome comparable to published data. We are planning for collaborative clinical research with institutions with extensive experience in this area to benefit our patients and further our knowledge and clinical research skills. A complete analysis of data will be presented at the meeting P854 Purging in vivo and auto-transplantation in patients with poor prognosis lymphoproliferative disease L. Pezzullo, O. Finizio, S. Rocco, L. Bene, L. Mettivier, G. Nunziata, C. De Rosa, V. Mettivier* A.O.R.N. A. Cardarelli (Naples, IT) Autologous stem cell transplantation is an efficacy therapy for limphoproliferative disease. However a concern with the procedure is the potential of malignant cells to reinfuse with stem-cell graft. Investigators have used rituximab to purge malignant cells in vivo without any manipulation in vitro. From April 2003 to December 2007 we have treated with Autologous stem cell transplantation, purged in vivo with monoclonal antibodies, 23 patients (7F; 16M median age:56 years) with limphoproliferative diseases to poor prognosis (2 Burkitt lymphoma; 3 Burkitt like; 4 mantle cells; 3 CLL; 2 NHL-T cells (1 peripheral and 1 lymphoblastic); 2 follicular and 7 large cells) and we have evaluated the results and the feasibility. In all patients, the purged in vivo, has been effected administering a dose of monoclonal antibodies (anti CD20 in B-NHL and anti CD52 in CLL and T-NHL) before the harvest and after the infusion of the stem-cells. To the transplantation 9 patients were in CR (2 Burkitt; 3 Burkitt like; 2 large cells; 1 NHL-T lymphoblastic and 1 mantle cells) 9 in PR (1 CLL; 3 mantle cells; 2 follicular and 4 large cells lymphoma) and 5 in resistant disease (2 CLL ; 2 large cells and 1 NHL peripheral T cells). All patients have harvest (median CD34:4 x106/Kg) and median minimal residual disease in the harvest has been < to 2%. All the patients have been conditioned with BEAM and the graft are documented in 21/23 patients (2 patients are dead to the day +4 and +10 for gastric haemorrhage and septic shock respectively) with neutrophils> 1000 in media to day + 14 (range 10-19 days). After transplantation 19/21 patients were in CR, a day +60 the MMR in bone marrow was <0, 5% (range 0-0, 3%). With a median follow-up of 18 months after transplantation (range 2-57) 14/21 (66%) patients are in CR. Two patients (1 large cells and 1 CLL) are died in remission at months +3 and + 7 for CMV reactivation and interstitial pneumonia respectively. The EFS and OS projected at 50 months are of the 50% and 57% respectively. In conclusion the purging in vivo effected during the harvest that immediately after the infusion of the stem-cells, allows to get besides a graft with least residual disease in this cohort and the preliminary results they are interesting.These data suggest treating in first line, with transplantation of stem-cells purged in vivo with monoclonal antibodies to eradicate the MRD, patients to poor prognosis or with chronic limphoproliferative disease. Gemcitabine and vinorelbine as a salvage regimen for patients with Hodgkin's disease who relapsed after autologous haematopoietic stem cell transplantation: a single-centre experience A. Czyz, D. Dytfeld, L. Gil, A. Nowicki, A. Lojko, M. Komarnicki University of Medical Sciences (Poznan, PL) An optimal therapy for patients with Hodgkin disease (HD) who experience relapse after autologous hematopoietic stem cell transplantation (autoHSCT) has not been established. Gemcitabine and vinorelbine have been shown to be effective in patients with relapsed or refractory lymphoma. We report our experience of gemcitabine with vinorelbine in combination (GV) in 10 patients (median age 32 years, range 24-54 years) with HD who relapsed at a median of 6 months (range 3-24) after autoHSCT preceded by a median of 3 (range 2-6) previous chemotherapy lines. The patients received a median of 2 (range 1-3) GV courses with filgastrim support, which consisted of gemcitabine 1000mg/m² and vinorelbine 25mg/m² on days 1, 8 and 15 of a treatment cycle repeated every 28 days. 8 of the 10 patients were treated with the intent to proceed to reduced intensity allogeneic HSCT (alloHSCT). Results: A toxicity of a total 24 GV cycles were analyzed. Granulocytopenia < 0,5 G/L occurred in 7/24 (29%) and thrombocytopenia < 20 G/L in 6/19 (31%) cycles. Two patients were thrombocytopenic before GV treatment. 6 of the 24 (25%) cycles were complicated by infection (neutropenic febrile-2 episodes in 1st and 1 episode in 3rd cycle, pneumonia-2 episodes in 1st and 1 episode in 2nd cycle). In 5/24 (21%) cycles one dose of gemcitabine and vinorelbine was omitted or delayed. Transient liver enzymes elevation (> 1,5x normal value) occurred in 2 cycles in 1 patient. The overall response rate was 60%, with 1 CR, 4 PR and 1 stable disease. After a median follow-up of 7 months (range 2-15) 8/10 patients remain alive. Two patients died due to progressive disease. Four patients with CR/PR proceeded to reduced intensity alloSCT. Two of the 3 assessable patients are in CR, 4 and 6 months after alloHSCT respectively. One patient experienced disease progression 6 months after alloSCT and has received DLI. OS for the whole group of 10 patients was 78% (95% CI 50-100) at 12 months estimated with the Kaplan-Meier method. A plateau in PFS curve was not observed. Conclusion: Our pilot study demonstrates that GV is a feasible regimen with acceptable toxicity profile and significant response rate in heavily pre-treated patients with HD relapsing after autoHSCT who are considered for alloHSCT. Favourable outcome of mantle cell lymphoma after autologous stem cell transplantation with in vivo or ex vivo purging Y. Kuwatsuka*, H. Kato, Y. Oki, H. Taji, K. Yamamoto, Y. Kagami, Y. Morishima Aichi Cancer Center Hospital (Nagoya, JP) Introduction: Prognosis of patients with mantle cell lymphoma (MCL) after conventional chemotherapy is poor. High-dose chemotherapy with autologous stem cell transplantation (ASCT) could be considered for MCL, but tumor cell contamination in the stem cell source remains problematic. We retrospectively analyzed the clinical outcome of patients with MCL who underwent ASCT with in or ex vivo purging methods, either upfront or for relapsed disease. Methods: Twenty patients with advanced stage MCL who received ASCT between September 1995 and August 2006 at Aichi Cancer Center were analyzed. Results: Median age was 57 (range 40 |67) years old. In 15 patients, ASCT was planned upfront, and 5 patients underwent ASCT after relapse. Of 15 patients undergoing ASCT upfront, 14 had stage IV, and one had stage III disease at the time of diagnosis. Fourteen of these patients responded (12 CR, 2 PR) after initial chemotherapy (regimen: bi-weekly CHOP, n=3; dose-intensified CHOP ± rituximab (R) plus CHASER [cyclophosphamide(Cy)/ara-C/DEXA/etoposide/ rituximab], n=12) and proceeded to ASCT (regimen: LEED [melphalan/Cy/etoposide/DEXA], n=12; Mel/TBI, n=2) immediately after. One had inadequate response to initial chemotherapy (dose-intensified CHOP-R plus CHASER) and salvaged with R-hyper CVAD, then underwent ASCT (regimen: LEED). Five patients underwent ASCT for relapsed disease. These patients proceeded to ASCT (LEED, n=4; MCEC [ranimustine/carboplatin/etoposide/Cy], n=1) in CR after first (n=2) or second (n=3) salvage therapy. Two patients who were planned for LEED did not complete the regimen because of toxicity but had stem cell infusion. Median count of infused CD34 positive cells was 5.8 (range 1.7-12.5)*10 6 /kg. Stem cell sources were either in vivo rituximab purged (n=15) or ex vivo CD34-positively selected (n=5). With median follow up of 37 (range 3-143) months, relapse occurred in 5 cases, and 4 patients (20%) died (from MCL, n=1; secondary MDS, n=1; infection, n=1; and other, n=1). Three-year overall survival and relapse free survival rates from the time of transplant of patients undergoing upfront SCT were 72% and 72%, respectively. Those of patients undergoing ASCT after relapse were 100% and 75%, respectively. (Table) Conclusion: Survival of MCL patients under going ASCT upfront or after relapse was favorable with intensive chemotherapy and purging of stem cell. Prospective clinical trial of upfront in vivo purging ASCT is ongoing. Treatment of high-risk diffuse large B-cell lymphoma with intensified induction therapy and high-dose sequential therapy B. Puccini, L. Rigacci, S. Guidi, L. Lombardini, R. Alterini, E. Bartalucci , F. Bernardi, C. Nozzoli, V. Carrai, R. Saccardi, A. Bosi* Azienda Ospedaliera Careggi (Florence, IT) The standard first-line treatment for diffuse large B-cell lymphoma (DLBCL) is CHOP, but its results in patients (pts) with aaIPI 2-3 is still not satisfactory. The use of high-dose sequential therapy (HDST) in first-line treatment led to discordant results. We treated young high-risk pts with DLBCL with an intensified induction therapy followed by HDST. Pts with DLBCL, age less than 55 years, and intermediate-high with bulky or high-risk IPI were eligible for this study. Treatment consisted of 3 phases: the 1st phase was constituted by 3 cycles of an intensified CHOP (cyclophosphamide 3 g/m²; doxorubicin 75mg/m²; vincristine 1,4 mg/m² and prednisone 100 mg for 5 days). Peripheral stem cells collection was performed after the 3rd cycle. The 2nd phase (HDST) consisted of cyclophosphamide 4g/m², methotrexate 8g/m² and etoposide 2g/m². The 3rd phase was HDT-ASCT, with a conditioning regimen composed of melphalan 180 mg/m² and mitoxantrone 60 mg/m². Since March 2002 to december 2004 we enrolled 13 pts, with median age of 37 years (range 22-49);8 had a stage III-IV (62%), 6 presented B-symptoms (46%), 11 had bulky disease (85%), 6 had more than one extranodal localization (46%). Two pts had a WHO performance status 2 (15%), and 10 had elevated LDH (77%). Twelve pts completed the scheduled treatment, while 1 died during the 1st phase for a sepsis. At the end of therapy 10 pts obtained a complete remission (CR) (83%), 2 pts were in partial remission (PR). After the 1st phase no pts have obtained a CR, 23% of pts obtained a CR after the 2nd phase and 10 pts (77%) have obtained the CR at the end of 3rd phase. After a median follow-up of 36 months (range 2-61) progression free survival was 79%, after a median observation of 51 months (range 2-64) 11 pts (82%) were alive; 2 pts died (one for a sepsis and one for disease progression). We can conclude that this protocol represents an attempt to improve the results of HDST adding an intensified treatment in the 1st phase. This therapy was feasible and effective in a high risk group of pts. Rituximab could further improve these results and could also be used in pts with involvement of bone marrow at diagnosis. The aim of the study was to prospectively evaluate the outcome of patients with aggressive non-Hodgkin lymphoma (NHL), undergoing autologous stem cell transplantation (ASCT) in first complete (CR1) or very high quality partial remission (PR), according to the age-adjusted International Prognostic Index (IPI). Thirty consecutive patients, aged less then 60 years (median of 34 years), with poor-risk aggressive NHL and at least a PR after induction chemotherapy (ACVBP regimen with or without rituximab), underwent ASCT after BEAM regimen. A median of cryopreserved CD34+ cell dose of 7.04 x 106 per kg was infused (range, 2.1-14.9 x106). Engraftment occurred in all patients after a median of 11 days post transplantation. A platelet count 20≥x 109/l was achieved after a median of 12 days post ASCT. There was no procedure related mortality and only minor morbidity was observed. Four patients relapsed at a median of 5 months (range, 3-18 months) post transplantation. The remaining 26 patients remain alive, well and in CR1 with a median follow-up of 34 months (range, 8-80 months). The overall survival (OS) and the disease-free survival (DFS) at 3 years for all patients considered for ASCT were 85% and 86% respectively. We conclude that ASCT for poor-risk aggressive NHL is safe and is associated with high survival when used as consolidation of first CR. , accounting for 220 alloSCT, were consecutive enrolled in the perspective trial on the role of combined intensive screening for circulating galactomannan (GM) and early high resolution chest CT (HRCT) for IA diagnosis. GM sampling were collected from admittance until day +365 ; HRCT and/or bronchoscopy with bronco-alveolar lavage (BAL) were performed when IA was suspected. Patients characteristics in table 1. EORTC/MSG criteria were considered for the analysis of IA incidence; in adjunction we registered every empirical antifungal treatments . Results: With a follow-up range of 12 to 34 months, 116 patient were alive and 93 had died. Relapse accounted for 57 deaths and transplant related mortality (TRM) for 58. We documented 2 proven, 22 probable , 10 possible invasive aspergillosis and 13 cases of empirical antifungal treatment (characteristics in table 2). Focusing on proven and probable IA, we observed an 10.9% overall incidence , with an IA mortality rate near 58%. GVHD on treatment was the strongest risk factor for developing IA. Positive GM test in body fluid was the microbiological factor in 23/24 patients GM elevation was the first sign of IA in 12 HSCT without fever. Considering false positive GM test when two consecutive samples were over the cutt off value the incidence of false positive test was near 11%. Conclusions: IA incidence remains high in the allo SCT setting. Diagnosis of IA is based primary on GM assays , and the time collection of samples and the time of execution of CT scans may have great influence on IA 'time diagnosis'. GM false positive test are present and may be evaluated. Many patients in the possible IA and empirical antifungal treatment may be spared to receiving antifungals due to the low probability to be affected by IA. Further tests are needed to ameliorate the diagnosis of invasive fungal infections. Background: Invasive fungal infections of immunocomprised patients with the opportunistic mycopathogen Aspergillus fumigatus show an increasing incidence for the last decade due to the growing number of stem cell transplantations. Many efforts have been undertaken to reveal immune mechanisms that control clearance of A. fumigatus from blood. Polymorphonuclear neutrophils (PMNs), as a part of the innate immunity, recognize fungal pathogens at an early step after infiltration. Besides phagocytotic mechanisms, PMNs attack pathogens by the release of reactive oxygen species (ROS). Methods: In the current study, human PMNs were isolated from fresh blood of healthy donors by the use of Biocoll separating solution and oxidative burst was determined in a kinetic measurement by the use of dichlorfluorescein. We could demonstrate that A. fumigatus germlings of the clinical relevant strain ATCC 9197 represented a strong stimulus for the release of ROS. At the same time, PMNs actively tracked germlings and directly attached to fungi as demonstrated by real-time microscopy. Results: Co-cultivation of PMNs with A. fumigatus germ tubes resulted in a strong upregulation of genes (hämoxygenase, heat shock 70kDa protein HSPA8, thioredoxin, HSPA1B, HSP90AB1, Ferritin), involved in self-protection against radicals, as identified by whole genome expression analyses (Affymetrix U133 Plus 2.0 Array). In total, unstimulated PMNs showed expression of approximately 7.500 genes (20% of all genes spotted on the chip). After 6h co-cultivation of PMNs and A. fumigatus germ tubes, 195 (1.273) genes showed an at least 4fold (2fold) altered gene expression. Therein, 4 genes encoding for modulating factors of inflammatory responses (IL-8, CCL3, CXCL2, IL1RN) were significantly upregulated. Luminex analysis was performed for TNF-alpha, IL-12, GM-CSF, IFN-gamma, IL-6, IL-8, IL-10 and IL-1beta to identify secreted cytokines, thereby confirming array data and revealing IL-8 to be strongly released (5fold) by PMNs after fungal co-culturing. Conclusion: In conclusion, A. fumigatus had a substantial effect on the activity of human PMNs when coming in close contact. In consequence, various defence strategies were activated, including phagocytosis, ROS release and mobilization of other immune effector cells by secretion of chemoattractant cytokines. A better understanding of innate immune defense mechanisms may provide new directions for antifungal therapies. Immunocompromised patients could suffer from clinical consequences of infection with the human herpesvirus 6 (HHV6). Using PCR methods, we are now able to detect viremia while serology results might be inconclusive in this cohort of patients. Phenomenon of chromosomal integration of HHV6 (CI-HHV6) however opened new era in interpretation of HHV6 PCR positivity. Since CI-HHV6 could be inherited from parent, viral DNA can be detected in every body cell. CI-HHV6 does not represent active HHV6 infection and its prevalence in population is described between 1-2%. In such situation, misinterpretation of PCR positivity may than lead to inadequate and potentially harmful therapy due to risks of unsubstantiated toxicity and side effects. In situation of allogeneic HSCT, it is possible to observe increase in HHV6 positivity caused by the engraftment of the cells from CI-HHV6 positive donor, or decrease in detected HHV6 DNA after HSCT in CI-HHV6 positive recipient transplanted with the donor without CI-HHV6. Between I/2002-XII/2007 we have tested for HHV6 2208 samples from 270 adult patients and 2807 samples from 136 children patients after allogeneic HSCT. DNA was isolated from whole blood and detection was performed using RQ-PCR. Results were normalised to 100,000 human genomic equivalents accessed by quantification of albumin gene. HHV6 was detected in 464 samples from 70 children and 178 samples from 46 adults. Within three weeks after HSCT, we have detected more then 1,000 normalised viral copies in 14 children and 11 adults. From these, we have confirmed CI-HHV6 by documenting positivity in the pre-transplant samples and the non-blood samples (hair follicles) in two children and one adult. In all three we have detected variant A of the virus. We are working on confirmation of CI-HHV6 in two patients with variant B with decrease of HHV6 shortly after HSCT and long lasting PCR positivity in post-transplant period. In none of the CI-HHV6 patients we have observed post-transplant complications attributable to HHV6. We excluded CI-HHV6 by the pre-transplant negativity in the remaining patients. Despite HHV6 is close related to human cytomegalovirus, in this particular situation patients are endangered only by misinterpretation of highly positive viral DNA load and side effects of virostatics. Therefore in case of high HHV6 DNA load we should be aware of this phenomenon and we should try to exclude CI-HHV6 as soon as possible. Methods: All patients were monitored for clinical features attributable to LPD. Nested or Real time PCR detected EBV-DNA copies in peripheral blood or serum. Biopsy specimens were classified according to the World Health Organization. EBV was detected in biopsy samples using latent membrane protein (LMP1) immunostaining and in situ hybridization for EBV-encoded RNAs (EBER). Results: Overall the cumulative incidence of LPD was 4%. 6 males and 5 females developed LPD at a median of 122 days (range 61-304) after matched (2/94) or mismatched (9/205) transplants. Clinical signs included palpable lymphadenopathy in 10/11 patients, fever in 7, tonsillitis in 5, diarrhea in 1, a rhino-pharyngeal mass in 1 and neurological symptoms in 1. PCR showed median EBV-DNA at diagnosis was 9.600 g/ml (range: 106-68.387 g/ml). Biopsy specimens were not obtained from 1 patient with rhino-pharyngeal mass, another with meningeal involvement and a third patient with rapid disease progression. Biopsy samples were positive for large B-cell non-Hodgkin lymphoma in 8/8 patients; 6 were positive for EBV. All 11 patients were treated with Rituximab, combined with Cidofovir in 10. 7 patients received donor infusion lymphocyte (DLI), 1 EBV-specific cytotoxic Tlymphocytes and 2 CHOP-like chemotherapy. Six patients (55%) achieved complete remission, 3 are alive in CR and 3 died (leukaemia relapse in 1; GvHD in 2), after LPD had resolved. Four non-responders died (LPD progression in 3; Hodgkin lymphoma relapse in 1). One patient is still under observation. Conclusions: Extensively T-and B-cell-depleted matched or mismatched HSCT avoids post-transplant immunosuppression and helps reduce the incidence of LPD. Early Rituximab treatment reduces LPD-related mortality. How to improve the application of a vaccine programme for allogeneic stem cell transplant recipients? Y. Hicheri*, S. Maury, C. Pautas, K. Debbache, M. Kuentz, C. Cordonnier Henri Mondor Hospital (Creteil, FR) Background: Both the EBMT and the Center for Disease Control and prevention have elaborated guidelines for immunization of SCT recipients. However, the practical application of these guidelines deserves to be assessed. For our JACIE application, we elaborated in November 2004 a vaccine program for the first year following SCT, including 3 conjugate anti-H.influenzae B vaccines (Act-Hib® 3, 4, 5 mo after SCT), 3 conjugate (Prevnar® at 3, 6, 12 mo) and one polysaccharide pneumococcal vaccines (Pneumo 23 at 9 mo). In 2006, we noted in the patient records that the application and the track of this program did not fully fit with the procedure. In order to improve the quality control of the vaccine program, we implemented in June 2006 a calendar in each patient chart on a single sheet. The aim of this study was to assess the improvement of the procedure application provided by this action. Patients and methods: We assessed the application of the vaccine program, before (Nov 2004 -May 2006 and after (June 2006-Aug 2007) the implementation of the calendar, by measuring the delay between the theoretical date and the effective date of vaccination for all consecutive patients during the first year of transplant. Patients were withdrawn in case of leukemia relapse or death. When patients had documented H influenzae or pneumococcal infection before the theoretical date of vaccine, they could be prematurely vaccinated. Results: 70 adult patients (M/F: 45/25; median age: 45y) underwent allogeneic SCT within the study period. Half of patients (n=35) were transplanted before the introduction of our vaccination calendar. There was no statistical difference between the 2 groups as for diagnosis, haematological status at transplant, standard vs. reduced intensity conditioning regimen, and early death before d100. As shown in the table below, the delay between the theoretical and effective date of vaccination was clearly shortened after the introduction of our calendar. When considering the 7 injections of the vaccination program, the implementation of the calendar-based prescription reduced this delay of meanly 36 weeks. Conclusion: The implementation of a vaccination calendar in the patient chart permits to highly reduce the deviation between procedures and their applications. Additionally, it allows an easily tracing of the vaccines effectively administered. Evolving pattern of microbial susceptibility to antibiotics in a HSCT unit C. Annaloro*, M.L. Ranzi, P. Usardi, A. Grancini, F. Onida, A. Della Volpe, C. Olivares, E. Tagliaferri, G. Lambertenghi Deliliers Fondazione IRCCS Ospedale Maggiore (Milan, IT) Over the last twelve years, 621 HSCT recipients (232 allogeneic and 389 autologous; median age 49 years, range 13-69) have entered a microbiological surveillance programme including weekly cutaneous and mucosal swabs. Until June 2002, 340 (138 allogeneic and 202 autologous) received no systemic antimicrobial prophylaxis, and febrile episodes were treated empirically with combined ceftazidime, amikacin and vancomycin (VM); since then, 281 HSCT recipients (94 allogeneic and 187 autologous) have received oral prophylaxis with levofloxacin (LF) and fluconazole, and febrile episodes have been treated with combined tazobactam/piperacillin and amikacin. In the first group, 131 Gram+ [53 fluoroquinolone(FQ)-sensitive] and 81 Grambacteria (68 FQ-sensitive) were isolated from surveillance cultures (one Streptococcus and two Enterococcus strains were VM-resistant, and seven Pseudomonas both ceftazidime-and amikacin-resistant); 119 Gram+ (24 FQsensitive) and 75 Gram-strains (59 FQ-sensitive) were isolated as the causes of febrile episodes (none of the Gram+ strains was VM-resistant; two Pseudomonas strains were both ceftazidime-and amikacin-resistant). In the second group, 42 Gram-(15 LF-sensitive) and 49 Gram+ (21 LF-sensitive) were isolated from surveillance cultures (one Enterococcus faecium strain was VM-resistant, and four Enterobacter cloacae and two Pseudomonas aeruginosa strains were both tazobactam/piperacillin-and amikacin-resistant); 99 Gram+ (8 LF-sensitive) and 32 Gram-strains (4 LF-sensitive) were isolated as the causes of febrile episodes (none of the Gram+ strains was VM-resistant, and three Pseudomonas strains were both tazobactam/piperacillin-and amikacin-resistant). Evolving concepts in managing febrile neutropenia do not automatically apply to HSCT recipients. Although there is still some debate about the effects of FQ prophylaxis, we have found that introducing LF has led to a striking reduction in Gram-colonisations and bacteriemias, with most of the febrile episodes being obviously sustained by FQ-resistant strains. The two-drug antibiotic regimen did not increase resistant strains; morever, VM resistance has been minimised. These results show the relevance of microbiological surveillance when analysing the effects of antimicrobial prophylaxis and treatment. Diagnostic driven antifungal strategy in patients submitted to allogeneic haematopoietic stem cell transplant: a prospective study C. Girmenia*, A.P. Iori, G. Gentile, A. Micozzi, S. Santilli, D. Ballarò, G.F. Torelli, F. Milano, B. Lucarelli, E. Arleo, S. Brocchieri, V. Guerrisi, R. Foà Ematologia, Azienda Policlinico Umberto I (Rome, IT) Preemptive antifungal strategies based on chest computed tomography (CT) findings and laboratory markers have been recently proposed in allogeneic hematopoietic stem cell transplant (HSCT) recipients. However, such strategies are at an exploratory level, do not have standardized criteria and may be demanding and expensive, particularly when applied to the entire transplant population for a prolonged time period. Since March 2006, we have prospectively assessed the feasibility of a simple approach based on two diagnostic levels: 1) a surveillance strategy (periodic nasal swab culture and serum galactomannan (GM) detection once or twice a week) limited to the phases at high risk for IFI: the engraftment phase for all cases, up to day 100 post-transplant in alternative HSCT and in all patients with acute or chronic GVHD; 2) a symptomatic diagnostic approach (GM serum detection over three consecutive days, thoracic CT scan and other exams as indicated) in patients with signs and symptoms possibly related to an IFI. In the event of a positive result of the surveillance strategy, a symptomatic diagnostic work-up was performed as above. A total of 56 HSCT patients were considered in the study. There were 34 transplants from a sibling donor and 22 from an alternative donor. Overall, 635 GM assays were performed (mean 11.3 per patient, range 8-27). One or more surveillance GM positive serum samples were detected in 14 patients during the high risk phases. Overall, a symptomatic diagnostic approach was applied to 24 patients. Ten cases of proven/probable IFI (17.8%) were documented and treated with antifungal drugs. The infection was first revealed by surveillance GM assay in 7 asymptomatic patients and by symptomatic diagnostic approach in 3 cases. In 7 of 14 patients with positive surveillance GM, the result was considered as a false positive. Seven patients with IFI (70%) died and the infection was considered the cause of death in 4. No empiric antifungal treatment was administered. No undetected cases of IFI were identified. Our diagnostic driven antifungal strategy in allogeneic HSCT patients proved to be effective, safe and easy to apply in the clinical practice. This strategy avoided the exposure to expensive and potentially toxic empiric treatments and offered effective antifungal control. Association of the single nucleotide polymorphisms and haplotypes of tumour necrosis factor receptor-1 gene with increased risk to develop invasive pulmonary aspergillosis J. Sainz*, E. Pérez, I. Salas-Alvarado, S. Gomez--Lopera, M. Jurado Virgen de las Nieves University Hospital (Granada, ES) Background: Invasive pulmonary aspergillosis (IPA) has become the most prevalent fungal infection in immunocompromised and hematopoietic stem cell transplantation patients. Patients with similar clinical conditions and extrinsic risk factors vary in susceptibility to IPA. Genetic factors that predispose individuals to IPA are not fully understood. Objective and design. The aim of this study was to determine whether TNFR1 -609 (C/T) and TNFR1+36 (A/G) polymorphisms are implicated in IPA pathogenesis. A candidate gene-association approach was used to analyze the role of TNFR1 gene in IPA pathogenesis. Methods: Subjects comprised 79 hematological patients and 123 healthy controls. TNFR1 -609 (C/T) and TNFR1+36 (A/G) polymorphisms were genotyped by PCR-RFLP. Results: Genotypic and allelic frequencies were similar between hematological patients and controls. IPA was diagnosed in 53 of the 79 patients according to consensus criteria published by the EORTC/IFICG. Individual locus analysis showed that TNFR1 polymorphisms were weakly associated with presence of IPA (p=0.065 and p=0.025; Table 1 ). As expected, TNFR1 polymorphisms were in linkage disequilibrium in hematological population (TNFR1 -609 (C/T) vs. TNFR1+36 (A/G): D'=0.4988 and r=-0.3264). Haplotype analysis with Expectation-Maximization (EM) algorithm revealed that GT haplotype was significantly associated with resistance to develop IPA infection (p=0.031) while GG haplotype showed a trend towards susceptibility to IPA (0.099). Global haplotype association p-value was 0.019. Further, G carrier patients (TNFR1 -609 (G/T+G/G) and TNFR1+36 (A/G+G/G)) had a higher positive serum galactomannan percentage versus patients with other genotypes. Conclusion: These findings support our working hypothesis that TNFR1 genotypes or haplotypes contain allele combinations that may participate in the pathogenesis of IPA infection. DC-SIGNR neck-region polymorphism has no impact on invasive pulmonary aspergillosis susceptibility J. Sainz*, E. Pérez, A. Romero, A. Moratalla, A. Comino, I. Salas-Alvarado, S. Gómez-Lopera, M. Jurado Virgen de las Nieves University Hospital (Granada, ES) Background: Invasive pulmonary aspergillosis (IPA), which is mainly caused by Aspergillus fumigatus, remains one of the leading causes of mortality in hematological patients. Research efforts are ongoing to understand inflammatory mechanisms in IPA and determine how microorganisms such as Aspergillus fumigatus can escape elimination by the immune system and cause fatal destruction of pulmonary tissue. The C-type lectins DC-SIGN and DC-SIGNR have been shown to interact with Aspergillus fumigatus. Our working hypothesis is that because DC-SIGNR interacts with Aspergillus, as well as with other pathogens, variation in this gene might have a broad range of influence in the pathogenesis of a number of infectious diseases, including IPA. DC-SIGNR possess a neck region, made up of a variable number of 23 amino acid tandem repeats. The length of the neck region can critically influence the pathogen binding properties of this receptor. Methods: We tested whether polymorphisms in the neck region of CD209L, the gene encoding DC-SIGNR, are associated with susceptibility to invasive pulmonary aspergillosis through genotyping analyses in a Spanish cohort. Subjects comprised 95 hematological patients and 158 healthy controls. IPA was diagnosed in 61 of the 95 patients according to consensus criteria published by the EORTC/IFICG and the remaining 34 showed no evidence of this infection. The IPA patients were also classified into two groups: proven/probable and possible IPA. DC-SIGNR polymorphisms were genotyped by direct PCR. Results: Proven/probable IPA was diagnosed in 25 patients and possible IPA was diagnosed in 36 patients. Genotypic and allelic frequencies were similar between hematological patients and controls. As expected, DC-SIGNR polymorphisms were in Hardy-Weinberg equilibrium in both hematological and control populations. Individual locus analysis showed that DC-SIGNR polymorphisms were not associated with presence of IPA (2x6 contingency table; IPA vs. non-IPA; p=0.333). Further, genotype and allelic frequencies of the DC-SIGNR gene polymorphism observed in both proven/probable and possible IPA groups were similar to that found in non-IPA group. Conclusion: These findings show that polymorphisms in the neck-region of DC-SIGNR gene is not a risk factor to develop invasive pulmonary aspergillosis infection. HHV6 reactivation: important risk-factor for poor outcome in myeloablative treated stem cell transplantation adult recipients A.P. J. de Pagter, E. Meijer, D. van Baarle, E. Fries, L.F. Verdonck, A.M. van Loon, R. Schuurman, J.J. Boelens* University Medical Centre (Utrecht, NL) Background: Haematopoietic cell transplantation (HCT) is frequently complicated by viral reactivations. The clinical significance of Human herpes virus type 6 (HHV6) reactivation after HCT remains unclear. In a recent pediatric study, we found that HHV6 reactivation, exceeding 1000 cp/mL, was associated with HCTassociated morbidity (e.g Graft versus Host Disease: GvHD) and -mortality (TRM). To compare these results with adult HCT patients, we studied the incidence and associations of HHV6reactivation with GvHD and TRM in our adult HCT patients. Methods: All patients transplanted between 2005 and 2007 and receiving a myeloablative (MA) preparative conditioning regimen were included. The control group consisted of 24 consecutively treated patients prepared with a nonmyeloablative (NMA) conditioning regimen. Epstein Barr virus (EBV) and cytomegalovirus (CMV) DNA loads were prospectively measured by realtime PCR; in retrospect HHV6 and Adenovirus were measured weekly (first 2 months post HCT) in the same samples. EBV and CMV reactivations were treated according to local guidelines. For the analysis HHV6reactivations were categorized in 3 groups: group I (no HHV6), group II (loads <1000 cp/mL) and group III (loads >1000 cp/mL). Results: 24 NMA patients and 25 MA patients were included (median age 40.9; range 18.8-65.8 years) . Median follow-up was 8.8 months (range 1. 3-35.8) . 13/25 (52%) MA patients had HHV6 reactivation (median 1010 cp/mL, range 59-800.000 cp/mL) of which 8/13 (62%) had high copy numbers (>1000 cp/mL HHV6 DNA). In the NMA group only 4/24 (17%) patients showed marginal HHV6reactivation (median 334 cp/mL, range 65-999 cp/mL). 11/48 (22%) patients developed EBV reactivation and 17/48 (35%) patients developed CMV reactivation. None of the patients developed Adenoreactivation. Median time of reactivation for HHV6 was 22 days (range 12-35), for CMV 56 days (range 11-124) and for EBV 44 days (range 4-186). In a univariate logistic regression analysis, MA conditioning was the only predictor for HHV6 reactivation (p=0.013). HHV6reactivation was the only significant risk factor for higher TRM (p= 0.014), and acute Gr II-IV GvHD (p=0.037). All GvHD episodes occured during or after HHV6reactivation. Conclusion: HHV6reactivation is common in patients receiving MA conditioning and associated with severe GvHD and lower survival rates, comparable with observations in children after HCT. HHV6reactivation occurs early after HCT, prior to all other viral reactivations. Characterisation of neurological syndromes after haematological stem cell transplantation: clinical findings, diagnostic assessment of viral and fungal infections and radiological images M.C.D. Fink (1), M.I.A. Madeira (2) , E.X. Lima (1) , C.E. Charbel (1), C.L.M. Do Canto (1) , B.P. Simoes (2) , A.C. Santos (2) , A.C. F. Dos Santos (3) , M.P. De Souza (3) Neurological alterations are common after haematological stem cell transplantation (HSCT) with frequency rates up to 70% in non-related transplants. Drug toxicity, central nervous system (CNS) infections, leukemia relapse and bleeding are the main causes of CNS symptoms. We conducted a prospective study to evaluate the neurological syndromes after HSCT, the prevalence of viral and fungal infections, and the spectra of radiological images during these episodes. Virus infections were diagnosis by PCR + RFLP targeting the T region of polyomaviruses and the DNA polymerase region of herpesviruses (panherpes). Fungal infections were diagnosed by PCR targeting a section of the 28S rRNA gene (panfungal). CT images were obtained from all patients at transplant admission. MRI was performed during the acute phase of symptoms. From June 2006 to November 2007, 298 transplants were performed and 28 patients developed CNS alterations (9.4%). Median time for CNS symptoms was 26.5 (-3 to 2,651) days. More than 90% of the patients had normal brain CT images before transplantation. CNS symptoms appeared abruptly and most of the patients were clinically well (median Karnovsky index = 90%). Convulsion was the main manifestation in 12 patients (42.8%), headache in 8 (28.6%), disorientation in 7 (25%), visual alterations in 4 (14.3%), memory impairment in 3 (10.7%) and dysarthria in 3 (10.7%) cases. Two patients had serum cyclosporin levels of 1,243 ug/mL and 449.6 ug/mL, respectively. Viral infection was diagnosed in 4 patients (14.3%): 2 cases of polyomavirus, one case of HHV-6 and one case of VZV infection. Fungal infection was diagnosed in 9 patients (32.1%) and serum galactomannan was concurrently positive in two of them. Twenty-one patients performed MRI or brain CT and 5 of them (23.8%) showed normal images during the acute phase of symptoms. Twenty-four patients were reassessed after 14 days, 16 were considered better (66.6%), three unaltered (12.5%) and 5 (20.8%) had progression of CNS manifestations. Disclosure information about the episode was available in 23 patients, 13 of them (56.5%) died at a median time of 17 (4 to 47) days after the beginning of symptoms. The prevalence of CNS alterations was low (9.4%) in this series. The prevalence of fungal and viral infections was 32% and 14%, respectively. Although many patients recovered in the first 14 days, neurological alterations may aggravate other conditions since more than 50% of the patients died within 50 days of the beginning of CNS symptoms. The impact of NOD2/CARD15, TLR, IL23R and KIR polymorphisms on the incidence of infectious complications in allo-HSCT recipients A. Holowiecka-Goral* (1), S. Giebel (1) , I. Nowak (2) , E. , T. Czerw (1) , J. Wojnar (1) , M. , M. Markiewicz (1) , P. Kusnierczyk (2) , J. Holowiecki (1) (1)Silesian Medical University (Katowice, PL); (2) Institute of Immunology and Experimental Therapy (Wroclaw, PL) Infections remain the major obstacle for successful allogeneic stem cell transplantation (alloHSCT). As specific immune response is profoundly suppressed during the first months after transplantation, the components of innate immunity are expected to play important role in anti-infectious responses. The goal of this prospective study was to evaluate the impact of NOD2/CARD15, IL23R, Toll-like receptor(TLR)4 and TLR5 gene single nucleotide polymorphisms (SNPs), as well as killer immunoglobulin-like receptor (KIR) repertoire, on the frequency of infectious complications after alloHSCT. One-hundred-two consecutive patients with hematological malignancies, aged 32 (18-58)y, treated with alloHSCT from HLA-matched related (n=34) or matched unrelated donor (MUD) (n=68) were included. The conditioning regimen was myeloablative, GVHD prophylaxis consisted of CsA, Mtx, and, in case of MUD-HSCT, pre-transplant ATG. Donors and recipients were tested for SNP8,12,13 of the NOD2/CARD15 gene, TLR4(299), TLR4(399), TLR5(stop codon C1174T) and Il23R(11209026) SNPs, as well as KIR genotype. We analyzed separately infections occurring in the early, cytopenic phase and those occurring after engraftment. Results: Presence of KIR2DS3 gene in the donor protected against bacterial pharyngitis (46% vs. 66%, p=0.05), whereas KIR2DS1 in the donor was associated with decreased incidence of pneumonia (12% vs. 32%, p=0.02). Presence of KIR2DS1 in the donor correlated with lower rate of EBV infection (0 % vs. 10,2%, p=0.04). SNP8 of the NOD2/CARD15 gene in the recipient tended to increase the risk of severe bacterial pharyngitis (100% vs. 57%, p=0.08) and neutropenic pneumonia (40% vs 7%; p=0.06). There was a lower incidence of urinary tracts infection in NOD2/CARD15 SNPs carriers (32% vs. 59%; p=0.04). As well, NOD2/CARD15 SNPs carriers tended to be protected against HSV infections (0% vs. 9.6%, p=0.08). No significant associations were found between TLR4, TLR5 and IL23R SNPs and infectious complications despite trend for higher EBV incidence int TLR5(1174) recipients. Conclusions: NOD2/CARD15 and KIR gene polymorhisms both influence the incidence of infections after alloHSCT. In particular, the effect of donor activating KIRs is generally protective while recipients with NOD2/CARD15 SNPs seem to be more susceptible to respiratory tract infections. The genomic analysis may be useful for prediction of infectious complications nd planning appropriate pathogen-specific prophylaxis. Epstein Barr virus (EBV) is an important cause of morbidity and mortality after allogeneic haematopoietic stem cells transplantation (HSCT) particularly in T-cell depleted transplant recipient. EBV reactivation is related to the intensity of immunosuppression and may evolve in post transplant lymphoproliferative disease (PLTD). A highly sensitive EBV screening by quantitative polymerase chain reaction (Q-PCR) allows a pre-emptive treatment with anti-CD-20 monoclonal antibody rituximab that seems to reduce the PLTD incidence in HSCT recipients. Since EBV detection by Q-PCR was available, we conducted a prospective trial in T-cell-depleted HSCT from unrelated or haploidentical donor to evaluate the incidence of EBV reactivation and the impact of the preemptive therapy on the development of PTLD. Since June 2002 we begun a prospective EBV surveillance program by Q-PCR on 50 recipients of T-cell depleted HSCT. 47 received an HSCT from an unrelated donor and 3 from an aploidentical family one. Diagnosis were: AML 22, ALL 11, lymphomas 4, SAA 2, CMS 4, MDS 4, MM 3; median age was 38 yrs (range 14-59); 22 were male/28 female; 36 pts conventional conditioning and 14 reduced intensity. 36 pts received a TBI based conditioning; 47 pts received ATG and 3 Campath-1H as part of conditioning regimen. Stem cell source was peripheral blood in 25 pts, bone marrow in 21 and cord blood in 4 pts (at least 4/6 mismatched). EBV DNA Q-PCR was weekly performed from the engraftment. Only pts with EBV DNA >1.000 copies/105/PBMC received as preemptive treatment rituximab ± cidofovir . 24/50 (48%) patients experienced EBV reactivation, with EBV DNA copies >1000 (range 1000-800.000 viral copies), 3 PTLD (6%) within a median time from HSCT of 59 days (range 27-144). Pts with EBV reactivation were treated with Rituximab 375 mg/m2 weekly (median number of cycles 3) and 10 pts also with cidofovir 5mg/kg i.v., till EBV Q-PCR negativity was detected. Treatment was well tolerated and induced clearance of EBV DNA in all patients, also in 3 PTLD pts. From statistical analysis (chi-square test) intensity of conditioning, stem cell source, donor relatedness, CMV reactivation, aGVHD and cGVHD occurrence did not influence the EBV reactivation. Moreover non relapse mortality was not correlated with EBV reactivation but only with cGVHD occurrence (p 0.001). In conclusion Q-PCR monitoring allows early detection of EBV reactivation and preemptive therapy avoids EBV mortality. Adoptive T-cell transfer of pp65-specific T-cells as a treatment of cytomegalovirus viraemia after allogeneic stem cell transplantation T. Feuchtinger* (1), S. Joachim (1) , W. Bethge (2) , C. Faul (2) , M. Schumm (1) , M. Scheible (1), R. Handgretinger (1), P. Lang (1) (1)University Children 's Hospital (Tubingen, DE) ; (2)University Hospital (Tubingen, DE) Following allogeneic stem cell transplantation (SCT) the host is at significant risk for viral infections until post-transplant immune reconstitution. Human cytomegalovirus (HCMV) infection is even in recipients with a HCMV-IgG positive donor still a potentially serious complication post SCT. Adoptive transfer of HCMV-specific T cells has the aim to restore longlasting, virus-specific immunity and clear HCMV viremia in recipients of allogeneic stem cell transplants. For successful transfer of a sustained T cell immunity, the T cell product should contain CD4(+) and CD8(+) HCMV-specific T cells. To achieve these requirements, a GMP-grade protocol was developed to generate CMV-specific T cells by using 500-mL blood from CMV-IgG positive donors. HCMV-specific T cells were stimulated ex-vivo with pp65 recombinant protein. Activated T cells were isolated with the interferon-gamma (IFN-gamma) secretion assay without any further in vitro expansion. Virus-specific donor T cells were isolated and adoptively transferred into 7 patients (4 children and 3 adults 10-59 years), with increasing HCMV viremia under antiviral chemotherapy. 1-25x10E3/kg T-cells were infused. Neither acute toxicity nor GvHD were observed in all seven patients. Adoptive transfer of HCMV-specific immunity was successful in four of six evaluable patients, documented by a doseindependent in-vivo expansion of HCMV-specific T-cells, associated with a clearance/decrease of viremia. In one patient HCMV infection could not be controlled by the adoptive T-cell transfer and another two patients died of pre existing pulmonary toxicity symdrome. In conclusion, specific T-cell immunotherapy as a new treatment approach was performed in 7 patients with systemic HCMV-infection after allogeneic SCT. Adoptive transfer of specific T-cell immunity was save and feasible. When performed early in the course of infection, induction of a specific T-cell response through adoptive transfer was effective, and may protect from HCMV-related complications. (1) Adenovirus infection after allogeneic hematopoietic stem cell transplantation (SCT) is an emerging pathogen causing relevant morbidity and mortality, with preponderance in children. During the last years, clinical and basic research regarding the specific T-cell response ameliorated the risk assessment and therapeutic strategies. Risk factors for infection have been described and have commonly an impact on the adaptive T-cell response. During the early phase after allogeneic stem cell transplantation the recipient has to protect himself mainly through the innate immunity. In addition emerging stem cell grafts contain only a limited number of gamma delta T cells, but high numbers of NK cells and possibly gamma delta T cells. These effector cells of the innate immunity have not been shown to induce GvHD and are therefore attractive tools for post-transplant immunotherapy. We investigated antiviral activity ex-vivo of gamma deltaT cells and NK cells against adenovirus in stem cell donors and recipients after allogeneic SCT. Adenoviral infected MHC-I+ and MHC-II+ human targets were used to analyse the influence of pro-inflammatory cytokines on the antiviral cytotoxic activity. Unstimulated NK cells showed a low and gamma delta T cells almost no antiviral activity. In contrast, stimulation with Il-15 significantly increased the response to infected target cells in both effector cell types. Alloreactivity of effector cells was not increased by Il-15. Il-2 increased the alloreactivity of NK cells as expected but had no effect on gamma delta T cells. The effect of a variety of activatory and inhibitory receptors on the activity was analyzed. In patients with adenoviral infection antiviral ex-vivo function of NK cells and gamma delta T cells could be detected. However, a sustained clearance of adenovirus in these patients was associated with a specific gamma delta T cell response. In conclusion, innate effector cells in healthy donors and recipients during the early phase after SCT show antiviral function and contribute to an anti-viral immune response post SCT. Especially Il-15 activated NK cells and gamma delta T cells have good ex-vivo antiviral activity. Significant reduction in deaths related to invasive fungal infections: a ten years' single-centre survey in patients with haematological malignancies J. Auberger* (1), , H. Ulmer (1) , J. Clausen (1) , E. Gunsilius (1) , H. Einsele (2) , G. Gastl (1) , D. Nachbaur (1) (1)Innsbruck Medical University (Innsbruck, AT); (2)Wurzburg University (Wurzburg, DE) Background: This retrospective study analysed the incidence and the outcome of invasive fungal infections (IFI) in patients with haematological malignancies over the past decade. Methods: 1095 patients who underwent treatment either with cytoreductive chemotherapy alone or subsequent haematopoietic stem cell transplantation at our institution between 1995 and 2004 were retrospectively evaluated. Results: Among 1095 patients investigated, IFI occurred in 167 (15%) patients; 54 (32%), 70 (42%) and 43 (26%) patients suffered from proven, probable, and possible IFI according to EORTC/MSG criteria. Both, A. terreus and A. fumigatus were predominant (70%) followed by Candida species (10%). The attributable mortality for invasive fungal infections was 35%. Assigning patients to an earlier and later cohort (treatment 1995-2000 and 2001-2004) fungus-related death decreased significantly in the last recent years (44% vs. 28%, p<0.0001). Overall survival (OS) was significantly better for patients having possible IFI compared to those having probable or proven IFI (3yr OS: 32% vs 25% vs 9%, p=0.019). Proven IFI turned out to be an independent risk factor for a dismal outcome in multivariate analysis (HR 1.67, p=0.018). Discussion: IFI remains a serious complication in haematological patients. Improvement of diagnosis and advances in the treatment of IFI over the past years resulted in a significantly reduced fungus-related mortality. The role of cytomegalovirus on reconstitution of gamma/delta T-cell subsets following allogeneic haematopoietic stem cell transplantation A. Knight* (1), S. Grace (2) , P. Kottaridis (2) , S. Mackinnon (2) , A. J. Madrigal (1) , P.J. Travers (1) (1)The Anthony Nolan Research Institute (London, UK); (2)Royal Free Hospital (London, UK) There are two major subsets of gamma/delta T cells in humans, a Vdelta1 subset predominantly present in the foetal and neonatal period and a Vdelta2 subset representing the major subset of gamma/delta T cells in adult peripheral blood. Currently the role of gamma/delta T cells during CMV infection remains undefined. It has been suggested in animal studies that gamma/delta T cells play a protective role in CMV infection. The expansion and cytotoxic function of gamma/delta T cells during CMV infection has been observed also in the peripheral blood of human recipients of renal and lung allografts. We studied the immune reconstitution of Vdelta1 and Vdelta2 T cell subsets in 40 patients during the first 24 months following allogeneic stem cell transplantation (SCT). The Vdelta1 subset reconstituted rapidly to median normal levels by 6 months post transplant; the Vdelta2 subset showed a slower recovery, particularly in the older patients, and remained at the lower normal range at 2 years post SCT. We detected naïve Vdelta1 and Vdelta2 T cells in younger and older age groups suggesting the ongoing production of gamma/delta T cells even in older patients. We observed an increase in the absolute numbers of the Vdelta1 subset in CMV+ healthy donors (p=0.0002), while absolute numbers of the Vdelta2 subset were not significantly altered (p=0.0550). CMV status also seemed to effect the production of new naïve Vdelta1 and Vdelta2 cells, as both subsets showed elevated absolute numbers in CMV+ donors. However this result only reached statistical significance for absolute naïve Vdelta1 cells (P=0.0162) compare to absolute naïve Vdelta2 cells (P=0.0616). These results suggest that new generation and expansion of Vdelta1 cells are due to immune response to CMV infection. Interestingly, both gamma/delta T cell subsets also showed higher numbers recovering post-transplant with significant expansion of the Vdelta1 subset detected in CMV+ recipients of graft from CMV-seropositive donors (n=14) when compared to CMVrecipients of graft from CMV-seronegative donors (n=20). These data demonstrate the profound impact of CMV infection has on gamma/delta T cells in normal healthy individuals and on CMV-seropositive recipients of SCT from such donors. Monitoring patients for CMV-immunity after HSCT with MHC-I-peptide-tetramers -implications for follow-up of CMV therapy S. Borchers*, S. Luther, J. Kontsendorn, B. Grabow, M. Stadler, H. Diedrich, S. Buchholz, M. Eder, A. Ganser, E. M. Weissinger Hannover Medical School (Hannover, DE) Risk factors for CMV reactivation include conditioning regimen for hematopoetic stem cell transplantation (HSCT), prophylaxis of graft versus host disease (GvHD) and development / treatment of acute or chronic GvHD. Moreover, transplantation of a CMV-seropositive recipient (R+) with cells from a CMV-seronegative donor (D-) bears the risk of recurrent reactivation of CMV. Treatment of CMV reactivations with antiviral drugs may fail. To control CMV virus-reactive T-cells are crucial. In order to improve understanding of restoration of CMV-immunity, we monitored absolute numbers of CMV-reactive CD8+ T-cells using MHC-I-peptide-tetramers commercially available for the major HLA class I groups. Tetramer-staining alone provides no information on the functionality of labelled T-cells and was therefore combined with functional assays. Screening points were prior to HSCT and on days +50, +80, +100, +180 and +365 or weekly in case of CMV reactivation. To date we included 83 patients (median age 53, range 18-69 yrs), 52 are currently in follow-up after HSCT, 17 died before or after HSCT, 14 are prior HSCT. The majority had AML (39%) or secondary AML (22%). From 07/2006 to 11/2007, 70% (67/96) could be monitored after HSCT with the commercially available tetramers. 40% had one matched tetramer, 46% two tetramers and 13% 3 or 4 matching tetramers. The mean follow up data is 224 days (19-440) after HSCT. 69% of the patients did not reactivate CMV, 21% reactivated once, 5% two times or more. In all patients of the R+/D+ setting (46%), CMV-reactive T-cells were detectable. Cut-off for protection against CMV seems to be around 10 cells / µL blood. Expansion of tetramer-positive cells (TPC) could be detected after an episode of CMV reactivation. Only 1 of 31 patients in R+/D+ setting reactivated CMV twice. Of 4 patients reactivating CMV at least twice, 3 were of the R+/D-(10%) setting. In one patient with a de novo CMV-infection emerging CMV-T-cells after in vivo priming were detected. Comparing tetramer screening and functional assays' data, we found that patients with TPC not reactivating or controlling CMV had functional T-cells as well. Our results indicate that tetramer-screening suitable to predict the risk of CMV-reactivation / protection against reactivation by providing the number, persistence and expansion of CMV-T-cells. Thus, for patients at risk of reactivation suitable therapies, like the infusion of CMV-specific T-cells, can be considered early on. Haematopoietic stem cell transplantation complicated by hepatitis B virus (HBV) infection M.B. Pinazzi*, S. Bernsaconi, S. Mancini, I. Majolino, A. Locasciulli Az. Osp. S. Camillo Forlanini (Rome, IT) Hepatitis B virus (HBV) ireactivation after Haematopoietic stem cell transplantation (HSCT) in patients HbsAg positive before transplant has been reported extensively. Interestingly, it may occur not only in known HbsAg carriers,but also in patients with HBV antibodies (antiHBc and/or antiHBs) before transplant.Its clinical manifestations range from anicteric hepatitis to progressive or fulminant liver failure. Several mechanisms,such as chemotherapy-enhanced viral replication, steroids, restoring of immunocompetence, may significantly concur to HBV flare-ups.Among 235 patients undergoing allogeneic HSCT in our unit between 2001 and 2006, 12 (5%), 1 female and 11 male,showed HBV active infection either before (4) or after transplant (8).The underlying disease was Acute Leukemia in4, Chronic Leukemia in2, MM in1, MDS in1 and Aplastic Anemia in4.One received autologous and 11 allogeneic HSCT (HLA identical sibling donor in 10 and alternative donor in 1) All pts were treated with Lamivudine.The drug was administered at the dose of 100mg/day as follows:treatment started concomitant to conditioning regimen in HBV carriers; pre-emptive therapy: at the onset of viraemia and/or HBV antigenemia during posttransplant follow-up. The duration of therapy varied according to serological response:patients showing seroconversion to HBV-DNA and HbsAg negative tests were treated for 3 additional months from seroconversion and then monitored monthly,while cases with ongoing infection and no evidence of viral mutation received at least 12 monthtreatment. Results: HBV infection= 8 patients (2 carrier and 6 with post HSCT reactivation) cleared the infection,showing seroconversion from:HbeAg to anti.Hbe, HBV-DNA+ to HBV-DNA-and seroconverted to anti-HBs within three months from the beginning of treatment. HBV-related liver disease:no patient developed fulminant hepatitis or severe flare-ups; 1/12 patients showed overt hepatitis concomitant toHBV reactivation, but transaminases returned to normal after 30 days of treatment, while11 had either a transient and mild increase in ALT values or normal liver function. We did not observe marrow or other relevant organ toxicity. In conclusion, Lamivudine proved to be safe and effective when given both in HBsAg carriers and in post-transplant reactivation as preemptive therapy. Its impact was evident not only in preventing HBV-related liver disease but also in the clearance of infection with complete seroconversion and disappearance of viremia, which remained stable long after therapy withdrawal. F.J. Vos*, N.M.A. Blijlevens, J.P. Donnelly, C.P. Bleeker-Rovers, W.J.G. Oyen Radboud University Nijmegen Medical Center (Nijmegen, NL) Objectives: To evaluate the usefulness of FDG-PET in detecting central venous catheter (CVC) related infection and deep venous thrombosis (DVT) in a prospective study of stem cell transplant (HSCT) recipients. Methods: Between November 2006 and November 2007 all patients who gave informed consent and were receiving an HSCT after myeloablative therapy were included. FDG-PET was to be performed during persistent neutropenia as soon as C-reactive protein (CRP) levels exceeded 50 mg/l. FDG-PET was assessed blindly Blood cultures were obtained twiceweekly and patients were evaluated for the presence of persistent bacteraemia due to coagulase negative staphylococci (CoNS) The presence of possible CVC related deep venous thrombosis (DVT) was investigated at the physician's discretion. Results: Thirty-four patients were included but FDG-PET was not performed in 5 cases because CRP-levels remained <50 mg/l, for technical reasons in 2 cases and 2 patients withdrew consent. Hence the results of 25 patients were analyzed. Increased FDG uptake was found in the CVC-tract of 9 of the 10 patients with persistent CoNS bacteraemia, while this was present in only 2 of the remaining 15 patients (p<0.001). Echo-Doppler of the subclavian vein was performed in 9 of 10 patients with persistent CoNS bacteraemia and showed a DVT in 4 of these cases with increased FDG uptake in the CVC-tract and in 1 FDG-PET positive patient without persistent CoNS bacteraemia. Blood cultures remained positive in 3 of them after removal of the CVC. In another two cases of FDG-uptake there was no DVT found though peripheral blood cultures remained positive after removal of the CVC. Echo-Doppler of the subclavian vein was undertaken in 3 cases without persistent CoNS bacteraemia one of which had shown FDG accumulation. DVT was only found in the FDG-PET positive patient. Conclusions: Increased FDG-uptake in the CVC-tract occurred significantly more often in patients with persistent CoNS bacteraemia, possibly due to septic thrombophlebitis. In the absence of FDG-accumulation in the CVC tract, CVC related thrombophlebitis appeared to be highly unlikely. The aim of this study was to compare the Abbott CMV realtime PCR based assay in the m2000 platform vs Roche PCR Cobas® Amplicor® CMV after DNA automatic extraction (TNAI-PCR) in plasma samples for the management of CMV infection in our allogeneic stem cell transplant (SCT) recipients. Methods: A total of 386 plasma samples obtained during 69 CMV infection episodes from 59 allogeneic SCT patients were prospectively studied with CINApool® pp65 antigenemia (Argene) (AG) and quantitative PCR COBAS® Amplicor® CMV Monitor test, Roche (CA-PCR). DNA manual extraction was used in 2005-2006 and automatic extraction COBAS® Ampliprep® TNAI kit (TNAI) was used in 2007. All samples were retrospectively tested using CMV Abbott real-time PCR (R-PCR) assay (m2000rt instrument). CMV DNA was extracted with m2000sp automated nucleic acids extractor, using 0.2ml as input volume. Data lower than 52 copies/ml were considered as negative. Samples from 2005-2006 were retrospectively tested with TNAI, using 0.35ml (TNAI-PCR). A positive sample was defined by antigenemia ≥2 cel/4x10 5 cel and/or CA-PCR ≥600 copies/mL. An episode was defined as the period between the first positive sample by antigenemia and/or CA-PCR, until the first negative sample by both techniques. Samples and CMV infection episodes were analyzed independently. Concordance between methods was determined by Kappa and Pearson coefficients. Results: R-PCR detected 79% of antigenemia positive samples vs 71% by TNAI-PCR. AG was not evaluable in 38 samples. Twenty-one (55%) were positive by R-PCR vs 14(37%) by TNAI-PCR. The median values for R-PCR were 274 copies/ml (range, 52-3.65 x 10 5 copies/ml) vs 2545 copies/ml for TNAI-PCR (range: 604-10 5 copies/ml). Concordance between assays was 81% (k=0.66). Correlation obtained by the R-PCR and TNAI-PCR assay was 0.97 (p<0.0001). As a whole, R-PCR detected 60(87%) episodes compare to 53(77%) with TNAI. Eight episodes (11.6%) were detected only by AG (all PCRs negative). Fifty-two off 69 episodes (75.3%) were detected by both PCR techniques, at the same time. Eight episodes (11.6%)were positive with rt-PCR; one episode (1.4%)was positive only with PCR TNAI. In 14 episodes (20.3%) R-PCR was earlier than TNAI-PCR (median=5 days), and in 10(14.4%) TNAI-PCR was earlier than R-PCR (median=10 days). Conclusion: R-PCR and TNAI-PCR have shown an excellent agreement. Both PCRs offered measurable results when AG was not evaluable and are good tools for management of CMV infection in allogeneic SCT patients. E. Distler*, E. Schnürer, C. von Auer, S. Kausche, B. Plachter, C. Huber, K. Kolbe, R. G. Meyer, W. Herr Johannes Gutenberg-University Mainz (Mainz, DE) Reactivated infection with varicella-zoster virus (VZV) can produce shingles that are associated with pain, scarring, and post-herpetic neuralgia. VZV reactivation occurs as the result of immunosuppressive disease or therapy, particularly in hematopoietic stem-cell transplantation (HSCT) patients. Although acyclovir medication effectively reduces VZV reactivation, strategies for long-term prophylaxis remain controversial, since antiviral protection is not complete and disease may occur several years after HSCT. Prevention and control of VZV infection critically depends on the presence of VZV-specific T cells. We developed an IFN-g ELISPOT assay that allows the rapid quantification of VZV-specific T cells in PBMC ex vivo. This easy-to-perform approach uses a commercially available VZVinfected cell lysate (and uninfected control lysate) as the antigen source. We validated this assay in 10 VZVseropositive healthy individuals, who showed a median frequency of 397 (range, 105-2404) VZV-specific T cells per nL blood. In line with published results, this anti-VZV memory response was dominated by HLA class II-restricted CD4+ T cells. We next used this assay to quantify circulating VZVspecific T cells in 4 patients who developed shingles 2-6.5 months after T-cell depleted allogeneic HSCT. Immediately before clinical symptoms, the numbers of VZV-specific T cells were extremely low (median, 33/nL; range, 17-161) . However, these counts increased dramatically by approximately 1-log (median, 496/nL; range, 297-931) during 1-2 weeks after reactivation, and remained at this level for several weeks. Again, VZV-specific CD8+ T cells were not detected at any time point. By analyzing pre-transplant donor PBMC, we confirmed that all allografts had transferred significant numbers of VZV-specific T cells. In conclusion, we describe a straightforward in vitro approach for monitoring VZV-specific T cells in peripheral blood. Although present in healthy donors at considerable numbers, VZV-specific T cells decrease substantially after T-cell depleted allogeneic HSCT. Viral reactivation can strongly boost VZV-specific T cells over 1-log. The frequency of VZVspecific T cells measured by this technology may serve as a surrogate marker (i) to assess the individual patient risk for VZV reactivation (ii) to decide on the necessity of continuous antiviral prophylaxis, and (iii) to optimize VZV vaccination strategies. These issues need further exploration in prospective clinical trials. Reduced relapse related death in alternative donor transplants with EBV/CMV reactivation A. Dominietto, E. Tedone, M. Soracco, A. Ibatici, A Raiola, M Van Lint, F Frassoni, A. Bacigalupo S.Martino's Hospital (Genoa, IT) Viral infections are a serious diagnostic and therapeutic problem in patients undergoing alternative donor transplants. We have analyzed 179 transplants with hematologic malignancies , ghrafted from unrelated (n=139) or family mismatched donors (n=40): Patients in this study were alive on day +30 and had been monitored weekly for cytomegalovirus (CMV) reactivation by CMV-antigenemia, and for epstein barr virus (EBV) reactivation by real time PCR. All patients received anti-thymocyte globulin 7,5-11 mg/kg for GvHD prophylaxis together with cyclosporin and methotrextae. The conditioniong regimen was a conventional CY-TBI (n=102 ) or a reduced intensity (RIC) thiotepa based regimen (n=77). The diagnosis was acute leukemia (n=126) or chronic lymphoid or myeloid disorder (n=53). Median age was 36 years (11-64) and transplants were performed between 2000 and 2006.Reactivation of CMV was seen in 78 patients (44%); EBV reactivation in 118 patients (66%) . The average time to CMV reactivation was day 49 (95%CI day 29-69) and for EBV it was day +78 (95%CI day 50-105). With an average follow up of 823 days (95%CI 717-929) the overall actuarial 5 year survival of the entire group was 47%. The 5 year survival of patients with (n=148) or without (n=31) CMV/EBV reactivation was respectively: 50%vs 28% (p= 0.02). This was due to a lower risk of relapse related death (RRD) in patients with reactivation 19% vs 42% (p=0.005) and similar transplant related mortality (TRM), 28% vs 27% (p=0.8). In multivariate analysis disease phase (≤CR2) predicted survival (RR 0.33) relapse related death (RR 0.50) and TRM (RR 0.72); CMV/EBV reactivation predicted survival (RR 0.69) ; and RRD (RR 0.61); RIC transplants were associated with inreased RRD (RR 4.56). In conclusion: reactivation of CMV/EBV is a common event in our transplant prgram . Possibly because of an early diagnosis and pre-emptive therapy , TRM is comparable in patients with and without infection. Viral reactivation seems to promote the graft versus leukemia effect of the transplant. Tetramer-based quantification of cytomegalovirus (CMV)specific CD4+ and CD8+ T lymphocytes after allogeneic stem cell transplantation may identify patients at risk for CMV infection/disease D. Pastore*, A. Mestice, M. Leo, M. Giannoccaro, A. Mazzone, A. Russo Rossi, N. Sgherza, C. Longo, R. Angarano, V. Liso, G. Specchia Hematology, University (Bari, IT) Recovery of cytomegalovirus (CMV)-specific T-cells after allogeneic stem cell (SCT) is critical for protection against CMV disease. In our study we used fluorochrome-conjugated tetrameric complexes of HLA-A101, HLA-A201, HLA-B702, HLA-B801, HLA B3501 to monitor recovery of CD4 and CD8 CMV-specific T-cells (according to the patient's HLA) in 45 patients after SCT; the patients were transplanted with unmanipulated peripheral blood stem cells from an HLA identical related donor (n=43) and an HLA identical unrelated donor (n=2). Median age was 38 years (range 18-61); diagnoses were acute myeloid leukaemia (n=36), acute lymphoblastic leukaemia (n=5), chronic myeloid leukaemia (n=2), lymphoma (n=1), myelofibrosis (n=1). Five patients were CMV seronegative (R-) and 3 of them received grafts from a CMV-seropositive donor; forty patients were CMV seropositive (R+). The absolute median number of CMVspecific CD4+ T-cells detected at 1, 3, 6, 12 months was 0,5 microL (range 0-6), 2 microL (range 0-15), 5 microL (range [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] and 8 microL (range 2-62), respectively. The absolute median number of CMV-specific CD8+ T-cells detected at 1, 3, 6, 12 months was 2 microL (range 0-15), 7 microL (range 0-36), 15 microL (range 3-62), 18 microL , respectively. Tetramer analysis showed that 27/45 (60%) patients reconstituted CMV-specific CD4+ and CD8+ T-cells at 3 months; in this group only 2/27 (7%) patients developed CMV infection. CMV infections were observed in 17/18 (94%) who failed to generate CMV-specific CD4+ and CD8+ T-cells response. In our experience no CMV infection/disease was observed with CMV-specific CD4+ T-cells > 2 microL and CMV-specific CD8+ T-cells > 5 microL. Recovery of both CMV-specific CD4+ and CD8+ T-cell immunity occurred in 39/40 (97%) in R+ patients within 6 months and 3/5 (60%) in R-patients within 12 months. The cumulative incidence of CMV infection was 19/45 or 42% at 1 year, with a median reactivation time of 45 days (range 28-96); one patient, without CMV-specific CD4+ and CD8+ T-cells recovery, developed CMV disease (colitis-pancreatitis) and died. In conclusion we suggest that failure to recover CMV-specific CD4+ and CD8+ T-cells after SCT was associated with the development of CMV infection/disease and we envision that this strategy may enable us to identify those patients who may benefit from preemptive therapy, in particular the adoptive transfer of CMV-specific T lymphocytes for prevention CMV disease. Oral valganciclovir as pre-emptive therapy for cytomegalovirus infection in allogeneic stem cell transplantation recipients D. Pastore*, P. Carluccio, F. Gaudio, T. Perrone, M. Delia, F. Albano, P. Manduzio, V. Fesce, P. Mongelli, V. Liso, G. Specchia Hematology, University (Bari, IT) In allogeneic stem cell transplantation (SCT) recipients, cytomegalovirus (CMV)-infections contributes to the increase in morbidity and mortality; monitoring is performed by detecting CMV-Ag or virus DNA in peripheral blood. The intravenous infusion of ganciclovir remains the first line treatment; in an attempt to improve patient's quality of life and reduce time and cost of hospitalization many groups have devoted much effort to discovering a drug that can be prescribed orally and prove at least as efficient and safe as iv ganciclovir. Valganciclovir hydrochloride (VGC) is a pro-drug of ganciclovir, orally available, that has been used in CMV infection in high-risk solid organs transplants (donor positive, recipient negative). The aim of our study was to retrospectively review the efficacy and safety of po VGC (900 mg bd) as pre-emptive therapy for CMV infection in a cohort of 20 (9 males and 11 females) allogeneic stem cell recipients treated in our Unit. Median age was 38 years (22-62) and 14/20 pts underwent SCT from matched related sibling, 2/20 from matched unrelated donor and 4/20 from haploidentical related donor. CMV infection which was diagnosed after a median time of 56 days (range 37-98) from the transplant. With regard to donor and recipient CMV serostatus, 18 D+/R+, 2 D+/R-combinations were observed. CMV prophylaxis consisted in acyclovir in matched related and unrelated transplantations, foscarnet, ganciclovir and acyclovir in haploidentical transplantation. The pp65 antigenemia assay was positive in all cases with a mean of positive nuclei of 35 (range 21-111). Overall 16/20 (80%) cases obtained a clearance of antigenemia; none of 4 haploidentical transplantation obtained clearance of antigenemia and they were treated with iv ganciclovir and foscarnet. The median duration of VGC therapy was 21 (range 14-28) days and no cases of CMV disease were observed. Mild anemia was reported in 4/20 (20%) pts, neutropenia in 9/20 (45%) pts and thrombocytopenia in 7/20 (35%) pts. Two patients developed a mild deterioration of renal function that required dose adjustment. In conclusion orally administered VGC seems to be an effective alternative option to iv ganciclovir as pre-emptive therapy for CMV infection after allogenic matched related sibling and matched unrelated SCT but not in haploidentical transplantation. Regular blood count and creatinine should be performed in all cases to make early detection of cytopenia or renal toxicity. Cytomegalovirus (CMV) reactivation constitutes severe and life-threatening complication after allogeneic haematopoietic stem cell transplantation (HSCT). Seventy adult patients transplanted from 33 HLA matched sibling (SIB) donors and 37 unrelated donors (MUD; matched at high resolution level for at least 9 HLA specificities) were investigated. They were typed for microsatellite polymorphism (CA)n within the first intron of the interferon gamma (IFNG) gene (by PCR-STR) and followed for immunological reconstitution (flow cytometry of blood lymphocytes including measurement of CD8high+ pentamer HLA-A*0201/NLVPMVATV (CMV pp65+ cells), post transplant complications and CMV reactivation (qPCR). CMV load in peripheral blood cells was determined in patients within the observation period ranging from day 7 to 1 year post HSCT (median: 8 months). Each patient was examined at least three times during 60 days post HSCT and then in three months intervals and when clinically indicated (median: 10 measurements). The highest level of viral copies seen during the observation period was considered as a representative one for a given patient. A higher CMV load was found in patients: (i) transplanted from MUD than from SIB donors (6491±2318 vs 547±249 CMV-DNA copies/10 5 cells, p=0.047); (ii) having than lacking aGvHD or/and extensive cGvHD (4786±1884 vs 1579±1049 CMV copies/10 5 cells, p=0.022). CMV reactivation (diagnosed if over 100 viral copies/10 5 cells was detected) was more frequent in patients having: (i) less than 10% of CD4+ lymphocytes as compared to those with more than 10% of these cells in blood mononuclear cell fraction (0.42 vs 0.21, p=0.005, Fisher's Exact Test) and (ii) IFNG 3/3 low producer genotype than in those with other IFNG genotypes (0.70 vs 0.20, p<0.001). Moreover, patients carrying the IFNG 3/3 genotype and having less than 10% of CD4+ cells were at higher risk of CMV reactivation as compared to HSCT patients with other IFNG genotypes independent on CD4+ lymphocyte proportions (0.75 vs 0.19, p=0.004). Proportions of CD8high+ CMV pp65 reactive cells were lower in patients with at least 10 CMV-DNA copies in blood cells than in those lacking CMV-DNA copies (0.30±0.06% vs 0.77±0.19%, p=0.008). In conclusion, a low number of CD4+ cells or CMV specific CD8high+ cells and the presence of IFNG low producer genotype are the main risk factors of CMV reactivation in patients after HSCT. Supported by the MNiSW N401 169 32/3358 and 2 P05B 085 28 grants. Increased risk for invasive aspergillosis in patients with lymphoid malignancies treated with autologous haematopoietic stem cell transplantation L. Gil, M. Kozlowska-Skrzypczak, A. Mol, D. Poplawski, K. Swierkocki, A. Czyz, D. Dytfeld, A. Nowicki, M. Joks, M. Komarnicki University of Medical Sciences (Poznan, PL) The risk of invasive aspergillosis (IA) has been considered to be low among autologous HSCT recipients, but an increase in the incidence is observed recently in this group of patients. The aim of the study was to asses the influence of immunomodulatory / immunosuppressive drugs (steroids, rituximab, fludarabine and thalidomide), used in treatment of lymphoid malignancies during 6 months of pre-transplant period, on incidence of IA after autologous HSCT. A total of consecutive 109 patients (median age 45; range 19-67) with NHL (39), HD (36) and MM (34) were analyzed prospectively between [2005] [2006] [2007] . Pts were conditioned with modified BEAM (75) or Melphalan (34) and transplanted with PBSC in dose 4.2 (2-8)x10 6 /kg of CD34+ cells. Antifungal prophylaxis included fluconazole. All patients were monitored with twiceweekly galactomannan (GM) test. HRCT and bronchoscopy with BAL were performed in case of positive GM test, persistent fever or pulmonary infiltrates. The diagnosis of IA was made according to EORTC/MSG criteria. Results: neutrophil recovery occurred in 107 pts at 14 (10-54) days, two pts died before hematologic reconstitution due to septic shock (Pseudomonas aeruginosa). Documented IA was diagnosed in 9 (8%) pts (3 proven, 6 probable) and possible in 35 (32%) pts. The median time to diagnosis of documented IA was 10 (range; 6-24) days. The incidence of documented IA was comparable in pts with NHL, HD and MM and was not influenced by age and conditioning regimen. The highest incidence of documented IA was observed in pts treated with fludarabine (3/9 vs 6/100; p=0.025) or rituximab (5/27 vs 4/82; p=0.040). The risk of IA correlated with the length of neutropenia. Factors significant for development of documented IA by univariate analysis were treatment with fludarabine (p=0.008, HR=6.5, 95%CI=1. 6-25.9 ) and treatment with rituximab (p=0.039, HR=4.0, 95%CI=1. 1-14.8) . Factor predicting documented IA by multivariate analysis was treatment with fludarabine (p=0.008, HR=6.5, 95%CI=1. 6-25.9 ). In summary, pts treated with fludarabine or rituximab in pre-transplant period are at high risk of IA and require close monitoring and/or anti-mould prophylaxis. Parvovirus B19 infection following umbilical cord blood transplantation in children N. Bellier, C. Jubert*, J. Hu, J. Tanner, P. Ovetchkine, M. Duval, M.A. Champagne, C. Alfieri Hopital Sainte-Justine (Montreal, CA) Background: Parvovirus B19 (Pv B19) causes acute erythroblastopenia in patients with chronic hemolytic anemia, as well as chronic and prolonged anemia in immunocompromised patients. Pv B19 is typically transmitted by respiratory secretions, but can also be transmitted by blood and blood products from infected donors. During pregnancy, transplacental transmission can also occur. Due to the specific tropism of this virus for erythroblasts and hence the bone marrow, we hypothesized that Pv B19 might be associated with an increased incidence of engraftment failure in patients receiving umbilical cord blood transplants (UCBT). The aim of this study was to assess the frequency and impact of Pv B19 infection in pediatric patients who recently underwent an unrelated UCBT at our institution. Patients and methods: From 2004 to 2007, consecutive children who underwent UCBT and consented (through their parents or legal guardians) to be enrolled in the study were tested weekly for Pv B19 beginning on day of transplant until 6 months after transplantation, using a sensitive qualitative PCR test. Patients were considered positive for Pv B19 if they had at least two weekly consecutive positive PCR results. All data (demographics, clinical signs, outcome) were collected on a standardized questionnaire. Statistical analyses were performed using the SPSS software package. Supportive care for our UCBT includes weekly IVIG (500 mg/kg) for the first 100 days, then monthly until discontinuation of immunosuppressive therapy. Results: Thirty-two patients, mean age 102 months (SD=64), were enrolled. Among these, 16 tested positive for Pv B19 during their first 6 months following UCBT. The median delay to observe a first positive PCR result after UCBT was 21 days (range: 1 day -3.9 months). Results are summarized in table 1 Conclusions: Positive PCR for Pv B19 seems to occur with a high frequency (50%) and early (21 days) after UCBT. The main clinical manifestation is a rash occurring concomitantly with the first positive PCR result. In our study, the occurrence of Pv B19 PCR positivity infection did not influence engraftment. Varicella zoster virus infection after haematopoietic stem cell transplantation M.K. Choi*, K. Kim, J.Y. Hong, K.H. Kim, C.W. Jung, W.S. Kim, J.H. Jang, W.K. Kang Samsung Medical Center (Seoul, KR) Varicella zoster virus infection frequently occurs in patients who have undergone hematopoietic stem cell transplantation and may lead to severe complications. We retrospectively analyzed the incidence, clinical outcome and risk factors of varicella zoster virus infection in 538 patients undergoing allogeneic or autologous transplantation between February 1996 and February 2007. All patients received acyclovir 15mg/kg daily, in divided doses three times a day 8 hourly from day 1 to engraftment for HSV prophylaxis. 151 patients (28.1%) developed VZV infection after transplantation. The median onset of infection was 149 days and 81.3% of patients developed within 1 year. One patient died of disseminated herpes zoster induced fulminant hepatic failure. Postherpetic neuralgia was the most common complication (n=27, 17.9%) and one patient developed VZV infection induced pancreatitis. Disseminated cutaneous involvement was observed in 20.8% of patients. Twenty-two patients (14.6%) with VZV infection required admission. No risk factor was discovered with VZV infection. The incidence of VZV infection after allogeneic transplantation (n=65, 28.3%) and autologous transplantation (n=86, 27.9%) was similar, but in patients undergoing autologous transplantation, VZV infection appeared earlier (119 days vs 228 days, p=0.001) and required shorter duration of treatment (9.5 days vs 12.8 days, p=0.001). VZV infections are associated with significant morbidity and mortality in patients following hematopoietic stem cell transplantation. Low mortality rate after diagnosis of invasive aspergillosis in 33 cord blood transplant recipients using direct intrabone injection M. Mikulska *, A.M. Raiola, A. Ibatici, F. Gualandi, D. Occhini, A. Dominietto, C. Di Grazia, S. Bregante, T. Lamparelli, C. Viscoli, A. Bacigalupo S.Martino's Hospital (Genoa, IT) Introduction: Invasive Aspergillosis (IA) is an important posttransplant complication with high morbidity and mortality in patients undergoing cord blood trasnplantation (CBT). Recently, we have been developing a novel technique of transplant by injecting CB cells directly into the bone (IBM). Despite the high rate of HLA disparity between donor/recipient pairs, the incidence of acute GVHD requiring treatment with high-dose steroids is quite low. In this study, we evaluated the incidence and clinical outcome of IA in this novel setting of IBM-CBT recipients. Materials and methods: Thirty-three patients with advanced haematological malignancies (CR1=6 , CR2=8, more advanced=19) received a single-unit graft CBT using direct intrabone injection. Median cell dose was 2.6 TNC/kg (1.5-4) . Median age was 35 years (18-62) and follow-up is 8 months (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) for the 27 survivors longer than 30 days after CBT. Twenty-eight pts received a myeloablative conditioning (TBI 1200cGy in 26/28 pts). GVHD prophylaxis consisted of CSA, MMF and pre-transplant ATG. All patients but with prior probable aspergillosis, were given prophylactic fluconazole at a dose of 400mg/day. No patients were treated with steroids as GVHD prophylaxis. Galattomannan test (GM) was performed twice a week. Patients underwent high resolution CT scan (HRCT) whithin 72h after the onset of FUO. IA was diagnosed according to the EORTC/MSG criteria. Results: Engraftment was evaluable in 26 pts. Median time to neutrophil recovery was 23 days. Grade II acute GVHD was seen in 7 cases (27%) and grade III in 1 case (4%). All but one pts (grade III aGVHD) were successfully treated with steroids at 1mg/kg/day. Probable pulmonary IA was diagnosed in 5 pts (15%). All the diagnoses were based on clinical signs and symptoms, lesions on thoracic HRCT and positive GM test. The median time of onset was 14 days (8-28) after IBM-CBT. Four patients were treated with voriconazole and one with caspofungin. Only one patient (20%) died from IA 8 days after the diagnosis, due to possible disseminated infection. Conclusions: In our series, the incidence of IA remains as a major post-transplant clinical issue. However, as comapred to literature, the rate of mortality seems to be lower than expected. Therefore, early diagnosis, prompt treatment and limited use of steroid therapy may result in encouraging outcome of IA even after in an immunodepressed transplant setting as CBT. T. Mori*, Y. Aisa, J. Kato, Y. Ikeda, S. Okamoto Keio University School of Medicine (Tokyo, JP) Introduction: Recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT) are at risk of developing invasive fungal infection. Voriconazole and itraconazole, which are effective against both yeasts and moulds, are becoming widely used particularly for patients at risk of developing mould infection. Although drug interaction between calcineurin inhibitors and voriconazole or itraconazole is well-known, it has yet to be fully elucidated in allo-HSCT recipients. Patients and methods: Recipients of allo-HSCT in whom administration of voriconazole (n=18) or oral solution itraconazole (n=9) was initiated while they were under the therapy with calcineurin inhibitors (cyclosporine A (CsA) or tacrolimus) were eligible. Underlying diseases were hematologic diseases including myelodysplastic syndrome and acute leukemia. The dose of voriconazole was 4 mgx2/kg/day i.v. or 200 mgx2/day orally, and that of oral solution itraconazole was 200 mg/day. Trough levels of calcineurin inhibitors were measured before and periodically after initiating voriconazole or itraconazole, and concentration/dose (C/D; (ng/mL)/(mg/kg)) ratio of calcineurin inhibitors was calculated to quantitatively evaluate the drug interaction. Results: Increase in C/D ratio of calcineurin inhibitors was 84.2% by voriconazole administration and 92.5% by itraconazole administration. Of note is a great interpatient variation in the increase in C/D ratio of calcineurin inhibitors, ranging from -10.6% to 307.6% with voriconazole, and from 24.7% to 328.2% with itraconazole. Conclusion: These results suggest that both voriconazole and oral solution itraconazole have a clinically significant drug interaction with calcineurin inhibitors in allo-HSCT recipients. However, dose of calcineurin inhibitors should not be reduced in a uniform manner on starting voriconazle or itraconazole as recommended because of a wide interpatient variation in the drug interaction. Individualized patient approach is required in dose modification of calicineurin inhibitors in this setting. Do the febrile neutropenic episodes during mobilisation have any effect on autologous stem cell collection yield in patients with myeloma and lymphoma? P. Topcuoglu*, A.U. Bilgin, E. Ayyildiz, K. Dalva, O. Arslan, O. Ilhan, M. Beksac, M. Arat, M. Ozcan Ankara University (Ankara, TR) High dose therapy followed by autologous peripheral blood stem cell transplantation (auto-PBSCT) has been demonstrated to improve survival and potentially ensure the curability of some hemological malignities such as lymphoma and myeloma. When we use a chemopriming regimen for mobilization of auto-PBSCs, a period of neutropenia and hazard of febrile attack are inevitable. Thus, the release of inflammatory cytokines during febrile neutropenic episodes (FNE) might have a pro-or contra mobilizing effect on hemapoietic progenitors. According this hypothesis we aimed to investigate retrospectively the effect of FNE on the stem cell yield and collection efficiency in patients, who received chemotherapy plus G-CSF for mobilization. Between August 2001-June 2007 total 84 patients (M/F: 52/32) with lymphoma (n=45) or myeloma (n=39) were evaluated. Median age was 47 ys (17-64). Mobilization regimen were mostly cyclophosphamide (Cy) ± etoposide (VP) followed by G-CSF (n=20 and n=43, respectively). FEN was diagnosed and treated according to published guidelines. Results: Median 10.36x10 6 /kg CD34+ cells (1.11-54.32) were collected following a median 1 cycle (1-3) of continuous flow leukapheresis. 56 % (n=47) of the patients experienced FNE. We were not able to find any effect of FNE on the quantity of preapheresis circulating CD34+ cells and WBC (Table) . FEN affected significantly the MNC content but mildly the total nucleated cell content of the harvest. The total CD34+ cells were not affected both in first procedure and in total. The rates of success to obtain 2x106/kg, 3x106/kg, 4x106/kg and 5x106/kg cells were similar in patients with or without febrile episode. We did not observe any effect of variables like disease subtype (myeloma vs lymphoma) and the mobilization regimens (Cy vs CyVP), on CD34+ content of the harvest. In conclusion, the FNE due to chemopriming regimen prior to apheresis of peripheral blood stem cells had no effect of the yield of stem cell collection. Incidence and onset of CMV infections following treosulfan based reduced-intensity conditioning and allogeneic stem cell transplantation M. Haversath, S. Rohde, G. Kundt, D. Wolff, I. Hilgendorf, J. Casper, M. Leithaeuser, C. Kahl, M. Freund, C. Junghanss* University of Rostock (Rostock, DE) Cytomegalovirus (CMV) infections contribute significantly to morbidity and mortality following allogeneic hematopoietic stem cell transplantation (alloHSCT). Since incidences and kinetics of CMV infections are depending on the type of immunosuppression applied during the pre-and posttransplantation period we analysed the incidences of CMV infections following a treosulfan based reduced-intensity conditioning (RIC) and alloHSCT. Methods: Recipients of an alloHSCT at our institution between 1/99 and 12/06 following a treosulfan (Treo) based RIC were retrospectively analysed with regards to CMV infections. Most commonly RIC consisted of Treo (8-14g/m 2 , days -6 to -4) and fludarabine (Flud; 30 mg/m 2 , days -6 to -2) for HLA-matched related donor (mVRD) HSCT. Prophylaxis for graft-versushost disease (GVHD) consisted of cyclosporine A (1.5 mg/kg BID, starting on day -1). Antithymocyte globulin (ATG; 10mg/kg, days -4 to 2) was added to this regimen for unrelated donor (VUD) HSCT. The CMV risk groups were defined as: low risk [recipient (R) and donor (D) serologically CMV negative], intermediate (R-/D+) and high risk (R+/D-, R+/D+). CMV infection was defined as a positive CMV antigenemia or CMV DNAemia, CMV disease was defined as earlier (Boeckh 2003) . Results: 112 patients (pts.) were eligible. Median follow-up was 289 days (mean 698 days, range 10 -2770 days). Median pt. age was 50.2 yrs (range 16.3 to 66.7yrs). Underlying diseases were ALL (n=6), AML (n=36), CLL (n=6), CML (n=18), MDS (n=13), MM (n=9), NHL (n=14), OMF (n=5), others (n=5). Donortypes were mVRD (n=34), mismatched (mm) VRD (n=2), mVUD (n=68) and mmVUD (n=8). Conditioning regimen were Treo/Flud (n=34), Treo/Cyclophosphamide (n=8), Treo/Flud/ATG (n=65), Treo/Flud/Campath (n=4), Treo/Thio/ATG (n=1). In the low risk group (n=23) 3 CMV infections occurred (13.0%), 1 pt developed CMV disease (4.3%). In the intermediate risk group (n=13) 1 CMV infection (7.7%) and no CMV disease occurred. In the high risk group (n=76) 45 CMV infections occurred (59.2%), 8 pts. developed CMV-disease (10.5%). The median time to CMV infections was 33 days (range 1 -207 days) after HSCT, to CMV diseases 99 days (range 21 -1574 days). Conclusion: CMV infections occur after treosulfan based RIC alloHSCT. Overall incidences are comparable to other RIC regimen. However, CMV infections seem to occur earlier compared to other RIC or nonmyeloablative regimen. Cytomegalovirus infection and disease in patients undergoing unrelated cord blood transplant receiving prophylaxis with valgancyclovir or gancyclovir. Singlecentre experience P. Montesinos*, J. Sanz, S. Weiss, S. Cantero, G. Sanz Hospital La Fe (Valencia, ES) Prophylactic antiviral treatment for prevention of CMV infection and disease with Gancyclovir (GAN) could be useful after unrelated cord blood transplant (UCBT), where the incidence of CMV infection is very high. There is scarce information about the efficacy and safety of oral Valgancyclovir (VAL) in hematopoietic transplant. This study aims to: 1) evaluate the efficacy and safety of a risk-adapted strategy of universal prophylaxis of CMV infection, and 2) compare the efficacy and safety of VAL vs GAN for prophylaxis and treatment of CMV infection and disease in patients undergoing UCBT. From May 1997 to November 2007 adults with hematologic malignancies underwent UCBT in a single institution. Twenty-eight CMV-seronegative patients (21%) received prophylaxis with intravenous acyclovir from day -5 until day +100. For CMV-seropositive patients, prophylaxis consisted of IV acyclovir from day -5 until engraftment followed until day +100 by IV GAN (5 mg/kg per day 3 to 7 days per week) in the first 38 patients (29%) and by oral VAL (900 mg per day) in the last 67 (50%). Conditioning regimen consisted of thiotepa (TT), busulfan (BU), cyclophosphamide (CY) and antithymocyte globulin (ATG) in 69 patients, fludarabine, BU, TT and ATG in 56, and reduced intensity (RIC) in 8. Median age was 31 yr (range, 15-55). The cumulative incidence (CI) of CMV infection at day 100 and 365 was 34% and 45%, respectively. The CI of CMV disease at day 100 and 365 was 2% and 10%, respectively. The CI of CMV infection was lower in CMV-seronegative patients compared with CMV-seropositive (14% vs 40% at day 100 and 14% vs 54% at day 365, respectively; P=0.001). No differences in the CI of CMV infection was observed between patients taking GAN or VAL (47% vs 34% at day 100 and 55% vs 53% at day 365, respectively; P=0.38). The CI of CMV infection and disease recurrence were 22% and 6%, respectively. The CMV-serostatus was also predictive for a lower risk of CMV recurrence (7% vs 24%, P=0.11), but no differences were observed between GAN and VAL groups (23% vs 27%, P=0.74). Two of the 12 patients who developed CMV disease died from CMV. Antiviral prophylaxis/therapy was adjusted or withdrawn due to toxicity in 5 patients with GAN and in 6 patients with VAL. The median visits to day hospital of patients with GAN were 53, higher than 22 observed in patients with VAL. These results suggest that CMV prophylaxis with oral VAL is as safe and effective than GAN after UCBT. Specific focus on mycophenolate and human granulocyte / dendritic cell function in Aspergillus fumigatusdirected immune response J. Löffler, M. Mezger, C. Blockhaus, H. Einsele* Labor Professor Einsele (Wurzburg, DE) Background: There is increasing data that certain immunosuppressive agents not only modulate lymphocyte function, but also interfere with other immune effector cells. Here, we investigated the influence of mycophenolate on the immune defence of human dendritic cells (DCs) and polymorphonuclear neutrophils (PMNs), directed against the pathogenic mould A. fumigatus. This mould can cause invasive aspergillosis in immunocompromised patients. Methods: Human monocytes were isolated by magneticassociated cell sorting followed by differentiation into DCs with GM-CSF and IL-4. Cells were either co-cultivated with mycophenolate (10 µg/ml), with A. fumigatus germ tubes (MOI=1) or both stimuli. Whole-genome microarray analysis (Affymetrix U133A) or quantitative real-time PCR assays (LightCycler, Roche) were performed to quantify differentially regulated genes. PMNs were obtained from blood using Biocoll separation. Release of reactive oxygen species (ROS) was detected photometrically by using dichlorofluorescein. Results: Incubation of fully differentiated DCs in the presence or absence of mycophenolate did not affect the genome expression pattern and allowed normal immune response to A. fumigatus. However, if cells were treated during early differentiation processes, DC development was influenced by mycophenolate resulting in increased apoptosis. We also observed subsequent reduced pro-inflammatory cytokine activity (TNF-a, CXCL10) of DCs towards fungi. Quantification of ROS release of PMNs after phagocytosis showed that A. fumigatus germ tubes represent a strong stimulus for the oxidative burst which was increased at least 2 fold by mycophenolate. Conclusion: Mycophenolate might trigger an acute inflammatory syndrome by enhancing the A. fumigatus induced oxidative burst of PMNs. In addition to inhibitive effects of mycophenolate on lymphocyte proliferation, induction of immune responses can be inhibited due to a reduced A. Nihtinen*, V.-J. Anttila, L. Volin, E. Juvonen, T. Ruutu Helsinki University Central Hospital (Helsinki, FI) Allogeneic stem cell transplant recipients are at high risk of invasive aspergillus infections (IA), pulmonary IA being the most common form of infection. The role of amphotericin B (amphoB) inhalations as prophylaxis against IA is controversial. Since the beginning of 2001 amphoB deoxycholate inhalation (25 mg daily) has been used as prophylaxis in all allogeneic stem cell transplant recipients receiving high dose (10mg/kg) methylprednisolone (MP) for acute graft versus host disease (aGvHD) in our centre. The inhalations are started simultaneously with the high dose MP and continued for 2-3 months. Prior to 2001, no antifungal prophylaxis was used. In a retrospective study, the incidence of IA was compared in patients transplanted during the last five years without the prophylaxis (1996 ( -2000 and the first five years with the prophylaxis (2001 -2005 . Matched unrelated donor was used in 87 cases in Period I (33.7%) and in 145 cases (40.9%) in Period II. AGvHD grade I-IV was seen in 96 patients (37.2%) in Period I and in 123 (34.7%) patients in Period II. High dose MP was given to 71 patients in Period I and to 98 patients in Period II. AmphoB inhalations were given to 95 patients for median of 81 days (range . No systemic prophylaxis was used in these patients. The tolerability of the inhalations was good, none of the patients interrupted the prophylaxis due to side effects. IA was diagnosed in 17/258 (6.6%) patients in Period I and in 9/354 (2.5%) patients in Period II (cumulative incidence, p = 0.021). There was a trend towards a delay in IA-infections in Period II compared to Period I, 205 days (median, range 55 -1067) vs. 95 days (median, range 20 -2005) respectively (p = 0.40). Of the 17 patients with IA in Period I, 12 had aGvHD. Of the nine patients in Period II with IA, eight had aGvHD. Eight patients in Period II had pulmonary aspergillosis and one patient central nervous system aspergillosis. In one patient, the IA was diagnosed during the prophylaxis. The five year overall survival in Period I and Period II was 47% vs. 60% (p < 0.001). In conclusion, the incidence of IA decreased significantly during the period when amphoB inhalations were used but there might be other contributing factors to this finding. Only one breakthrough infection was seen. The timing of the IA infections shifted from 3 months to almost 8 months post transplantation indicating the protective effect of the inhalations. Profound inhibition of antigen-specific T-cell effector functions by dasatinib R. Weichsel (1), C. Dix (1) , L. Wooldridge (2) , M. Clement (2), , A. Sewell (2) , E. Greiner (3) The dual BCR-ABL/SRC kinase inhibitor dasatinib (Sprycel®, Bristol-Myers Squibb) entered the clinic for the treatment of CML and Ph+ ALL. As SRC kinases are known to play an important role in physiological T cell activation, we analysed the immunobiological effects of dasatinib on T cell function. The impact of dasatinib on multiple T cell effector functions was examined at clinically relevant doses (1-100nM) ; the promiscuous tyrosine kinase (TK) inhibitor staurosporine, lead substance for many drugs, was used as a comparator. Purified human CD3+ cells and virus-specific CD8+ T cells from healthy blood donors were studied directly ex vivo; antigen-specific effects were confirmed in defined T cell clones. Functional outcomes included cytokine production (IL-2, IFNgamma and TNFalpha), degranulation (CD107a/b mobilization), activation (CD69 upregulation), proliferation (CFSE dilution), apoptosis/necrosis induction and signal transduction. Both dasatinib and staurosporine inhibited T cell activation, proliferation, cytokine production and degranulation in a dose-dependent manner. Mechanistically, this was mediated by the blockade of early signal transduction events and was not due to loss of T cell viability. Overall, CD4+ T cells appeared more sensitive to these effects than CD8+ T cells, and naïve more sensitive than memory T cell subsets (activation IC50=10nM for CD4+ and 15nM for CD8+ T cells; proliferation IC50=10nM for CD4+ and 13nM for CD8+ T cells) while no differences in sensitivity were observed for staurosporine (IC50=4-5 for activation and proliferation in both CD4+ and CD8+ T cells). Of note, gamma/delta T cell functions were also inhibited by dasatinib. Dasatinib resulted in significant upregulation of TCR/CD8 at the T cell surface of EBV specific T cell clones indicating that the drug blocks TCR downregulation and recycling at the T cell surface and therefore confirming that the drug exerts its effects by blockade of proximal signalling pathways. The inhibitory effects of dasatinib were so profound that all T cell effector functions were shut down at therapeutically relevant concentrations. These findings indicate that caution is warranted with use of this drug in the clinical setting and provide a rationale to explore the potential of dasatinib as an immunosuppressant in the fields of transplantation and T cell driven autoimmune diseases. Invasive fungal infection (IFI) are associated with increased risk of death. Risk factors include recent use of corticosteroids and presence of graft versus host disease (GVHD). Interferon gamma (IFN-g) has been shown to augment cellular immune response and restore its cytokinemediated fungicidal activity. Material and methods: We present four patients who received a combination of antifungals and IFN-g for refractory or rapidly progressing fungal infection. Patient 1 (Pt 1) had relapsed acute lymphoblastic leukaemia (ALL) and IFI with liver involvement. Patient 2 (Pt 2) had ALL and history of three lines of treatment for autoimmune disease, including steroids and anti-tumour necrosis factor therapy and rapidly progressing fungal sepsis with involvement of skin, kidneys and central nervous system. Patient 3 (Pt 3) had 2 allogeneic stem cell transplants (SCT), one for ALL, 2nd for a myelodysplastic syndrome, complicated by GVHD. Patient 4 (Pt 4) had completed treatment for acute myeloid leukaemia. Three of the patients required transfer to the Intensive Care Unit (ICU). The implicated fungi were Trichosporon beigelii (Pt 1), Aspergillus flavus (Pt 2), Cryptococcus sp. (Pt 4). Patient 3 had probable fungal infection based on lung computed tomography (CT) picture. IFN-g was given for 6 to 16 weeks at doses of 100 µg three times per week together with ongoing antifungal therapy. All patients had at least 2 antifungals and at least one antifungal combination prior to IFN-g. All patients received granulocyte colony stimulating factor. Results: All patients responded clinically to the treatment and there was a clear time correlation with the initiation of IFN-g. At week 12 survival was 100%. Pt. 1 completed chemotherapy and underwent SCT. Pt. 2 had a very good response but required several lines of chemotherapy and SCT for refractory disease and died on day + 47 due to progression. Patients 3 and 4 continue on prophylactic antifungal therapy, the latter receives ongoing immunosuppression for immune thrombocytopenia. Conclusions: We demonstrated efficacy of addition of IFN-g to antifungal treatment in patients with IFI. Excellent responses were observed in patients who had not responded to 2 prior lines of treatment including antifungal combinations. Herpes virus reactivation is one of the serious complications affecting the recipients of allogeneic haematopoietic stem cell transplants (HSCT). We have previously reported that the risk of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) reactivation is associated with the IFNG microsattelite polymorphism. We have also found that EBV reactivation is influenced by the RANTES/CCL5 and CCR5 gene polymorphisms. In the present study the (801 G/A) polymorphism within the 3'UTR of the stromal cell-derived factor-1 (SDF-1/CXCL12) encoding gene was analysed and related with CMV and EBV load. Fifty-nine (31 SIB, 28 MUD) HSCT recipients and 55 donors were investigated for SDF-1 genotypes. Numbers of viral DNA copies in peripheral blood cells were determined in patients within the observation period ranging from day 7 to 100 post HSCT with 5 measurements per patients in an average. SDF-1 alleles and viral DNA copies were analysed by PCR-RFLP and qPCR, respectively. The higher numbers of CMV copies were detected in patients homozygous for the SDF-1-3'A allele as compared with recipients carrying G allele (mean+SEM: 6815±5151 vs 279±150 CMV copies/10 5 cells, p=0.0234, U Mann Whitney test). The significant associations of the SDF-1-3'AA genotype with higher CMV load were also seen in patients receiving reduced intensive conditioning regimen (10190±7412 vs 3905±2009 CMV copies/10 5 cells, p=0.0417) and those receiving transplants from HLA-matched siblings (13570±9328 vs 2423±1468, p=0.0236). In contrary to CMV, EBV reactivation was not found to be associated with the recipient SDF-1 genotype. No relationship was observed for donor SDF-1 polymorphism and either CMV or EBV reactivation in patients after transplantation. Summarizing, CMV reactivation was found to be associated with the presence of the SDF-1-3'AA homozygosity in HSCT recipients. Supported the MNiSW grants No. 2 P05B 085 28 and N401 169 32/3358. J.Y. Hong*, J.E Uhm, K.H. Kim, M.K. Choi, J.H. Jang, K. Kim, W.S. Kim, C.W. Jung Samsung Medical Center (Seoul, KR) Lamivudine, the pyrimidine nucleoside analogue, is known to effectively suppress hepatitis B (HBV) viral replication without significant clinical toxicity. Furthermore, lamivudine prophylaxis has demonstrated the efficacy in the prevention of HBV reactivation in Hepatitis B surface antigen (HBsAg)positive patients receiving chemotherapy or hematopoietic stem cell transplantation (HSCT). This study was performed to evaluate the efficacy of lamivudine prophylaxis and the incidence of lamivudine resistance during and after HSCT. Thirty-two (6%) cases among 528 cases who underwent allogeneic or autologous HSCT at Samsung Medical Center from 1996 to 2006 had identified HBsAg positivity prior to HSCT. We reviewed the medical records of 32 cases with HBsAg, retrospectively. Median follow-up duration was 48 months (range, 6-101 months). Sixteen (50%) of 32 cases underwent allogeneic HSCT. Twenty-nine cases had started lamivudine prophylaxis before conditioning chemotherapy of HSCT (median 148 days before; range, 1-1298 days). The median lamivudine maintenance duration after HSCT was 42 weeks (range, 3-84 weeks). There was no mortality related to HBV reactivation. Two of 29 cases had developed the viral breakthrough with YMDD mutation and lamivudine resistance and they needed to change anti-viral therapy from lamivudine to adefovir at post-HSCT day 1292 and day 635. Seven of 29 cases could discontinue lamivudine at median 342 days after HSCT (range, 97-10 days). There was no HBV viral breakthrough after discontinuance of lamivudine. One out of 3 cases who did not receive lamivudine prophylaxis had developed HBV reactivation at 66 weeks after HSCT and was treated with lamivudine successfully. In conclusion, lamivudine prophylaxis is effective for prevention of HBV reactivation in HSCT recipients. However, further investigations are needed to determinate the optimal duration and guideline for discontinuation of lamivudine prophylaxis considering to prevent lamivudine resistance. Use of KGF (palifermin) but not of G-CSF is associated o a reduced rate of infections after high-dose chemotherapy and autologous PBSC transplantation G Milone (1), K. Battiato (1) , P. Murgano (1) Introduction: Reduction of infections after PBSC autologous transplantation is an important clinical goal. Use of G-CSF has been shown to shorten neutropenia without improving infectious risk. KGF (Palifermin) has been shown to reduce febrile neutropenia when used during TBI-containing regimens, however, efficacy of Palifermin has not been determined when used in association with non TBI containing regimens. Methods: We have studied factors associated to infections in a group of 156 patients, all received a non TBI-containing eradicating regimen and were treated with either G-CSF and/or Palifermin. G-CSF was used according to two randomised studies run in our institution while PALIFERMIN was used sequentially in a cohort of 29 patients. Mean age was 49 years, underlying diagnosis was MM (77 pts), Lymphomas (66 pts), other dx (13 pts), 32% of patients were in advanced phase of disease, conditioning regimens were: L-PAM (n.77), BEAM (n.60), other (n.19); dose of infused CD34+ was 5.5 x10 8 /Kg. Anti-infectious prophylaxis was standardised and comprised in all patients systemic antibiotic, intestinal decontamination, acyclovir, fluconazole. Results: Neutrophil engraftment was reached in a mean of 12 days, Febrile neutropenia of Unknown cause (FUO) was diagnosed in 45% of patients, Gram-negative Bacteraemia in 4.5%, Pneumonia in 5.1%, CVC-associated Bacteraemia in 16% of patients. TRM at 1 year was 0%. Results of association of various factors to Severe Infections, defined as diagnosis of "FUO or GRAM-neg. BACTERAEMIA or PNEUMONIA" in univariate and in multivariate logistic regression are reported in the table 1. Discussion: In conclusion use of PALIFERMIN is the only modifiable factor that reduces significantly the risk of "Severe Infections" after high dose non TBI-containing regimens and PBSC transplantation 1. Posaconazole is superior to itraconazole for antifungal prophylaxis in allogeneic stem cell transplant recipients: a single-centre experience M. Arnan *, B. Patiño, T. Peralta, C. Gudiol, R. Parody, A. Fernández de Sevilla, R.F. Duarte Institut Catalá d'Oncologia (Barcelona, ES) Antifungal prophylaxis with oral posaconazole has recently shown good tolerance and overall improved efficacy when compared with fluconazole and/or itraconazole in patients with prolonged neutropenia or GVHD. Whether such encouraging results translate to other groups at high-risk of invasive fungal infections (IFIs) has not yet been studied. Here, we present our experience with posaconazole for the prevention of IFIs in allogeneic stem cell transplant (alloSCT) patients. Twenty six consecutive recipients of a peripheral blood alloSCT, who received either posaconazole (POSA; n=13; 200mg/8h oral; from May 2007 onwards) or itraconazole (ITRA; n=13; oral and/or iv; consecutive cases up to May 2007) were analyzed. Patients with prior history of IFI or receiving other antifungal treatment prior to admission for alloSCT were excluded. More patients in the POSA group received a T-cell depleted alloSCT (4 vs 0; p=.04). Otherwise, patients age (median 53, 20-67), sex (15 males), disease (12 AML, 4 MDS, 4 CML, 3 lymphomas, 2 ALL, 1 myeloma), conditioning (15 reduced intensity, 11 myeloablative), type of donor (20 related, 6 unrelated), days to neutrophil engraftment (17, , and incidence of acute GVHD (23%) were similar in both groups. All patients received antifungal prophylaxis at least for 100 days post-alloSCT. ITRA patients received oral suspension or iv drug based on their tolerance. POSA patients had no problems with oral compliance. They required, however, a median 50% cyclosporine dose-reduction to maintain its therapeutic levels within range. No cases of significant toxicity or prophylaxis failure as a result of drug toxicity occurred in either group. However, the probability of prophylaxis efficacy failure was more common in ITRA patients than in the POSA group (p=.04). Four ITRA patients, but none of the POSA ones, discontinued prophylaxis in order to receive empiric antifungal treatment (3; 2 caspofungin, 1 lipid complex amphotericin) or IFI-directed voriconazole treatment (1) . In addition, the probabilities of early IFI-free survival at day +100 was superior in the POSA patients (85% vs 46%; p=.03), and the probability of overall survival at day +100 showed a statistical trend towards an improved outcome in the POSA group (85% vs 69%; p=.08). In conclusion, our single-centre series shows that posaconazole antifungal prophylaxis in alloSCT recipients is well tolerated and appears significantly superior to itraconazole in terms of efficacy. Toxoplasmosis after haematopoietic stem cell transplantation -a single-centre experience Z. Rusinakova, L. Raida, E. Faber*, J. Bednarikova, B. Paucek, K. Indrak University Hospital (Olomouc, CZ) The incidence of toxoplasmosis (T) after HSCT is low however the mortality may be as high as 60%. We have observed 3 cases of T in allo-HSCTs (2.6% of 116 patients after allo-HSCT since 2000) and one case in autologous HSCTs (0.3% of 395 patients after auto-HSCT since 1997). All allografted patients went through unrelated HSCT after conditioning Flu+CTX+ATG indicated for Hodgkin´s lymphoma, T-PLL and B-CLL. The indication for auto-HSCT was DLCL treated with sequential immuno-chemotherapy and BEAM. Manifestations of T were neurologic symptoms, cerebral salt wasting syndrome (hyponatremia and hypoosmolality), organic psychosyndrome and signs of respiratory infection. In the first case the diagnosis was done at autopsy. Symptoms manifested at day +139 after HSCT. T was suspected but repeated CSF PCR exams for T were negative. There was a significant improvement after steroids. Planned brain biopsy was not performed due to rapid deterioration and death. In the second case hyponatremia and hypoosmolality was the first manifestations of T 12 days after HSCT in the patient with history of posttraumatic epilepsy. Changes on brain MRI were unspecific but PCR for T were positive both from CSF and plasma. Treatment with pyrimetamine, clindamycine and cotrimoxazole (PCC) was started immediately with prompt symptomatic relieve and achievement of PCR negativity. The third case was diagnosed using CSF PCR exam indicated for respiratory infection, paraparesis and incontinency occuring 92 days after HSCT. Treatment with PCC resulted in clinical remission of T however repeated courses of therapy were needed due to the graft failure. The final case of T manifested on +85 day after auto-HSCT with bilateral pneumonia. Diagnostic exams including BAL were not helpful and broadspectrum antibiotics and antimycotics were unsuccessful. Organic psychosyndrome developed with somnolence, agitation, arm paresis and incontinency. T was confirmed using PCR CSF exam. Treatment with PCC was successful. With the growing use of highly immunosupressive agents (including rituximab, fludarabine and ATG) there is a need of awareness of T risk even in auto-HSCT. Detailed questioning about pets and pre-transplant serology should be a routine. Variable manifestations, non-specific results of MRI or CT scans and the possibility of PCR negativity are the main obstacles for successful diagnosis. Supported by MSM 6198959223 and MSM 6198959205. (1), M Debiaggi (2), M Parea (1), L Zenone Bragotti (1), P Bernasconi (1), D Caldera (1) Policlinico San Matteo-Pavia were enrolled in a prospective study aimed investigate the incidence of Human Metapneumovirus infection (hMPV) infection and related diseases. Thirty-six patients had an autologous transplant, 14 an allogeneic transplantation, six patients from related and 8 from unrelated donor. Detection of hMPV RNA in NPA samples was performed by specific reverse transcription PCR assay, in 123 samples collected before admission to transplant center, and at day +15, +30, +60, +90, and +180 from HSCT. Clinical data were recorded and examined for: type of hematological malignancy, type of HSCT, presence of fever, signs or symptoms suggestive for respiratory tract infection (i.e.: rhinorrhea and sneezing, nasal/sinus congestion, pharyngitis or dry cough, pulmonary infiltrates), chest radiographs, computed tomographic scans, isolation or identification of other pathogens, antiviral prophylaxis or therapy. At the median follow of 19 months (range: 10-24 mo) and 8 months (range: 1-24 mo.) for patients receiving autologous and allogeneic transplant respectively, no specific respiratory symptoms or signs were documented. In only in 2 asymptomatic pts, MPV was detected; the genotype analysis showed that infection was sustained from the genotype A. In this study the prevalence of hMPV infection is similar to the adult healthy population. A.A. Colombo, L. Zenone Bragotti, M. Gotti, F. Ripamonti, D. Caldera, P. Bernasconi, M. Lazzarino, E.P. Alessandrino* Fondazione IRCCS Policlinico S.Matteo (Pavia, IT) One-hundred-seven adult patients naturally immunized against HBV with anti HBc antibodies transplanted with an HBsAg and HBV-DNA negative donor, were studied during the post-transplant follow-up to evaluate the incidence of HBV S274 reactivation. After a median follow up of 16,1 months (range 0,2-154) a reversion of HBV sieroconversion was observed in 8 patients (7,5%), all patients were on therapy with Cyclosporine A and/or methilprednisolone because of extensive cGvHD. The prevalence of HBV reinfection in patients with c-GVHD was 15,7 % (p=0,021) with a probability of 17,3 at 5 years. HBV-DNA copies/ml were 68.000/ml (2.000-1x10 9 ). There were no evidences of correlation between hepatic necrosis index and viral genomic copies and ALT and AST medium values were 276 IU (range 15-1708) and 68 UI (21-1708) respectively. After HBV reactivation, all but two patients had lamivudine antiviral treatment; only three of 8 patients recovered anti HBV immuno-competence at four, six, and forty-four months: two patients in absence of specific antiviral therapy. No cases of fulminant hepatitis were reported. In one case (UPN 298), despite of a antiviral treatment, hepatic biopsy documented advanced liver cirrhosis related to chronic hepatitis. In this report dealing with HSCT recipients with natural immunisation against HBV before transplant, immunological imbalance and persistence of cGvHD contribute to HBV reactivation. Delayed NK-cell reconstitution is associated with a high risk of CMV-antigenemia after allogeneic SCT M. Schmidt-Hieber* (1), S. Schwarck (1) , E. Thiel (1), S. Ganepola (1) , H. Tillmann (2) , P. Reinke (2) , I.W. Blau (1) , L. Uharek (1) ( Identification of novel CMV-risk factors might be the basis for risk-adapted strategies, including adoptive NK-and T-cell transfer after allogeneic stem cell transplantation (SCT). We prospectively analyzed the influence of early immunoreconstitution as well as CMV-serostatus, donor type and conditioning on the risk of CMV-antigenemia after allogeneic SCT. Seventy-four patients (pts) with a median age of 50 years and various hematological diseases were included in this analysis (AML: 37 pts, ALL: 9 pts, MDS: 5 pts, NHL: 7 pts, other: 16 pts). Donors were matched related (MRD) in 25 pts (34%) and matched unrelated (MUD) in 49 pts (66%), respectively. Serum IgG concentration and peripheral blood counts for CD4-, CD8-, NK-cells, and CMV-specific cytotoxic Tlymphocytes (CMV-CTLs) were monitored monthly, starting on day +30. CMV-antigenemia was assessed every two weeks by PCR or detection of the pp65-antigen. Overall, 27 patients (36%) showed at least one period of CMV-antigenemia. The donor/recipient CMV-serostatus and the peripheral NK-cell count on day +30 were identified as risk factors for cumulative incidence of CMV-antigenemia after allogeneic SCT. The cumulative incidence of CMVantigenemia was 58% for CMV-seropositive patients transplanted from a CMV-seronegative donor, but only 5% if recipient and donor were CMV-seronegative (p=0.009; Fisher´s exact test). Patients with a peripheral NK-cell count ≤ 200/µL on day +30 had a significant higher risk for CMVantigenemia than those with a NK-cell count of > 200/µL (56% and 25%; p=0.04). Using the Kaplan-Meier method, the estimated cumulative incidence of CMV-antigenemia one year after SCT was also significantly higher for patients with a NKcell count ≤ 200/µL on day +30. No significant difference of the cumulative incidence of CMV-antigenemia was observed between patients transplanted from a MRD and those who had a MUD (25% and 44%, p=0.19) . The risk of CMVantigenemia was also comparable in terms of conditioning type (38% for reduced-intensity and 29% for conventional conditioning, respectively; p=0.60). We demonstrated that the early NK-cell count predicts the risk of CMV-antigenemia after allogeneic SCT and might thus be included as a novel risk factor in an algorithm of prophylactic or preemptive strategies. A novel algorithm to apply prophylactic intravenous immunoglobulins in patients at high risk for CMV after allogeneic SCT M. Schmidt-Hieber*, S. Schwarck, S. Ganepola, E. Thiel, L. Uharek, I.W. Blau Charite-Campus Benjamin Franklin (Berlin, DE) Cytomegalovirus (CMV) is still a major cause of morbidity and mortality in patients who underwent allogeneic stem cell transplantation (SCT). We developed an algorithm to apply high-dose prophylactic intravenous immunoglobulins (IVIG) in periods of high risk for CMV. Immunoreconstitution and graftversus-host disease (GvHD) were assessed monthly and IVIG were administered in a dose of 0.5 g/kg BW (twice monthly) if one of the following criteria was fulfilled: 1. Serum-IgG concentration < 4 g/L. 2. CD4-cell count < 100/µL. 3. NK-cell count < 100/µL. 4. Acute (grade II-IV) or chronic (extensive disease) GvHD. Immunomonitoring and IVIG application according to the algorithm was started on day +30 and continued to the maximum of one year after SCT. In a first phase (June 2005 until October 2006) patients received prophylactic IVIG according to the algorithm (intervention group), whereas in a second, subsequently evaluated cohort patients did not receive prophylactic IVIG (November 2006 until December 2007 . Primary endpoints of this analysis included cumulative incidence of CMV-antigenemia and -disease of the intervention group compared to the control group. The analysis encompassed 74 patients in total (44 intervention and 30 control group) with a median age of 50 years. In the intervention group, stratification was done to IVIG application in 47%, whereas 32 patients (73%) received at least ones prophylactic IVIG. The cumulative incidence of CMV-antigenemia did not differ significantly between intervention and control group (43% and 23%, respectively; p=0.08, Fisher´s exact test). Since median follow-up was shorter in the control than in the intervention group (142 and 254 days, respectively), we further analyzed `time to CMV-antigenemia´ using the Kaplan-Meier method. Also this approach did not reveal any significant difference between intervention and control group (estimated cumulative incidence of CMV-antigenemia one year after SCT 44% and 32%, respectively; p=0.20, log-rank test). CMV-disease was rare in both groups (9% intervention and 10% control group; p=1.00). In three patients (7%) of the intervention group, IVIG administration was terminated early due to toxicity (shivering). We conclude that prophylactic high-dose IVIG application in patients at high-risk for infectious complications is feasible and associated with low toxicity, but does not reduce the frequency of CMV-antigenemia and -disease after allogeneic SCT. T. Widmann*, U. Sester, B. Gärtner, J. Schubert, M. Pfreundschuh, H. Köhler, M. Sester University Hospital of Saarland (Homburg/Saar, DE) The control of latent CMV reservoirs by antigen-specific Tcells is impaired after stem cell transplantation (=SCTx). Available studies have measured CMV specific T-cells at limited intervals following SCTx. By routinely monitoring CMV specific CD4 and CD8 T-cells and CMV DNA in 40 allogeneic S275 SCTx recipients, we observed highly dynamic intra-individual levels of CMV specific CD4 T-cells, with a clear augmentation in recipients experiencing CMV DNAemia (p<0.001). Episodes of CMV DNAemia were characterized by a drop of CMV specific CD4 T-cells during viremia and an increase after viral clearing (p<0.001). In contrast, cumulative levels of CMV specific CD8 T-cells were not related to viremia (p=0.451). Furthermore, levels of CMV specific CD4 T-cells inversely correlated to peak viral load (p=0.011). As episodes of graftversus-host disease (=GvHD) require increased immunosuppressive therapy and frequently trigger CMV reactivation, we studied lymphocyte effector functions upon incubation with cyclosporine and corticosteroids in a whole blood assay. Both, proliferation and production of IFN-a by CMV specific CD4 T-cells was severely impaired during combined immunosuppressive therapy (p=0.007 and p=0.003 respectively) indicating a mechanistic link to CMV reactivation. In conclusion, levels of CMV specific CD4 T-cells inversely correlate with CMV DNAemia and may be a parameter to guide anti-CMV therapy. T. Widmann*, U. Sester, J. Schubert, B. Gärtner, H. Köhler, M. Pfreundschuh, M. Sester University Hospital of Saarland (Homburg/Saar, DE) Virusspecific T-cells are critical to withstand CMV viremia following stem cell transplantation (=SCTx). So far, the minimal inter-individual time interval for CMV specific T-cell reconstitution posttransplantation has not been reported. Therefore, we frequently measured CMV specific T-cell responses by cytokine flow cytometry and HLA class I specific pentamers in 51 SCTx recipients. CMV specific CD4 T-cells were reconstituted after a median of 2.4 weeks (autografting, n=6) and 4.0 weeks (allografting; p=0.006). Factors prolonging the time to initial reconstitution of CMV specific CD4 T-cells included a negative recipient serostatus (p=0.016) and CMV viremia before virus specific Tcell reconstitution (p=0.026). Frequencies of CMV specific CD4 T-cells increased over time, reaching a plateau after 90 days (p=0.043). The minimal time for reconstitution of CMV specific CD8 T-cells was similar to their CD4 counterparts (p=0.580). In long term SCTx transplant recipients, the absolute levels of CMV specific T-cells were indistinguishable from healthy controls. However, considering the persistent lymphopenia of long term SCTx recipients, the ratio of CMV specific CD4 T-cells to total CD4 T-cells was increased in SCTx recipients (p<0.0001). In conclusion, we show a rapid reconstitution of CMV specific T-cells and an augmented CMV specific CD4 T-cell response in long term SCTx recipients. EBV-associated problems following allogeneic stem cell transplantation: a study on 28 cases with lymphoproliferative disease and reactivation S. Ocheni*, N. Kröger, T. Zabelina, R. Erttmann, H. Kabisch, A. Zander, U. Bacher University of Hamburg-Eppendorf (Hamburg, DE) Objectives: Posttransplant lymphoproliferative disease (PTLD) is a fatal complication of allogeneic stem cell transplantation (SCT) with mortality rates of 50-90%. Although the frequency of PTLD is reportedly <1% of all transplantation cases, higher rates are seen in high risk settings such as intensive immunosuppression or T-cell depletion. Most PTLD cases are associated with the oncogenic Epstein Barr Virus (EBV). Currently there are several therapeutic strategies in the management of EBV reactivation and PTLD, but till date, the outcomes are very poor. The objective of this study was to determine incidence, risk factors, and outcomes of EBVreactivation and -PTLD in this Center. Methods: We performed a retrospective analysis in 28 consecutive adult and pediatric EBV-reactivations and PTLD between 1999 and 2007 at the University Medical Center of Hamburg-Eppendorf. Results: 28 patients (3.3 %) had EBV-reactivation or PTLD (18 adults; 10 children) out of a total of 854 transplantation cases (713 adults; 141 children). Specifically, there were 12 cases of EBV-associated PTLD in 7 adult and 5 pediatric patients, whereas EBV reactivation without lymphoproliferation was seen in 16 patients. Median age was 46.0 years (r. 17.0 -66.0) and 11.0 years (r. 1.0 -15.0) in the adult and pediatric patients. The median viral loads were 138 Geq/ml (r. 84 -10.400) and 250 Geq/ml (r. 100-100.000) in patients with only EBV reactivation and those with EBV associated PTLD, respectively. There was a high rate of bacterial, viral, fungal and parasitic infections (15/28; 54%) at the time of EBV reactivation / -PTLD. Treatment modalities included anti-CD20-regimen (Rituximab or Mabthera), cidofovir and foscavir either as monotherapy or in combination. 19/28 (68%) patients who had EBV-reactivation / -PTLD died after a mean of 210 days (r. following the manifestation of EBV disease. The mortality rates were 11/16 (69%) and 4/12 (33.3%) in patients with EBV-reactivation and PTLD respectively. Only 9 out of 28 patients (32%) are alive after a mean follow-up of 1289 days (r. 24-2751). Conclusion: EBV reactivation is frequently accompanied by other infectious complications in recipients of hematopoietic SCT. The mortality rates following EBV reactivation and PTLD are still very high in spite of the various current treatment modalities. Prospective multicenter studies should be initiated to identify the best treatment approaches. Assessment of CMVpp65 specific CD8+ T-cell frequencies and adoptive T-cell transfer to post transplant patients by streptamer technology A. Schmitt* (1), J. Yao (1) , H. Einsele (2) , G. Grigoleit (2) , D. Busch (3) Objectives: Cytomegalovirus (CMV) disease constitutes serious complications after allogeneic peripheral blood stem cell transplantation (PBSCT). Besides neutralizing CMV antibodies, the frequency of CMVpp65 specific CD8+ T cells is pivotal for the clearance of CMV after PBSCT. CMVpp65 specific CD8+ T cell frequencies can be measured using tetra-, penta-and streptamer technologies. The frequency in donors might allow us to define the best available donor in addition to the mere serostatus. Methods: The specificity of all three above mentioned multimer technologies was evaluated and correlated to the serostatus. As streptamers are available clinical grade, we performed an adoptive transfer of CMV specific CD8+ T cells selected by this technology to a patient with acute lymphatic leukemia suffering from high-grade CMV antigenemia resulting in life-threatening neutropenia after allogeneic PBSCT. For the adoptive T cell transfer a donor leukapheresis was performed followed by an CMVpp65 streptamer positive selection. The patient received one transfusion of 2x10 5 CMV specific CD8+ T cells per kg body weight. 23 samples from CMV seropositive healthy volunteers (HV) and 10 samples from CMV seropositive patients before and after allogeneic stem cell transplantation were stained with tetra-, penta-or streptamer in combination with CD4/8 and analyzed. Results: Frequencies of 0.05% up to 2.87% CMVpp65 specific CD8+ T cells were detected. Surprisingly, only in 48% (11/23) seropositive HV CD8+multimer+ T cell frequencies could be detected. The ALL patient developed a foscarvir resistant CMV antigenemia with a maximum of 959/500,000 CMVpp65 positive cells. After a switch to ganciclovir/valganciclovir and an adoptive transfer of CMV specific T cells, the antigenemia was cleared. Valganciclovir was discontinued, but the CMV antigenemia remained controlled. The frequency of CMVpp65 specific CD8+ T cells increased dramatically from 0.0% to 19.8% of all CMV specific CD8+ T cells. All of these T cells were donor derived as defined by STR chimerism analysis. The patient did not develop CMV disease at any time point. Conclusion: Donors should be screened for CMVpp65 specific CD8+ T cell frequencies. The streptamer technology offers the option to select CMVpp65 specific CD8+ T cells at GMP level for adoptive T cell transfer and can induce long-lasting CD8+ T cell responses effectively clearing even a high virus load. Patients suffering from hematologic malignancies, especially those treated with stem cell transplant, are at high risk of S pneumoniae infections. The epidemiology of S pneumoniae in the community may vary from one country to an other. Few data are available for hematology patients on the clinical relevance of the two available vaccines -PPV23 (Pneumo 23®), and heptavalent conjugate vaccine Prevnar® (PCV7)according to the serotypes encountered in this population. Objective: The aim of this retrospective study was to look at the serotype distribution of S pneumoniae in pneumococcal infections observed in hematology patients in Ile de France, and their proportion covered by PPV23 and/or PCV7. Methods: All the patients with S pneumoniae infections occurring between Jan 2004-Dec 2006 in 2 hematology departments (Henri Mondor and Saint-Louis hospitals) were identified from the laboratory registries. Serotyping was performed at the CNRP in Paris. Among 40 consecutive cases, serotype was performed in 25 cases occurring in 22 patients. The remaining 15 strains were either not available or did not regrow. Results: The 22 patients had acute leukemia (n=6), CML (n=1), myelodysplastic syndrome (n=1) or lymphoproliferative disease (n=14) (M/F: 1.4; median age: 54 y). Seventeen had received a stem cell transplant (SCT) (allo: 9; auto: 8) a median of 12 months before S pneumoniae was isolated either from blood cultures (24%) or respiratory samples (76%). Three out of the 22 (14%) patients died from pneumococcal infection. The most frequent serotypes were 9V (n=7) and 19F (n=6), then 14 and 23F (2 of each). 19/ 25 (76%) of the strains were covered both by PCV7 and PPV23, and 4/25 (16%) were covered only by PPV23. Only 2/25 (8%)strains were not covered by any vaccine. Six out of the 25 (24%) strains were penicillin R. Conclusion: We found that most strains of S pneumoniae responsible for infections in hematology patients from Ile de France, including a majority of transplant recipients, were covered by PPV23 (92%), and PCV7 (76%). Only 2/25 strains were covered neither by PPV23 nor by PCV7. These data make relevant the development of prospective studies and vaccination programs with both PCV7 and PPV23 in the high risk hematology patients. Cytokine response to adenovirus infection in children after allogeneic stem cell transplantation L.M. Haveman*, B.J. Prakken, W. Jager, M. Bierings Wilhelmina Children's Hospital (Utrecht, NL) Introduction: Despite improvements of supportive care after stem cell transplantation (SCT) both non-infectious complications as infectious complications occur frequently. In pediatric SCT recipients adenovirus (Adv) infection is an important infectious complication with a high morbidity and mortality rate. In clinical practice it is often difficult to distinguish between different complications. Hence determining cytokine production seems to be a relative easy way to distinguish SCT related complications. Innovation in technique for detection of proteins resulted in the development of particle based multiplex immunoassay (MIA). With this method we measured 17 cytokines involved in the inflammation process with the aim to identify a panel of cytokines to use as a predictor for Adv infection. Methods: During a 3 year's prospective study 47 pediatric SCT recipients were included. Adv screening included weekly culture of faeces, throat swabs and urine and PCR on plasma samples. From 7 out of 8 patients (17%) with a positive PCR in total 22 samples were analyzed. From 4 out of 7 patients (15%) with a positive Adv-culture without a positive PCR in total 13 samples were measured with MIA. Cytokine production was compared with patients with Epstein-barr virus (EBV) infection and GvHD. Results: Cytokines produced in patients with Adv or EBV infection looks similar, but Adv infected patients produced more IL1b and IL17 and less IL10 and Rantes. In comparison with GvHD patients Adv infected patients produced more IL17, OSM, MIP1a and IP10 (p<0.05) (Figure 1 ). Comparing cytokine production during a positive Adv PCR with patients with a positive Adv culture and with the same patients without an infection all proinflammatory cytokines are produced in larger extent, notable IL17, IL18, OSM, Mip1a and IP10. From the patients with a positive Adv PCR 1 patient could not clear the infection. Patients who could clear the infection produced more IL1b, IL6, IL12, IFNg and TNFa (p<0.05). The patient who did not clear the infection produced more IL10, IL18 and IP10; similar to the cytokines produced in EBV infected patients. Conclusion: After SCT it seems possible to use a cytokine profile to make a distinction between different complications. In Adv infection besides production of proinflammatory cytokines well known for infection, the cytokines IL1b, IL17, OSM and IP10 seems to be Adv specific. In case of a positive Adv PCR cytokine profile may also predict clinical outcome. Levofloxacin prophylaxis decreases the incidence of BK polyoma virus-induced haemorrhagic cystitis in patients after allogeneic haematopoietic stem cell transplantation M. Lache, F. Jacob, A. Schmitt, D. Michel, T. Mertens, H. Döhner, D. Bunjes, M. Schmitt* University Clinic of Ulm (Ulm, DE) Objective: BK polyoma virus (BKV) can cause hemorrhagic cystitis (BKV-HC) in patients after allogeneic stem cell transplantation (allo-SCT). Levofloxacin can hamper the proliferation of BKV in patients after kidney transplantation even resulting in less rejections of transplants. We therefore wondered whether the prophylactic use of levofloxacin might also reduce the incidence of BKV-HC in patients after allo-SCT. Methods: 148 patients undergoing SCT at our institution were included in this study. 46 patients received a BKV-HC prophylaxis with 500 mg/d levofloxacin administered orally till d+50 while 102 patients did not. Patients developing clinical symptoms of cystitis were screened for BKV in the urine by PCR. Results: 31/102 patients from the non-prophylaxis group tested positive for BKV after developing clinical symptoms of cystitis. The severity of BKV-HC was classified clinically as follows: grade I (no microhematuria) = 10, grade II (micro-, but no macrohematuria) = 11, grade III (macrohematuria) = 9, and grade IV (intravesicular blood clots) = 1. Patients with BKV-HC grade I were treated symptomatically by oxybutynin. Grade II patients received levofloxacin alone, grade III patients additionally leflunomide. The patient with grade IV BKV-HC received in addition bladder instillations with prostaglandin. BKV-HC was not associated with higher mortality (p=0.77). Differences according to the conditioning regime could be observed: 29 % of patients receiving a radio immune therapy (RIT) developed BKV-HC, vs. 26 % of patients after reduced intensity conditioning (RIC), vs. 19 % of patients after standard or FLAMSA regimen, vs. 7 % of patients after mini-RIT. In contrast, six of 46 patients with levofloxacin tested positive for BKV in the urine. These patients showed mild or moderate clinical symptoms with microhematuria. Oxybutinin and leflunomide were added and the patients recovered. As for the conditioning, BKV-HC occurred in two patients each after RIC, FLAMSA, standard regimen. Of note, the difference between the prophylactic group with a BKV-HC incidence of 17% vs. 30% in the non-prophylaxis group tested significant (p<0.05). Summary: Incidence of BKV-HC depends on the type of conditioning, and can be reduced by levofloxacin prophylaxis. Bloodstream infection after reduced-intensity umbilical cord blood transplantation versus other reduced-intensity allogeneic haematopoietic stem cell transplantation S. Takagi*, K. Ishiwata, H. Araoka, M. Tsuji, H. Yamamoto, D. Kato, Y. Matsuhashi, E. Kusumi, S. Seo, N. Matsuno, T. Matsumura, N. Uchida, K. Masuoka, A. Wake, S. Miyakoshi, S. Makino, M. Matsuzaki, A. Yoneyama, S. Taniguchi Toranomon hospital (Tokyo, JP) Blood stream infection (BSI) is a major cause of transplantrelated mortality (TRM) following allogeneic hematopoietic stem cell transplantation, and to overcome it, reducedintensity preparative regimens were developed in recent years. However, little information has been reported on BSI after reduced-intensity cord blood transplantation (RI-CBT). To clarify the characteristics of BSI after RI-CBT, we compared the incidence of microbiologically documented BSI before day 100 between RI-CBT and reduced-intensity noncord blood allogeneic hematopoietic stem cell transplant (RInon-CBT) recipients in Toranomon hospital, Japan. RI-non-CBT group includes related bone marrow (rBM), related peripheral blood stem cell (rPBSC) and unrelated bone marrow (uBM) transplantation. We retrospectively reviewed the first events of BSI in 211 consecutive adult patients between Jan 2004 and July 2006. One hundred and fifteen patients received RI-CBT and 96 patients received RI-non-CBT (4 from rBM, 34 from rPBSC and 58 from uBM). The median ages of patients in both groups were 55 years. All of the preparative regimens were fludarabine-based and prophylaxis against GVHD was tacrolimus alone in most of the RI-CBT recipients and combination of calcinurin inhibitor and short-term methotrexate in most of the RI-non-CBT recipients. The median time to achieve neutrophil engraftment was delayed in RI-CBT group (day 20 vs. day15). The cumulative incidence of engraftment at day 60 was 73.0 % in RI-CBT group versus 90.6 % in RI-non-CBT group. The cumulative incidence of BSI was 39.3 % at day 100 and RI-CBT group tended to have more BSI compared to p=0 .0122), particularly at the early points after transplantation. Median day of positive culture for bacteremia was earlier (day 9 vs. day 14) in RI-CBT group. In spite of reduced-intensity preparative regimen, RI-CBT in adults is associated with higher rates of BSI at early time points after transplantation. none of them developed invasive fungal infections while receiving the prophylactic treatment. Conclusions: These findings suggest that the dosage of cyclosporine should be reduced when posaconazole therapy is started and that plasma levels of the immunosuppressant should be monitored during and at the discontinuation of posaconazole therapy. Conditioning regimens, acute graft-versus host disease (aGvHD) prophylaxis and supportive policies were uniform for all patients. The incidence of engraftment was 94%. Grade III-IV aGvHD was observed in 5 cases. The mother's serum was tested for CMV at delivery and, in all cases, resulted IgM negative. Before transplant 26 pts (76%) were CMV seropositive. All blood samples were tested for CMV by both quantitative CMV-PCR (Cobas Amplicor CMV Monitor) in plasma and pp65-Ag at weekly intervals, from day -7 to day 100 post transplant Results: 16 out of 26 pts (61%) showed a CMV reactivation, none developed a CMV disease, no CMV related mortality was observed. In 13 pts (81%) CMV was detected by both methods, in the remaining 3 cases by PCR assay alone because of the low numbers of white blood cells. Pre-emptive therapy was started at the first detection of pp65-Ag positive cells and/or quantitative CMV-PCR. Fifteen patients were treated by pre-emptive-therapy, 11 with Foscarnet in an early post transplant period and 4 with Ganciclovir after engraftment Conclusions: We concluded that in paediatric population, plasma PCR and pp65-Ag assay were effective in detecting CMV infection after UCB-T. No significant discordance between both methods was observed. Plasma PCR and pp65-Ag are complementary for diagnosis and management of CMV infections. In a pre-engraftment period, Foscarnet is safe and effectiveness for pre-emptive therapy against CMV. Beneficial effects of intensive glucose control after allogeneic haematopoietic stem cell transplantation: a retrospective matched-cohort study S. Fuji*, S. Kim, T. Fukuda, S. Mori, S. Kamiya, K. Yoshimura, H. Yokoyama, S. Kurosawa, B. Saito, T. Takahashi, S. Kuwahara, Y. Heike, K. Tobinai, R. Tanosaki, Y. Takaue National Cancer Center Hospital (Tokyo, JP) Background: Van den Berghe et al. (NEJM 345:1359 ,2001 showed that the intensive glucose control (IGC) improved the outcome of patients treated in the ICU. We previously reported that hyperglycemia during neutropenia was associated with increased risks of acute GVHD and nonrelapse mortality (NRM) after a myeloablative allogeneic HSCT (Fuji S, et al. Transplantation 84:814,2007) . To evaluate the benefit of IGC in a HSCT setting, prospective study was conducted with this procedure to compare clinical outcomes with a matched-cohort. Only one patient in the IGC group developed severe hypoglycemia with faintness, but no patient developed seizure or loss of consciousness. There was no difference in the duration of neutropenia, but the incidence of documented infections and bacteremia was significantly lower in the IGC group, respectively, 14% vs 46% (HR 0.17, 95% CI 0.04-0.75, P=0.004, Figure 1 ) and 9% vs 39% (HR 0.10, 95%CI 0.01-0.74, P=0.002). This trial was not powered to demonstrate statistically significant changes, but we disclosed that there was a trend that IGC reduced the incidence of renal dysfunction (18% vs 38%, HR 0.59, 95%CI, P=0.36), increased inflammatory markers (19% vs 37%, HR 0.45, P=0.13 ) and grade II-IV acute GVHD (28% vs 37%, HR 1.05, 95% CI 0.38-2.91, P=0.93). The incidence of NRM within 100 days was low in both groups (1 vs 1 patient). Conclusions: This is the first prospective study performed in oncology field that suggested a beneficial effect of IGC, and this warrants a large prospective randomized study. Antibiotic prophylaxis in haemopoietic stem cells transplantation patients. Results from two consecutive cohorts study (2002-04 versus 2005-07) D. Serrano*, R. Carrion, G. Rodríguez-Macías, J. Gayoso, M. Kwon, P. Balsalobre, J. Anguita, A. Gómez-Pineda, I. Buño, J. Díez-Martin Hospital Gregorio Marañón (Madrid, ES) Introduction: Recently, two large controlled trials and a metaanalysis have showed evidence of benefits for prophylactic quinolone in patiens (pts) treated with chemotherapy, in allcause mortality, infection-related mortality, febrile episodes and bacteriemias. In order to analyze the impact of quinolone prophylaxis in the outcome of infectious complications in haemopoietic stem cells transplantation (HSCT) -pts, we have studied, in a retrospective way, two consecutive cohorts-pts: with vs without prophylaxis. Methods: 172 pts were considered evaluable for this study (febrile neutropenia, no active treatment with antibiotics). During the first period (July-02 til December-04, cohort 1) 88 pts were treated with ciprofloxacin from the first day of conditioning therapy til fever (temperature >37.8 ºC). Cohort 2 (January-05 til June-07) included 84 pts did not receive antibacterial prophylaxis. Both cohorts were comparable for the most relevant features: age, diagnoses (acute leukaemia, lymphoma and myeloma), kind of HSCT (allogeneic -sibling and no-related donor-and autologous) disease status pre-HSCT, time to myeloid engraftment and G-CSF treatment postransplantation. Results in cohort 1 compared to cohort 2: 82/88 pts (93.2 %) presented febrile neutropenia vs 82/84 (97.6%). The median time postransplant to fever were 5 vs 4 days. Bacteriemic episodes were diagnosed in 19/88 (22%) vs 28/84 (33%). Gram negative were isolated in 10/19 (53%) and 16/28 (57%) with Quinolone resistance in 9/10 (90%) vs 4/16 (25%) (p=0.001)respectively. Gram positive bacteria were isolated in 9/19 and 12/28. Septic shock was seen in 7/88 (8%) and 2/84 (2.4%) (p=0.1). Infection-related mortality preengraftment rates were 2/88 vs 0/84. In addition, mortality at 100-days rates were 10/88 (11.3%) and 9/84 (10.7%)due to: conditioning related toxicity 4 vs 3, tumor progression 1 vs 3, postrasplant linfoproliferative disease 1 vs 1, and postengrafment infections: bacteriemic 0 vs 1, fungal 0 vs 1, viral 1 vs 0, and toxoplasmosis infection 1 vs 0. None of these analyzed variables were statistically different. Conclusions: In our BMT Unit, quinolone prophylaxis was not associated with a reduction in neutropenic fever rate. We found a similar number of bacteriemic episodes, but more Gram negative organisms quinolone-resistants, were seen in prophylaxis group. Quinolone prophylaxis was not associated to a reduction in infection-related mortality preengrafment. In addition, overall mortality was not different between both cohorts. Introduction: Pseudomonas aeruginosa (PA), an ubiquitous aerobic gram-negative bacterium and opportunistic pathogen, rarely causes disease in healthy persons but is one of the leading gram-negative organisms associated with nosocomial infections. In particular, PA bacteremia is a serious and lifethreatening infection in the immunocompromised host. Reported mortality rates in adults range from 20% to 70%, depending on patient-and infection-related factors. The increasing frequency of multi-drug-resistant Pseudomonas aeruginosa (MDRPA) strains is concerning as effective antimicrobial options are limited. Since only few data are available on MDRPA in children, we started a multicenter survey in the AIEOP centres. Study design: the participating centres were asked to review all cases of PA bloodstream infections occurred in their patients with childhood cancer undergoing chemotherapy or HSCT between 2000 and 2007. Data on demographics, diagnosis, chemotherapy, as well as details on isolates and antibiotic in vitro sensitivity, treatment and outcome were collected. Results: 87 patients with PA septicaemia, documented by isolates from blood, were reported from 8 centres. Of them, 26 developed PA septicaemia during HSCT. Six (23%) isolates were MDRPA. Fatal outcome was observed in 4 of these 6 patients (66%), compared with only 2 of the remaining 20 non-MDR (10%). In one patient, antibiotic therapy was effective to resolve septicemia occurring during initial chemotherapy for AML, but unfortunately later on, during HSCT, infection by the same strain reactivated with fatal outcome. Conclusion: MDRPA, occurring in about one quarter of PA septicaemia, is associated with fatal outcome in the majority of cases. Patients with MDRPA should receive prompt and aggressive treatment. In survivors, following cancer-directed therapy should be tailored to avoid MDRPA reactivation under profound immune suppression, as induced by the conditioning regimen for HSCT. Adenovirus fulminant hepatitis following allogeneic stem-cell transplantation: report of two cases R. O. Vallansot, C. Martínez, R. Miquel, F. Fernández-Avilés, P. Abrisqueta, G. Gutiérrez-García, M. Rovira, E. Montserrat, E. Carreras Hospital Clinic (Barcelona, ES) Background: Adenovirus (ADV) infection occurs in 5-21% of allogeneic stem cell transplants (alloSCT). Whereas symptomatic enteritis and hemorrhagic cystitis are rarely fatal, mortality rates of up to 75% are reported for ADV pneumonia or hepatitis. Although hepatitis has been frequently described as part of a disseminated ADV infection, fulminant hepatic failure is extremely rare and usually fatal. We herein report two cases of such a complication. Case 1: A 46-year-old male with a 7 months history of NHL received an alloSCT conditioned with BU and CY from an identical sibling donor. CSA and MTX were used as GVHD prophylaxis. During the first year he developed several bacterial and viral infections that resolved with treatment. Thirty-two months after alloSCT he presented with fever unresponsive to antibiotics. At hospital admission he had moderately abnormal liver function tests with normal coagulation parameters. Afterwards, a profound fall of hepatic function with a prothrombin time of 14% and a raise in transaminases (AST 5940 IU/L; ALT 1830 IU/L) was observed. Acyclovir treatment was initiated but the patients developed a multiorgan failure and died 72 h. after his admission. A liver biopsy showed massive necrosis due to viral hepatitis. PCR study confirmed ADV infection. Case 2: A 51-year-old male with a 4-year history of CMML received an alloSCT conditioned with BU and CY from an identical sibling donor. MMF was used as GVHD prophylaxis. On day +71 he developed acute cutaneous GVHD that resolved with corticosteroids, and on day +108 gastrointestinal GVHD that required ATG. On day +205 he was admitted because of probable CNS fungal infection that improved upon treatment with antifungal agents and steroids. On day +258 he developed fever with cytolysis and mild cholestasis (AST 5800 IU/L;ALT 1840 IU/L;bilirrubin 5.5 mg/dL) with progressive hepatic failure. Foscarnet and high dose steroids were initiated because of a suspected hepatic GVHD vs. CMV hepatitis. In spite of this treatment, the patient developed clinical signs of intracranial hypertension with diffuse encephalic bleeding confirmed on CT-scan, dying 24 h. later. The necropsy showed viral hepatitis with extensive necrosis and nuclear inclusions consistent with adenovirus infection. Conclusion: ADV infection is an extremely infrequent complication with a very poor prognosis in alloSCT recipients and that should be taken into account in the differential diagnosis of hepatitis in such patients. Success of sequential therapy with amphotericin B (AMB) and posaconazole combined with hemimaxillectomy as treatment of rhinosinus zygomycosis in an allogeneic stem cell transplantation recipient E. Simeone* (1), F. Costa (2) , M. Robiony (2) A 46-year-old man was diagnosed with follicular lymphoma. The patient showed partial remission (PR) after 6 cycles of CHOP-rituximab, autologous stem cell transplantation, and radiotherapy. In July 2006, the patient relapsed with multiple pulmonary nodules and he was treated with Y90-Ibritumomab tiuxetan achieving a second PR. In October 2006, he underwent HSCT from a fully matched UD with a RIC regimen. Cya and MTX were administered as GVHD prophylaxis. He was isolated from day -7 before HSCT and received prophylactic anti-fungal treatment with Fluconazole iv 400mg/day. On day +6 he developed a blackened area of the left posterior maxillary mucosa, fever and local pain. On day +8, the blackened area had involved on both palatal and vestibular side. A facial CT scan showed thickness of the whole left maxillary sinus and of part of the right maxillary sinus without bone erosion. The biopsy showed infiltration of the mucosa by non septate hyphae that were morfologically compatible with Zygomycosis. Fluco was discontinued and 5 mg/kg/day Liposomal AmB was started. Fever and pain improved and a PMN count above 1x106/l was reached on day +11. The patient underwent a left hemimaxillectomy on day +21. The histological examination showed large areas of necrosis involving the bone, muscular, fat, and mucosal tissues and invasion by Mucor hyphae; the resection borders were infiltrated. When the patient was able to initiate oral feeding, AmB was withdrawn (total dose 10.5 g) and Posaconazole oral suspension was started 800 mg/day (for 5 months). At present the patient was in CR, without clinical signs of chronic GVHD and no evidence of fungal disease recurrence. This case is interesting because: a)The IFI occurred early post HSCT and pts was probably previously colonized; b)Posaconazole after AmB allowed a long-lasting and welltolerated therapy able to eradicate the infection. c)The new antimycotic drugs combined with surgery have improve the prognosis of these severe mycotic infections. Respiratory syncytial virus infections following allogeneic haematopoetic stem cell transplantation: palivizumab as a new promising approach for combination therapy with ribavirin and immunoglobulin? S. Fritsch, M. Bergmann, F. Mumm, G. Jäger, G. Ledderose, J. Tischer, H.J. Kolb Ludwig-Maximilians-University Munich (Munich, DE) Introduction: Respiratory syncytial virus (RSV) is a frequent cause of serious infectious complications following allogeneic bone marrow transplantation. Among these immunocompromised patients RSV infections often progress from upper respiratory iIlness (URI) to fatal interstitial pneumonia with high morbidity and mortality rates. Apart from treatment with aerosolized ribavirin and intravenous immunoglobulin there was up to now no promising approach to provide a better outcome for this patient group. Palivizumab is a neutralizing humanized monoclonal antibody against the epitope A of the RSV fusionprotein, which is essential for fusion of viral and cellular membranes. In animal models palivizumab has shown potential to reduce RSV load and replication as well as ability to decline acute disease severity by modulation of the immune response. Case report: A 46 year-old patient with high-risk acute lymphatic leukemia underwent allogeneic haematopoetic stem cell transplantation. Three days after transplantation the patient developed URI followed by infection of the lower repiratory tract six days later with initial pulmonary infiltrates in the CT scan and need of oxygen substitution, but no ventilar support. Microbiological analysis of nasopharyngeal secret and bronchoalveolar lavage (BAL) showed an infection of RSV, with positivity of antigen and PCR. The patient received an antiviral combination therapy with intravenous ribavirin for fourteen days, immunoglobulin several times and palivizumab 500mg on day 16 and 37 post transplantation. Results: Beginning on day 18 following transplantation, according to day 7 of antiviral therapy, the patient´s clinical condition improved together with engraftment on day 19. RSV-PCR and RSV-antigen became negative after first application of the combination therapy, but relapsed again. Finally by administration of a second course of palivizumab and ribavirin the RSV detection by PCR became and remained continuously negative for more than seven months until now. Conclusions: Palivizumab seems to be an effective inhibitor of RSV replication and virus load by interacting with the RSV fusionprotein of both RSV subtypes. Combination therapy with intravenous ribavirin and immunglobulin provides a hopeful strategy against RSV infections in immunocompromised patients even under progression to pneumonia. Further clinical trials will be needed to establish palivizumab as a therapeutical approach in RSV infected patients. Result: Five consecutive cases of adenoviral pneumonitis treated with antiviral agents were reviewed among 280 stem cell recipients (incidence 4.46). Three patients received antiviral agents for pneumonitis occurring at median 212 days (range 18-448) post hematopoietic stem cell transplant. Two patients treated with rivavirin as the first line therapy. Among those patients, rivavirin changed to cidofovir as second line therapy in one patient. Of the four patients receiving cidofovir, there was clinical improvement in three patients. The median duration of therapy with cidofovir was 56 days (range 30-98), with a median of 5 doses given (range [3] [4] [5] [6] [7] [8] . No case of dose-limiting nephrotoxicity was observed in association with cidofovir therapy. Furthermore, none of the other previously reported side effects attributable to cidofovir were observed. Conclusions: We concluded that cidofovir therapy may be useful for adenoviral pneumonitis in stem cell transplant recipients. However, large and controlled prospective trial is warranted to confirm the encouraging results in this report. Incidence of febrile episode during stem cell mobilisation after high-dose ciclophosphamide chemotherapy and G-CSF (filgrastim or lenograstim) administration in multiple myeloma patients: interim data E. Orciuolo* (1), G. Buda (1), K. Battiato (2) , E. Mauro (2) , D. Pastore (3) Introduction: Clinical use of G-CSF in pts with high grade chemotherapy (CT) induced neutropenia does not conduce to a reduction of the incidence of febrile episodes (FE). This paradox may be explained by the acquisition of a defective chemotaxis by neutrophils (PMN) exposed to filgrastim (Fil), due to a higher adhesivity and cytoscheletric alterations. Lenograstim (Leno), a glicosilated form of G-CSF, is able to stimulate PMN production, manteining in vitro all the functional capabilities. On these bases, we hypotized that Leno may prevent FE and reduce their lasting in pts with CT derived neutropenia. Patients and methods: starting from April 2005, 67 MM pts achieving HD-CTX for SCM were enrolled in 10 Centers. Treatment plan consisted in: HD-CTX (3-4 g/sqm) on day 1, G-CSF (random: Fil or Leno) 30 MU/day from day +4 to +9, 60 MU/day from day +10 to the achievement of an optimal CD34+ cell count for staminoapheresis. Random, 1:1, was effectuated on the base of a generated random list. FE, significant if equal or higher than 38 oC for at least 2 different determinations, were recorded till day +30. Primary endpoint is the incidence of FE; secondary endpoints are the duration of FE, efficacy in the CD34+ cell mobilization, time to mobilization. Results: 67 pts were enrolled. All pts underwent post-CT grade 4 neutropenia and G-CSF was administred starting from day +4. FE were recorded in 21 pts, 12 in the Fil arm (33 total pts) and 9 in the Leno arm (34 total pts). The global fever incidence was 31.3%, 36.4% with Fil and 26.5% with Leno, with a 9.9% difference. Average days with fever are 4.25 with Fil and 3.67 with Leno. Related to the neutropenia grade, 7 FE are recorded with Fil and 1 FE with Leno with absolute PMN count >500/µL (grade 4); 6 episodes with Fil vs 1 with Leno when PMN are >1000/µL (grade 3-4). CD34+ SCM occurs after an average time of 10.6 day with Fil and 10.0 day with Leno, with an higher absolute count with Leno when compared to Fil: 142.5 CD34+/µL (range 40-640) vs 124.5 (range 40-616) CD34+/µL. Conclusions: Leno is associated with a reduced incidence (9.9%) of FE in MM patients undergoing HD-CTX and SCM when compared to Fil. FE are recorded with Fil even in presence of PMN confirming the functional block by Fil on PMN documented in vitro. CD34+ mobilization occurs shorter and with higher efficiency with Leno when compared to Fil. On these evidences, patients' enrollment will continue to 180 to validate these data. Respiratory viral infections in adults after haematopoietic stem cell transplantation: a retrospective study in 133 patients V. Pérez Andreu, J.B. Nieto, A. Jerez, E. López, C. Castilla, F. de Arriba, M.J. Candela, I. Heras, V. Vicente* Hospital Morales Meseguer (Murcia, ES) Community respiratory viruses (CRVs) have been recognized as a potencial cause of pneumonia and death among hematopoietic stem cell transplantation (HSCT) recipients, and in patients with hematologic malignancies. Objectives: The aim of this study was to determine the prevalence, risk factors, therapy effects and clinical outcome of CRVs infection in HSCT recipients in our institution. Methods: We included 133 patients who underwent a HSCT from March 2005 to March 2007 and identified those who had respiratory specimens positive for CRVs. Patients with an episode of symptomatic upper respiratory tract had undergone a detailed clinical evaluation and were screened for CRVs with inmunofluorescense techniques to identify respiratory specimens positive for influenza A and B viruses, respiratory syncytial virus (RSV), parainfluenza virus and human adenovirus (ADV). Results: During this period 18 HSCT recipients with CRVs infection were identified (13.5%). Median age at diagnosis was 42 years (18-65). Primary diseases were acute myeloid leukemia (n=4), acute lymphoid leukaemia (n=3), chronic myeloid leukemia (n=1), non-Hodgkin lymphoma (n= 4), Hodgkin lymphoma (n=1), multiple myeloma (n=3), myelofibrosis (n=1) and prolymphocytic leukaemia T (n=1). Were identified 11 (61%) CRVs infections in allo-HSCT recipients and 7 in auto-HSCT recipients. The graft consisted in peripheral blood (n=14) and bone marrow (n=4). Seven patients had active GVHD and inmunosuppresive therapy at time of diagnosis. CRVs was diagnosed at a median of 1 year after HSCT. Most clinical signs were cough (n=13), fever (n=2), dyspnea (n=2) and pneumonitis in 1 patient ( with moderate respiratory distress, active GVHD, therapy with three inmunosuppresive drugs and bronquiolitis obliterans radiological findings). Influenza A infection was accounted in 14 patients, RSV in 3 and parainfluenza in 1 patient. Only three patients, received specific antiviral therapy, zanamivir (n=2) and oseltamivir (n=1). Clinical outcome was favorable in all cases. Conclusion: Community respiratory viruses infection is relatively common after HSCT and is associated with a high incidence of transplant-related morbility and mortality. Our observations confirm that nasopharyngeal culture is an easy diagnostic test with moderate rentability. In our retrospective evaluation, risk stratification is very important to decide what type of HSCT recipient is the most appropiate candidate to receive specific antiviral treatment. A unique case of PTLD after BMT for BCL-ABL positive ALL O. Moser* (1), W. Woessmann (1) , A. Reiter (1) , A. Schulz (2) , A. Hunold (1) , B. Goertz (2) , P. Bader (3) , H. Wagner (1) ( We report a unique presentation of an Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disease (PTLD) in a 13-year old boy who underwent two subsequent bone marrow transplantations (BMT) from two different donors for BCR-ABL positive acute lymphoblastic leukaemia (ALL) and relapse of the leukaemia, respectively. Case report: The boy was diagnosed of a BCR-ABL-positive ALL at the age of one year. After remission induction, he received a BMT from his HLA-identical brother. At the age of 10 years he suffered a first relapse of the BCR-ABL-positive ALL. He was treated with chemotherapy, imatinib, as well as donor lymphocyte infusions (DLI) and achieved 2nd remission, but developed chronic graft-versus-host disease (GvHD). The 2nd relapse of the ALL 2 years later responded to imatinib, but progressed within 6 months. Following remission induction, the patient received a 2nd BMT from an 8/10 matched unrelated female donor after TBI-based myeloablative conditioning. The BMT was complicated by cytomegalovirus disease leaving a poor graft function. 4 months after the 2nd BMT the patient presented with fever, painful swelling of tonsils, and marked eosinophilia. A biopsy proved an EBVinduced PTLD of the tonsils. The patient was treated successfully with 6 doses of rituximab, partial tumor resection, radiotherapy (20 Gy to both tonsils plus 10 Gy boost to the PTLD site) and DLI from the 2nd donor (infusions of 2 x 10 4 -4 x 10 6 CD3+ cells/kg body weight). PTLD tissues from the biopsy and the partial resection were analysed for the origin of both the B-and the T-cells of the lesion. Chimerism analysis performed with short tandem repeat systems revealed no autologous signals; approximately 50% of the cells were from the first and second donor, respectively. XY-fluorescence-insitu-hybridisation demonstrated a B cell PTLD originating from the first donor surrounded by T-cells from the second donor. The EBV load was excessively elevated at diagnosis of the PTLD and reflected the course of the disease. The boy is in complete remission of PTLD with undetectable EBV load in his peripheral blood for 18 months now This case is noteworthy and unique, because it demonstrated for the first time that EBV-infected B cells from a first donor can be the source of a PTLD after a second myeloablative BMT from a different donor. This B cell lymphoproliferation was under control by T cells from the second donor. The association of BK polyomavirus (BKV) infection and hemorrhagic cystitis (HC) after stem cell transplantation (HSCT)has increased with the use of potent immunosuppressive drugs. 40-50% of SCT patients with persistent BKV viruria remain free of HC. BKV viremia is shown to be strongly associated with the risk of developing HC, the suggested threshold being viral load >10 4 copies/ml. Our patient is a 16-year-old boy. His mother had defects in cell mediated immunity (e.g. multiple warts), and later myelodysplastic syndrome (MDS). His sister had died because of MDS, and brother because of generalized varicella. The patient had also defects in B-and T-cell cooperation and multiple warts. The definite diagnosis of the family is open (e.g. WHIM-syndrome was genetically excluded). MDS was diagnosed in December 2006, and progressed to AML. In CR1, PBSCT from an HLA-matched (15/16) URD was performed in June 2007. Conditioning was with busulphan, cyclophosphamide and melphalan. Prophylactic ATG, methotrexate, and cyclosporin-A (CyA), and standard inf.prophylaxis were used. Immunoglobulin infusions were given until day(D) +99. Engraftment occurred on D+13. On D+31 he developed hematuria (HU) with pain. BKV was detected in serum and urine. Previous serum and urine samples were retrospectively analyzed, and the follow-up samples were taken weekly. Nucleic acids were extracted using Nuclisense easyMag automated extractor. BK virus genomes were quantitated using duplex real-time PCR with universal polyoma virus primers for large T-antigen gene and fluorogenic hydrolysis probes specific for BK or JC virus sequences. Plasmids with cloned viruses were used as standards. The detection limit of the assay was 20 copies/ml urine and 50 copies/ml plasma. Viral load, symptoms, and treatment are shown in fig.1 Objectives: Most people have had a primary infection with human herpesvirus 6 by age 2-4 and virus is wide-spread in the population. HHV-6 has been also recognized as a potential significant pathogen in haemopoietic stem cell transplant (HSCT) recipients. Different clinical manifestations have been described including fever, skin rash and bone marrow suppression. Allogeneic SCT, advanced malignancy, younger age, gender mismatch between donor and recipient are commonly reported risk factors associated with HHV-6 infection after SCT. We have determined to measure prevalence of the HHV-6 DNA in a group of allogeneic SCT recipients hospitalized in the Hematology, Oncology and Internal Medicine Department of the Medical University of Warsaw from 2004 till 2006. Methods: A retrospective review of a group of 294 plasma samples taken from 26 adult recipients of allogeneic SCT was made. Collection of specimens for PCR investigations begun at a median of 3 days after transplantation (range, 1-7 days) and lasted until a median of 105 days (range, 30-180 days). HHV-6 DNA was tested by quantitative real-time PCR (ALPCO®) in plasma samples obtained once a week until +100 day after allogeneic SCT, thereafter once every 2 weeks. Results: Most of the recipients (73%) were immune to HHV-6 and the virus was not isolated from anyone at the time of SCT. HHV-6 DNA was detected in plasma samples in eight (30%) of the 26 recipients between day 18 and day 41 of transplantation. All of them developed fever of unknown origin, and 50% had aGvHD features. Three individuals from this group died during detectable HHV-6 viremia. Clinical features observed in recipients during active HHV-6 infection after SCT are presented in table 1. Conclusions: There is a high frequency of detectable HHV-6 viral load in SCT recipients in Poland. Limited sensitivity of serological methods along with the necessity of rapid introduction of antiviral treatment has forced the development of molecular diagnostics. Further investigations to monitor HHV-6 reactivation in graft recipients will be important to improve the outcome for these patients. Valganciclovir hydrochloride (VGC) is a pro-drug of ganciclovir, orally available, that is approved for CMV infection in high-risk solid organ transplants (1800 mg/day as standard dose). The suitable dosage of this drug in allogeneic stem cell transplant (SCT) has not been established. The aims of our study were the assessment of efficacy and safety of two dosages of VGC (1800 mg/day and 900 mg/day) as preemptive therapy of CMV disease after SCT. VGC was administered to 30 consecutive outpatients with a CMV infection which was diagnosed after a median of 86 days (59-480) from SCT; 18/30 pts underwent SCT from UD and 12/30 from RD; 17/30 (57%) received a RIC regimen. Antiviral prophylaxis consisted in acyclovir in all pts. At the onset of CMV infection 21/30 (70%) pts had an acute or chronic GVHD for which were received immunosuppressive therapy. The pp65Ag assay were positive in all cases with a mean number of positive nuclei of 22,5±43. The first 15 pts were treated with VGC 1800 mg/day (GROUP A) and the following 15 pts with VGC 900 mg/day (GROUP B); treatment was continued in all cases until the CMVAg became negative in two consecutive samples or until the treatment failure (increase of Ag in two consecutive samples). The two groups resulted well balanced. Overall 27/30 cases obtained a clearance of CMVAg (14/15 in the GROUP A and 13/15 in the GROUP B). The median duration of VGC therapy was 18 (8-64) and 22 (8-61) days, respectively. No cases of CMV disease were reported. Fifteen pts experienced one or more recurrence of CMV infection without significant differences in the two groups (8/15 vs 7/15) and seven cases required intravenous therapy with foscavir. Only three pts developed a mild deterioration of renal function that required dose adjustment (VGC 450 mg/day in 1/3 and VGC 900 mg/day in 2/3); mild and transient neutropenia was reported in 12/30 (40%) cases, transient anemia in 9/30 (30%) and transient thrombocytopenia in 12/30 (40%) pts without significant differences between two groups. In conclusion: preemptive therapy with VGC after SCT seems to be safe and effective with a rapid clearance of CMVAg; the optimal dose and duration of VGC therapy, in this setting, need to be established but in our experience the low dose of VGC (900 mg/day) has comparable efficacy than the higher dose (1800 mg/day) supporting the low dose use; regular blood counts and creatinine should be performed in all cases to early detect cytopenia or renal toxicity Objective: Retrospective evaluation of IFI incidence, risk factors and timing in our pediatric population. Patients and methods: Between October 1991 and November 2006, 110 allogeneic HSCT were performed in 103 children. Mean age was 6,4 years. Indication for HSCT was high risk hematological diseases in 68 (61.8%), 28 (25.4%) storage diseases and immunodeficiencies, severe aplastic anemia in 9 , Fanconi Anemia in 4 and autoimmune disorders in 2. The donor was a matched unrelated in 54 HSCT (49%), an identical sibling in 41 cases (37.2%), haploidentical grafts were use in 12 HSCT and 3 pts received a graft from a familial phenoidentical donor. Six pts were transplanted twice and 1 pt 3 times. We also made comparative analysis of the HSCTs done between October 1991 and December 1999 and the HSCTs performed between January 2000 and November 2006. Gut decontamination with, oral amphotericin B, was prescribed in all patients. All pts received antimycotica if they had persistent febrile neutropenia despite broad spectrum antibiotics, after work up with CT-thorax. IFI was defined according the EORTC/IFICG criteria in possible, probable en proven . Results: The incidence of IFI was 10.9% (12 transplants), 2 pts suffered already from proven invasive Aspergillus infection before HSCT. One pt developed 2 different IFI at different time points). There were 4 possible, 4 probable and 5 proven IFIs. The timing of IFI diagnosis had a median of 93 days (range 3-1700). The IFI were seen in 3/12 cases after chemotherapy due to relapse, in 5 out of 12 was in pts under immunossupression due to GvHD > grade II and in 2 cases were 2nd HSCT. The transplant related mortality in this group was higher than in the group without IFI (p= <0.0001). Between the period of 1991-1999 and 2000-2006, there was not statistical difference in incidence of IFI. Conclusion: Gut decontamination with oral amphotericin B seems to be effective in our population and the incidence of IFI remains stable over the time. IFI risk factors are reinduction chemotherapy, 2nd HSCT and immunosuppressive therapy for GvHD. Adequate fungal prophylaxis should be use in this high risk group of patients. CMV retinitis is a frequent complication of immunocompromised patients, especially in AIDS patients whilst it has been scarcely reported in patients who underwent haematopoietic stem cell transplant (HSCT). The largest study on CMV retinitis after HSCT showed an incidence of 1.4%. We report a 14-year old boy diagnosed with a T-cell acute lymphoblastic leukaemia (T-ALL) in 2005, who underwent PB-HSCT from his HLA-haploidentical father for early relapse. R/D CMV serostatus was pos/pos. The patient received a myeloablative conditioning regimen with TBI, VP16 and ATG. Pre-engraftment toxicity was limited to WHO grade III mucositis. PMN engraftment was at day +13; PLT engraftment was at day +14. Frequent CMV reactivations (by blood antigenemia or CMV-DNA) were diagnosed starting from day + 20. This required multiple course of pre-emptive treatment with foscarnet, ganciclovir or cidofovir until day + 239. Moreover, 2 infusions of CMV-specific CTLs were performed on day +88 and +117. On day +251, 12 days after last therapy with foscarnet, he complained a reduction of visual acuity. Ophtalmologic investigations and fine needle cytology on right eye anterior chamber were consistent with the diagnosis of CMV retinitis. Therapy with foscarnet and then cidofovir was resumed and a complete regression was documented after 4 weeks. The antiviral treatment was continued for other 7 months. A second relapse of CMV retinitis occurred 3 months later in the same eye. He was successfully treated with foscarnet followed by valgancyclovir resulting in a complete regression of active lesions and a returning to an almost normal visual acuity. We conclude that CMV retinitis is a late complication of HSCT and requires a prolonged antiviral treatment. Moreover, local CMV reactivation may occur despite a satisfying post-HSCT immune recovery. We suggest that ocular screening is part of routine monitoring for late complication in pediatric patients after haploidentical PB-HSCT. Background: Patients surviving an invasive fungal infection (IFI) in need of further cytoreductive chemotherapy are at high risk for recurrent fungal infection. In the absence of randomized controlled trials on this issue, applied prophylactic regimens are diverse and rely primarily on the personal experience of the attending physician. Methods: From 670 patients registered with the Multinational Case Registry of Secondary Antifungal Prophylaxis we evaluated patients who had received secondary antifungal prophylaxis with caspofungin or itraconazole. All patients had a diagnosis of haematological malignancies and were diagnosed with an earlier episode of proven or probable IFI according to EORTC/MSG criteria. Data collected comprise demographics, underlying disease, first episode of IFI, antifungal prophylaxis, incidence and outcome of breakthrough IFI, and survival. Results: A total of 75 patients were evaluated, 47 receiving itraconazole and 28 receiving caspofungin. Patient characteristic at beginning of secondary prophylaxis was similar in both groups. There were more patients with hepatic (0 % and 25 %, P= 0.001) or splenic (0 % and 14 %, P= 0.017) involvement of 1st IFI, with uncontrolled 1st IFI (0 % and 10.7 %, P= 0.049), or uncontrolled malignancy (8.5 % and 32.1 %, P=0.012) in the caspofungin group. Stem cell transplantation was more prevalent in patients of the caspofungin group (21.3 % vs 50 %, p=0.01). There was no significant difference in the occurrence of breakthrough IFI between both groups (17.0 % and 28.6 %, P=0.186). Treatment outcomes for recurrent IFI, underlying disease, and overall mortality did not differ between both groups. Conclusions: Both itraconazole and caspofungin performed equally efficacious as secondary antifungal prophylaxis in high-risk patients. While there was a trend towards higher risk of recurrent IFI and overall mortality in the caspofungin group, these patients were significantly sicker at start of prophylaxis. In spite of antifungal prophylaxis, risk of breakthrough IFI was high. Prospective controlled trials are warranted to define the best strategy of secondary antifungal prophylaxis. Subclinical Epstein-Barr virus (EBV) reactivation by Q-PCR commonly developes after allogeneic stem cell transplantation (allo-SCT). Preemptive Rituximab has been proposed to prevent development of EBV-related post-transplantation lymphoproliferative disease (PTLD). From October 2002 85 patients (pts) underwent allo-SCT in our centre. We found EBV reactivation in 32 pts (M/F 23/9, aged 10-64 years). 25/32 pts were submitted to myeloablative peripheral blood (PB) SCT from siblings in 17 cases, matched unrelated donor (MUD) in 7 cases, cord blood (CB) in 1 case; 7/32 pts received a reduced intensity conditioning (RIC) PB-SCT from sibling in 4 cases and MUD in 3 cases. 11/32 pts (34.4%) received ATG in conditioning regimen, while of the remaining 53 pts who did not reactivate EBV only 12 received ATG (22.6%). Thirty out of 32 pts showed only PCR +, with a maximum of 106080 copies/ml, but no evidence of PTLD; 12/30 pts presented hypergammaglobulinemia, with a IgG monoclonal component in 7/12 cases, but 13/30 presented a peak in gamma-globulines region. 6/30 pts achieved spontaneous regression of viral load; 11/30 received preemptive rituximab for a viral load higher than 1000 copies/ml (375 mg/m²/week for 1-4 doses): 9/11 obtained regression of viral load, but 1 presented a new reactivation. 2 pts died before EBV reevaluation. Two out of 32 pts developed PTLD. 1 pt presented EBV reactivation at day +15 post MUD-SCT, and subsequently PTLD of rhynopharinx at day +37, without monoclonal component but with hypergammaglobulinemia and gamma-peak; she received 5 doses of rituximab, without achieving clinical nor laboratoristic remission. The pt died because of PTLD two months after S286 SCT. One pt presented PTLD on a bowel biopsy at 11 months from CB-SCT, with a negative viral load nor hypergammaglobulinemia, but with IgG kappa monoclonal component. He was treated with 4 doses of rituximab, achieving complete remission. The pt is actually alive and in complete remission at 21th month from SCT. EBV reactivation is documented after both T-cell depleted and unmanipulated allo-SCT. In our experience, hypergammaglobulinemia and/or monoclonal component and/or gamma-peak are not diagnostic but frequently associated to EBV reactivation or PTLD; moreover, treated pts obtained viral load and hypergammaglobulinemia regression, but not resolution of monoclonal component and gamma-peak. It could be useful and unexpensive to associate monthly electrophoretic monitoring to Q-PCR for early detection of EBV reactivation or PTLD development. Use of ciprofloxacin to prevent bacterial infection in patients with AML and patients receiving high-dose chemotherapy and autologous stem cell transplants fot NHL, Hodgkin's disease and solid tumours H. Menzel*, T. Szislo, M. Neuenhahn, C. Peschel, J. Duyster Klinikum rechts der Isar (Munich, DE) Background: Antimicrobial prophylaxis with ciprofloxacin (cipro) is used for patients (pat.) at high risk for neutropenic fever after chemotherapy. 2005 Bucaneve et al. published data about the use of levofloxacin (levo) vs. placebo to prevent bacterial infection in cancer pat. with neutropenia, concluding that levo is effective in preventing febrile episodes, lowers the rate of microbiologically documented infections (MDI) and bacteraemia especially with gram-negative germs. To reassess these data with the standard use of cipro we evaluated pat. with AML and with solid tumours, lymphoma and Hodgkins Disease receiving HD-CTx and auto-SCT. Methods: We retrospectively analyzed 44 AML and 56 HD-CTx pat. which were at risk for prolonged neutropenia due to intensive chemo. One cycle per pat. was analyzed. Pat. with allo-SCT, fever, antimicrobial therapy or infections were excluded. All pat. received 500 mg cipro orally TID until they developed fever or neutropenia resolved. All pat. had a central venous line (CVL). Using the IDSA-guidelines we analyzed the reason of fever and classified pat. with fever of unknown origin (FUO), clinically (CDI) or microbial documented infections (MDI). Results: In the AML group (mean age 57y.) the mean duration of leukopenia <1G/l was 16d. 41/44 pat. (93%) developed fever. Of these 24% were FUO, 17% CDI and 59% MDI. 9/24 MDI were CVL infections and 10/24 MDI were bacteraemia. In the group with HD-CTx (mean age 44y) mean duration of leukopenia <1G/l was 8d. 42/56 (75%) developed fever. 60% FUO, 10% CDI and 31% MDI. 3/13 MDI were CVL-infections and 2/13 MDI bacteraemia. 1 AML-pat. died of gram+ sepsis. 1 pat. had gram-bacteraemia, 23/100 had gram+ bacteraemia. Mean duration of cipro application was 13d. Conclusions: The low mortality rate of 1% and the low incidence of gram-bacteraemia of 1% with the use of cipro is not inferior to the published levo-data (Mortality-rate: 3%, gram-bacteraemia: 6%). AML-pat. had an incidence of neutropenic fever of 93% and HD-CTx pat. 75%. This reflects the different duration of neutropenia in the two groups. Compared with the levo-data (incidence of neutropenic fever of 67% in AML, 62% in HD-CTx-pat.) our results differ unfavourable. The incidence of gram+ bacteraemia and CVLinfections was higher with cipro, presumably reflecting the higher efficacy of levo to gram+ germs. Optimisation of catheter management may improve the outcome with regard to gram+ bacteraemia and CVL-infections. Immunosuppression, including that which occurs following bone marrow transplantation (BMT), potentially results in the reactivation of infections that are otherwise latent or controlled by effective immune surveillance. Although development of surface and core antibodies and loss of surface antigen following acute hepatitis B virus (HBV) infection is thought to represent clearance of the virus, evidence exists to support the possibility that the virus may remain latent within the liver. From May 2001 to December 2007 we have auto transplanted in our division 129 patients (35 AML; 4 ALL; 3 CLL; 13 HD; 34 NHL; 39 MM and 1 TTP) they have been conditioned with: BEAM: 57; BuCy2: 5; BuMel: 21; MEL200: 38; others conditioning: 8 patients. In all patients we have effected the tests serologic for the hepatitis B before the transplantation and they are results: HBsAg+: 5 (4%); HBsAg-/HBsAb+/HBcAb+: 39 (30%); HBsAg-/HBsAb-/HBcAb+: 9 (7%); HBsAg-/HBsAb+/HBcAb -: 56 (43%) and HBsAg-/HBsAb-/HBcAb -: 20 (16%) patients. In total in our cohort we have 48 patients (37%) with precedent infection and apparent clearance of HBV. In all the 48 patients the HBV-Dna test has been effected and they are only 2 patients results positive for low replication. After a median of observation of 24 months after transplantation (range 3-54 months) 6 patients (12.5%) (1 AML; 1 CLL; 1 LNH-T and 3 MM) have developed infection B it activates and they have reverse seroconversion (HBsAg+/HBV-DNA with high replication of virus). 2 patients have developed an acute hepatitis and 3 a reactivation subclinic. In 4/6 patients has been initiated treatment with lamivudine and 1 patient (has not been treated with lamivudine) it is dead at month +4 after transplantation for the hepatitis. The risk of reactivation of HBV infection is important when considering BMT and the associated immunosuppression. The potential for poor outcomes (including chronic active hepatitis and fulminant hepatitis) following reactivation of HBV infection should be noted. Preemptive treatment with hepatitis B specific antiviral therapy may potentially have a role, but this remains to be studied. In addition, monitoring of hepatitis B serological test results and/or HBV DNA levels, as well as clinical evidence of reactivation, may allow for the early detection (and therefore treatment) of this potentially very serious complication. Kaposi's sarcoma (KS) is a rare complication in recipients after allogeneic stem cell transplantation (SCT) and is associated with compromised immune system. Whereas skin disease only has a good prognosis, patients with systemic disease usually die from KS. We report the history of a 17 years old African boy born in Mosambique diagnosed for very severe aplastic anaemia. An HLA identical donor could not be identified, so the patient received immunosuppressive treatment for 4 years. As his general conditions decreased a decision was made to use CD3/CD19 depleted peripheral stem cells from an only 4/10 HLA-mismatched unrelated donor according to 4 digit high resolution typing. The patient was prepared with reduced conditioning Flu(200)-Mel(140)-THIO(10)-Okt3(10days) and showed prompt engraftment of neutrophils and platelets at days 13 and 7, respectively. He was complete chimera in all of his cell subpopulations and showed also early and rapid T-cell engraftment with more than 100/µl CD3 cells at days +32. Post transplant course was complicated by unusual repeated periods of septicaemia. At day +240 after SCT he presented with a red-bluish solid indolent nodular lesion on his left thigh and lower leg and enlarged axillary, inguinal and paraaortal lymph nodes. Biopsy of skin lesions and lymph masses revealed a typical histology pattern of KS. HHV-8 was detected by PCR in biopsy specimens. Treatment was started with cidofovir, ribavirin and alpha-interferon (1.8 Mio/d 5 x week), supplemented by IL-2 (200.000 UI/d) applied subcutaneously. He showed an initial good response of the lymph nodes, but mixed response to skin lesions. To further improve immune competence he received 1x106 CD3+ cells/kg DLI at day + 370. Disease progressed and at day + 386 lung metastasis was detected by CT and proven by biopsy. Around day +410 continues reduction of skin lesions as well as the pulmonary infiltrations was noticed. Analysis of T-cell repertoire showed a steadily increasing diversification of T-cell clones. In the following, all skin lesions including the visceral metastasis disappeared. The patient is doing well and back into normal live 680 days post transplant. Cluster of acyclovir-resistant herpes simplex virus reactivations after allogeneic bone marrow transplantation H. Wagner*, A. Hunold, O. Moser, R. Bluetters, A. Reiter, W. Woessmann Pediatric Hematolgy and Oncology (Giessen, DE) Herpes simplex virus (HSV) reactivations can cause serious problems in immunocompromised patients after bone marrow transplantation (BMT). Resistance to acyclovir (ACV) due to mutations in the thymidine kinase gene or less frequently in the DNA polymerase gene of HSV is a growing concern. We report on four children with severe mucocutaneous herpes simplex virus (HSV) infections despite of an otherwise effective ACV prophylaxis (3 x 250 mg/qm starting on day +1) after BMT. Patient 1: An 18-year-old girl presented with a therapyresistant relapse of an acute lymphoblastic leukaemia (ALL). Remission of ALL was achieved with an AML-type chemotherapy regimen (Dauno-Fla: fludarabine, high-dose cytarabine, liposomal daunorubicin). She received an allogeneic BMT from a matched unrelated donor (MUD) after conditioning with total-body irradiation (TBI), VP 16 and ATG. GvHD-prophylaxes included short-course MTX and cyclosporine (CsA). She developed HSV-induced glossitis on day +17. Patient 2: A 16-year-old girl received an allogeneic BMT from a MUD for refractory cytopenia with partial trisomy 8. The reduced intensity conditioning regimen included thiotepa, fludarabin and ATG as well as GvHD-prophylaxes with shortcourse MTX and CsA. She developed a severe oral HSV infection on day +16. Patient 3: A 16-year-old boy received an allogeneic BMT from a matched sibling donor (MSD) for therapy-refractory acute myeloid leukaemia (AML-M1). Remission of AML was achieved with AML-relapse chemotherapy courses Dauno-Fla and FLASMA (fludarabine, high-dose cytarabine, amsacrine), followed four days later by reduced intensity conditioning (RIC: TBI (4 Gy), cyclophospamide and ATG). He also developed severe oral HSV-infection day on day + 7. Patient 4: A 5-year-old boy had an early relapse of an AML-M2. Remission of AML was achieved with chemotherapy regimens Dauno-Fla and FLASMA. He received RICconditioning and a MSD-MMT four days after FLAMSA. He developed oral HSV-infection on day + 14. In all patients, the HSV infections caused a high degree of morbidity requiring prolonged courses of systemic foscarnet and local cidofovir therapy. Of note, all four patients received fludarabine for induction of remission of their leukaemia (3 patients) or for reduced conditioning (1 patient). If the potent immunosuppression supplied by fludarabine can act as a cofactor for predisposing acyclovir-resistant HSV reactivations after allogeneic BMT remains to be shown in larger trials. Chronic myelogenous leukemia (CML) is rare in children. Although the long-term benefit of imatinib has already been determined in adults, sufficient data are not available for pediatric patients. Allogeneic hematopoietic stem cell transplantation (HSCT) is still the only curative treatment in the imatinib era, but there is limited data on its use for children. Here, we report the results of the recent 11 years of HSCT for pediatric CML patients in Japan. We retrospectively analyzed 94 patients diagnosed with childhood CML from 1996 to 2006 who underwent HSCT in Japan. Among the patients, 47 were males and 47 were females; the median age at first diagnosis was 10 years (range: 11 months to 17 years) and the median interval between diagnosis and transplant was 9 months (range: 1 to 65 months). All the patients except one received pretransplant therapy and of these, 36 received imatinib (with or without other drugs). At the time of conditioning, 64 patients were in first chronic phase (CP), 2 were in accelerated phase (AP), 7 were in blastic crisis (BC) and 21 were in second or higher CP. The stem cell sources were BM (n = 78), PBSC (n = 10) and cord blood (n = 6). Donors were HLA-identical sibling (n = 31), matched unrelated (n = 37), mismatched related (n = 14) and mismatched unrelated (n = 12). Thirteen patients received a reduced intensity conditioning regimen. Eighty-nine patients achieved engraftment. Sixty-eight patients had acute GVHD ( ‡T: 30, ‡U: 24, ‡V: 6 and ‡W: 8), and 37 had chronic GVHD (16 limited and 21 extensive). Ten patients relapsed (4 BC and 6 CP) and 23 patients died (21 from TRM causes and 2 from disease progression). Five-year overall survival (OS) rates were 83% in first CP at the time of HSCT, 45% in AP or BC and 75% in higher CP. The OS rates of the patients in CP undergoing transplantation from HLAidentical sibling were better than those undergoing transplantation from other donors (96% vs 76%; P = 0.02). Influence of imatinib as pre-transplant therapy was not significant (OS 83% with imatinib, 75% without imatinib). The OS rate of patients who received the reduced intensity conditioning regimen and myeloablative regimen were 92% and 77%, respectively. The results obtained in this study were better than those in previous studies and sufficiently encouraging, especially for children in the CP. We should investigate these results more specifically and compare long-term benefits of HSCT with continuing imatinib without HSCT. Use of clofarabine for stem cell transplantation in paediatric patients with refractory disease P. Lang*, I. Mueller, H.M. Teltschik, T. Feuchtinger, M. Pfeiffer, M. Schumm, R. Handgretinger University Children's Hospital (Tubingen, DE) We report the results of a clofarabine containing conditioning regimen for allogeneic stem cell transplantation. Several studies have shown the efficacy of clofarabine in resistant leukemias. Thus, we replaced fludarabine in a melphalan based regimen by clofarabine for pediatric patients with refractory disease after standard treatment (n=7) or after previous transplantation (n=3). 10 patients with ALL (NR 1=3, NR 2=3), AML (NR 1=2) and metastatic rhabdomyosarcoma/neuroblastoma (PR =2) received clofarabine 4x50mg/m² (day -8 to -5), thiotepa 10mg/kg (day -4), melphalan 2x70mg/m² (day -3 to -2) and OKT3 (0,1mg/kg day -8 to +14; n=9) or ATG (10mg/kg; n=1), followed by infusion of haploidentical stem cells (n= 9) or bone marrow from MUD (n=1). In MMFD, depletion of T and B cells was carried out with CD3/CD19 coated magnetic microbeads. Primary engraftment was observed in 9/10 patients. One patient experienced graft rejection but was successfully retransplanted with stem cells from a second parental donor. Thus, sustained engraftment could be obtained in all patients. Median time to ANC >500/ l with G-CSF stimulation was 10 days (9-11). Independence from platelet transfusion was reached after 9 days. Median T cell count at day 100 was 138 cells/µl (n=5). 6/10 patients are disease free with a median follow up of 0.6 years (0. 3-1.3) . Single cause of death was relapse (median time to relapse: 90 days (70-245)). The regimen was well tolerated. No TRM and no toxicity grade 4 according to NCI-CTC grading system occurred apart from mucositis. The profile of toxic side effects was: gastrointestinal: diarrhea grade 0-2 (n=10), stomatitis grade 3-4 (n=10); skin: none; pulmonary: hypoxia and pneumonitis grade 3 in 2 and 1 patient, respectively; cardiac: none; hepatic: GOT/GPT or bilirubin elevation grade 1-2 in 4 patients, grade 3 in 6 patients; renal: creatinine (-clearance) grade 1 in 1 patient; neurological: none; infection: grade 2 (n=8), grade 3 (n=2). Conclusions: clofarabine was well tolerated as part of a conditioning regimen. Compared to our standard regimen with fludarabine, thiotepa, melphalan, the introduction of clofarabine did not result in any unexpected toxicity. Immunosuppressive effects appeared to be similar to those of fludarabine, since primary engraftment was observed in 90%. Further studies are warranted to evaluate, whether clofarabine can contribute to reduce the risk of relapse in patients with refractory disease. Low transplant related toxicity after haploidentical stem cell transplantation with reduced-intensity conditioning in children P. Lang *, I. Mueller, H. Teltschik, T. Feuchtinger, M. Pfeiffer, M. Ebinger, M. Schumm, R. Handgretinger University Children's Hospital (Tubingen, DE) Transplantation of haploidentical, positive selected stem cells in combination with TBI or Busulphan can result in significant mortality. New graft manipulation methods and a reduced intensity conditioning helped to minimize TRM. We present results of a direct depletion procedure using antiCD3/antiCD19 coated magnetic microbeads and melphalan (2x70mg/m²), fludarabine (4x40mg/m²), thiotepa (10mg/kg) and OKT3 (0.1mg/kg, day -8 to +14). All patients (n=27) underwent intensive pretreatment according to current study protocols; 11/27 already received previous autologous or allogeneic transplantations. Diagnoses: AML/MDS (n=9), ALL (n=5), relapsed neuroblastoma / Ewing-/ rhabdomyosarcoma (n=11), SAA (n=2); remission status: CR2 =6, CR3 =3, NR/PR =16. Primary engraftment occurred in 85%. After reconditioning and second stem cell donation, final engraftment was achieved in 100%. Median time to reach >500 neutrophiles/µl and independence from platelet substitution was 10 (9-12) and 8 (6-188) days respectively. GvHD grade 0-1 occurred in 63%. 30% and 7% had GvHD grade II and III, respectively. TRM occurred in 4% (follow up:0.8 years (3 months -3.0 years)). The regimen was well tolerated without severe side effects. The profile of toxic side effects according to NCI-CTC grading system was: gastrointestinal: diarrhea grade 0-2 (n=25), grade 3 (n=2); stomatits grade 3-4 (n=27); skin: grade 0-2 (n=27); pulmonary: hypoxia grade 0 (n=24), grade 3 (n=3); pneumonitis (n=1); cardiac: lethal myocardiopathy (n=1), none (n=26); hepatic: GOT/GPT or bilirubin elevation grade 0-2 (n=19), grade 3 (n=4), grade 4 (n=4), VOD: none; renal: creatinine (clearance) grade 0-2 (n=27); neurological: none; infection: grade 0-2 (n=17), grade 3 (n=7), grade 4 (n=3), EBV-LPD: none. Overall survival at 1 year was 47%. Causes of death were myocardiopathy (n=1) and relapse (n=13). Pretransplant disease status was predictive: 1 year EFS in patients with leukemias in CR and SAA was 83%, whereas all patients with active disease relapsed. Conclusions: the regimen helped to minimize TRM, despite intensive pretreatment of the patients. Fast recoveries of neutrophiles /platelets were achieved as well as engraftment rates similar to that of patients with myeloablative conditioning and positive selected stem cells. Relapse remained an unsolved problem in patients with active disease. Thus, further therapeutic elements have to be evaluated. Profound depletion of T and B cells is a fundamental prerequisite for haploidentical transplantation and allows to minimize GvHD despite HLA incompatibility. However, posttransplant recovery of donor derived T cells is delayed after various graft manipulation procedures and may result in severe infections. Methods to improve this recovery are of great importance. Here we present immune reconstitution data in patients who received CD3/CD19 depleted stem cells in combination with melphalan based or standard conditioning regimens. 32 patients with ALL (n=14), AML/MDS (n=17), CML n=1) were included. T-and B-cells were directly depleted using antiCD3/antiCD19 coated magnetic microbeads and the CliniMACS device. The patients received either TBI or Bu i.v. (n=9) or an reduced intensity conditioning ("RIC": Mel 140mg/m², Flud 160mg/m², TT 10mg/kg, OKT3, n=23). Absolute numbers of lymphocyte subsets per microliter on day 90 were compared within these both groups and with a historical control group (patients with leukemias who received CD34 selected grafts and TBI or Bu based standard conditioning regimen, n=28). CD3+4+, CD3+8+ and total numbers of CD3+ of patients after CD3/CD19 depletion were significantly higher in the RICgroup than in the TBI/Bu-group (mean numbers: 85.83 vs. 38.84; 133.46 vs. 19.69; 270.27 vs. 63.99; p<0.05, unpaired ttest In the last ten years, the incidence of HVOD among pediatric patients treated with the busulfan -thiotepa (BU-TTP) conditioning regimen, appeared to increase, without change being brought to the protocol. Thus, the aim of the present study was to identify the risk factors of HVOD which can explain such a change during the last decade. Methods: a retrospective analysis of record cases of all pediatric patients having received their first high-dose chemotherapy by BU -TTP, followed by autologous haematopoietic stem cell transplantation (AHSCT), treated between May 1988 and December 2005 in the Pediatric Transplant Unit of Institut Gustave Roussy was carried out. One hundred sixteen patients fulfilled these inclusion criteria. The demographic, clinical, biological and therapeutic parameters have been evaluated in uni-and multivariate analyses. Results: according to McDonald's clinical criteria, HVOD was diagnosed in 31% of this population. As assumed, the incidence of HVOD had increased during these 17 years. Between 1988 and 1993 it was 24%, as opposed to 45.2% between 2000 and 2005. Uni-and multivariate analyses showed a significant correlation between the previous administration of carboplatin and the risk to develop HVOD post-transplant (p = 0.028). Similarly for etoposide (p = 0.048). Moreover, the risk increased with the cumulative dose of carboplatin (p = 0.019) and of etoposide (p = 0.031). A significant parallelism between the increase of the incidence of HVOD (p= 0,053) and the increase, during the last seventeen years, of the use of etoposide -carboplatin (VPCARBO) combination in previous conventional chemotherapy was demonstrated (p < 0,001). Finally, the analysis highlighted the correlation between HVOD and the risk of death post transplantation, due to its association with other organs' failures (p = 0.029). Conclusions: this study demonstrated that previous administration of VPCARBO, used in conventional chemotherapy, significantly increased the risk of HVOD among patients who later received consolidation by BU -TTP followed by AHSCT. This risk factor must be taken into account in the discussion of the strategy for the treatment of relapsed and high risk tumors. The BK virus loads in serum but not in urine correlate with symptoms of haemorrhagic cystitis in children after allogeneic stem cell transplantation M. Ussowicz*, E. Gorczynska, B. Rybka, R. Ryczan, I. Bil, K. Kalwak, J. Owoc-Lempach, A. Dyla, J. Musial, A. Chybicka Wroclaw Medical University (Wroclaw, PL) BK polyomavirus (BKV) is a prevalent pathogen causing haemorrhagic cystitis (HC) and nephropathy in immunocompromised recipients after hematopoietic stem cell transplantations. The hypothetic mechanism of HC requires tissue damage by conditioning regimen, uroepithelial BKV replication and alloimmunologic response against BKVinfected uroepithelial cells. Material and methods: 24 patients after allogeneic stem cell transplantations were prospectively studied. During 11 month observation five patients developed haemorrhagic cystitis. 114 urine and 89 serum samples were collected from patients with symptomatic haemorrhagic cystitis and during scheduled follow-up visits. DNA was isolated from blood with QIAgen DNA blood kits and from urine with QIAgen Viral RNA kits. The DNA was tested for BK virus load using the Q-BKV Ampliprobe (Amplimedical) real-time quantitative PCR assay. The assay allowed a specific quantification over a 10-log range. Results: The median BKV load in the analysed group was 22,5*10 6 genomic copies/mL in urine (ranged 0-96428*10 6 genomic copies/mL), and 140 genomic copies/mL in serum (ranged 0-7.25*10 6 genomic copies/mL). The BKV loads found in serum were 4 logs lower than in urine. In children with haemorrhagic cystitis the BKV loads in serum were significantly higher than in asymptomatic patients (5.98log(10) versus 2.9log(10), p=0,000038). There was no difference in the urine BKV loads between symptomatic and asymptomatic patients (9.39log(10) versus 9.29log(10), p=ns). On cidofovir treatment, the BKV loads decreased significantly but replication and urine excretion of the virus remained even after therapy. Conclusions: The RQ-PCR based approach to monitoring of HC showed that BKV load in serum correlates better with symptoms of the disease than urine virus loads. The role of BK viremia in pathogenesis of HC remains unclear and might be only a secondary phenomenon resulting from uroepithelial barrier damage. Lack of correlation between BKV urine loads and HC occurrence supports the hypothesis of multistep pathogenesis of the uroepithelial damage. Results: All patients but one in oral-Bu group reached hematopoietic engraftment in Auto-SCT while in Allo-SCT there were 10 engraftment failure (6 in oral-Bu and 4 in IV-Bu). In Auto-SCT 8 children (12.3%) with oral-Bu and 3 (7.7%) with IV-Bu developed hepatic venoocclusive disease (HVOD) (p=ns). There were no statistical differences when compared HVOD related mortality (2 children in oral-Bu and none in IV-Bu) or 100-day transplantation related mortality (6 patients in oral-Bu and 1 in IV-Bu). In Allo-SCT, 5 children (16.6%) who received oral-Bu and 3 (7.8%) who received IV-Bu developed HVOD (ns). HVOD related mortality was higher in oral-Bu group (4 patients, 13.3%) compared with IV-Bu group (1 patient, 2.6%) (p=ns). Transplantation related mortality was significantly higher in oral-Bu group (10 children, 33.3 %), compared with IV-Bu group (5 children, 12.8%) (p=0.04). Other non-hematological toxicities (neurological, metabolic and cutaneous) were higher in IV-Bu group mainly non-severe grades. Finally, in Auto-STC overall survival was higher in IV-Bu group compared with oral-Bu group (p=ns) and in Allo-SCT overall survival was significantly higher in IV-Bu compared with oral-Bu (p=0.05). Conclusions: In this study the incidence rate of HVOD and mortality related HVOD is lowered in children treated with IV-Bu as part of Auto and Allo-SCT conditioning regimens and consequently the 100-day survival rate significantly improved. Sequential high-dose chemotherapy with autologous stem cell rescue for children with high-risk medulloblastoma and supratentorial primitive neuroectodermal tumours C. Dufour*, V. Minard-Colin, J. Grill, E. Benhamou, G. Goma, O. Hartmann, C. Kalifa, D. Valteau-Couanet Institut Gustave Roussy (Villejuif, FR) Background: Prognosis of high-risk meduloblastoma (HR MB) and supratentorial primitive neuroectodermal brain tumours (sPNET) is poor. In an attempt to improve outcome, we have developed strategies based on sequential high-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) and radiotherapy for thirteen years. Patients and methods: Seventy-nine children (median age, 3.95 years) were treated with HDC/ASCT (59 HR MB; 20 sPNET). All patients received induction chemotherapy with one or two cycles of carboplatin-etoposide. Treatment strategy was modified with time and according to the age. Eighteen children were treated by two courses of high dose melphalan (100mg/m 2 ) and one course of busulfan thiotepa combination (regimen 1). Two myeloablative courses of melphalan (100mg/m 2 ) with ASCT were performed in twenty-one children (regimen 2). Nine patients were planned to receive two courses of high dose thiotepa (600mg/m²) with ASCT (regimen 3). Thirty-one children received five sequential courses of chemotherapy followed by ASCT : melphalan (100 mg/m 2 ) at day 43, cisplatin (100 mg/m 2 ) at day 64, melphalan at day 85, cisplatin at day 106 and thiotepa (720 mg/m2) at day 127 (regimen 4). Treatment was completed by ageadapted craniospinal irradiation plus local boost in HR MB and age-adapted local irradiation only in sPNET. Results: Two hundred forty two cycles of high-dose chemotherapy with stem cell rescue were administered. At the end of chemotherapy, 6 pts were in CR in regimen 1, 8 CR in regimen 2, 3 CR in regimen 3 and 14 CR in regimen 4. The visceral toxicity of the regimen 1 was unacceptable with 3 toxic deaths secondary to multiorgan failure and 7 hepatic veno-occlusive disease. The toxicity was manageable in regimen 2, 3 and 4. Conclusion: Sequential high-dose chemotherapy improves survival rates in patients with HR MB and sPNET. This approach can be used to alleviate the use of craniospinal radiotherapy particularly in young children. Sequential high-dose chemotherapy and reduced craniospinal irradiation in young children with metastatic medulloblastoma C. Dufour *, D. Couanet, D. Figarella-Branger, C. Carrie, F. Doz, D. Frappaz, J. Gentet, P. Leblond, P. Chastagner, A. Bertozzi, C. Sainte Rose, M. Raquin, A. Laplanche, J. Grill, C. Kalifa on behalf of the SFCE Brain Comitte Introduction: Prognosis of disseminated medulloblastomas (DMB) is poor and treatment remains a major challenge in young children because of the late toxicity of standard dose craniospinal irradiation (CSI). We developed therefore a new strategy based on sequential high-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) and reduced dose CSI. Patients and methods: Thirty-four children were enrolled in this phase II study. Median age at diagnosis was 3.2 years (range, 0.8 to 7). Chang stages were M1 (6 pts), M3 (28 pts). After initial surgery, children received two courses of carboplatin-etoposide; then peripheral blood stem cells were harvested. In absence of disease progression, five sequential courses of chemotherapy followed by ASCT were performed: melphalan (100 mg/m²) at day 43, cisplatin (100 mg/m²) at day 64, melphalan at day 85, cisplatin at day 106 and thiotepa (720 mg/m²) at day 127. Children with resectable tumour residue were operated at the end of chemotherapy. Treatment was completed by age-adapted CSI. Results: At the end of the chemotherapy 11 pts were in CR, 7 in PR. Out of the 19 pts who completed the treatment, 13 children are alive in CR1 (median follow-up, 26 months). Delivered doses of CSI were 50 Gy to the posterior fossa and 18 Gy to the brain and spinal canal in 3 pts, 50 Gy and 24 Gy in 6, 50 Gy and 35 Gy in 1.The overall survival is 50% at 30 months. The haematological toxicity was high but manageable; no toxic death was observed. Conclusion: This chemotherapy regimen combined with reduced dose CSI produced a survival, which compares favourably with published data. A longer follow-up is necessary to assess long-term quality of life. Clinical results of autologous or allogeneic stem cell transplantation following a new intravenous busulfan dosing as part of conditioning regimen in children: clinical outcomes, toxicity and compliance A. Prete* (1), R. Rondelli (1) (HVOD, modified Baltimore criteria) and engraftment (ANC >0.5 x 109/L and PLT >50 x 109/L) were evaluated. In 18 pts Bu was followed by cyclophosphamide. No HVOD prophylaxis was given. To prevent Bu induced seizures, all pts received sodium valproate 20 mg/kg/die from 24 h before the first Bu dose, until 6 h after last Bu dose. Results: Only 1 (0,2%) patient developed severe HVOD and resolved. One pt evidenced seizures 24 h after last dose of Bu and 16 h after the last dose of sodium valproate. One pt, that received a mismatched unrelated graft, died before engraftment for DIC. Forty eight out 49 valuable pts engrafted. Median time to neutrophil engraftment was 15 days (range, 10-19) . Median time to platelet engraftment was 22 days (range, 12-25) . Overall survival at day +100 was 96% (47/49). Regarding the alloSCT procedure, all the 18 valuable pt evidenced a Donor Complete Chimeras since day +30. After a median follow up of 21 months (range, , 34 patients (69%), are alive and well. Conclusions: i.v. Bu in children, administered on accrual pt weight, is safe and feasible. There was very limited toxicity and a rapid and sustained bone marrow function recovery. No valuable pts underwent to alloSCT rejected nor evidenced primary or secondary graft failure. Moreover allows to avoid assumption difficulties in younger patients. Introduction: This case control multicenter study was designed to compare iv Busulfan (Bu) versus oral Bu as part of conditioning regimen in autologous stem cell transplantation (ASCT) in terms of toxicity, 100 days TRM, engraftment kinetics, relapse rate (RR), survival (SUR) and event free survival (EFS), in the treatment of high risk neuroblastoma (HRNB). Methods: Between 2004 and 2007, 12 paediatric patients (Group 1) with HRNB underwent ASCT and received iv Bu every 6 h for a total of 16 doses, + Melphalan (L-PAM). Iv Bu doses were based on accrual patients weight: <9 kg 0.95 mg/kg/dose; 9-16 kg, 1,2 mg/kg/dose; 16-23 kg, 1.1 mg/kg/dose; 24-34 kg, 0,95 mg/kg/; >34 kg, 0,8 mg/kg/dose. 24 paediatric patients (Group 2), underwent ASCT between 1993 and 2007 conditioned with oral Bu, 1 mg/kg/dose every 6 h for a total of 16 doses, + L-PAM + other were available for selection as appropriate control for matched pairs analysis. Matching criteria were sex, centre, age, disease status, stem cell source, conditioning regimen, days of PMN and PLT engraftment, number of CD34+ infused and grade of toxicity according to Bearman scale. Results: Between the two groups of patients PMN (p = 0.490) and PLT (p = 0.230) engraftment were comparable. 100 days TRMp was 0.00 in Group 1 and 0.04 in Group 2 where 1 patient died for transplant related complications. Two year SURp , EFSp and RRp in Group 1 were 0.77, 0.64 and 0.23 respectively. Two year SURp , EFSp and RRp in Group 2 were 0.69, 0.51 and 0.35 respectively, without significant statistical differences in front of Group 1. After a median follow up of 21 months (range, 3-36), 9/12 patients in Group 1 are alive and well. Conclusions: i.v. Bu in children grafted because affected by HRNB, administered on accrual pt weight, is safe and feasible and in comparison with oral Bu seems less toxic. On the observation of a better outcome in terms of SUR, EFS and RR it is possible to suppose a more efficiency in disease control. Outcomes after unrelated cord blood transplantation for children with malignant and non-malignant indications. An Utrecht-Prague collaborative study J. Boelens* (1), M. Bierings (1) , M. Tilanus (1), J. Lie (2) , P. Sedlacek (3) ( In pediatrics, stem cell transplantatations are performed for various (non-)malignant diseases (leukemias, inborn errors of metabolism; IEM, (severe) combined immunedeficiencies; (S)CID, bone marrow failure syndromes). Unrelated-cord blood (UCB) is suggested to be a good alternative cell source. We have analyzed all children that received an UCB transplantation (UCBT) from 2001 to 2007 in Utrecht and Prague and studied outcome. 44 (21 malignant and 23 non-malignant) patients received an UCBT and were included. The malignant group contained the following patients: 2 ALL-CR1, 7 ALL-CR2, 1 ALL-CR3, 2 ALL-Ph+, 2 infant-ALL, 2 AML-CR2, 3 MDS/AML and 2 JMLL. The non-malignant group contained the following patients: 16 IEM, 3 (S)CID, 3 hemophagocytic-lympho-histiocytosis and 1 Fanconi anemia. Median age at UCBT was 2.9 (0.2-18.2) yrs, median follow up was 18 (1-84) mths. The donor was HLAidentical (HLA-A and B by low resolution and HLA-DRB1 by high-resolution) in 8 cases (18%) and incompatible in 36 cases (82%: most with 1 (66%) and 2 (16%) HLA disparities). The median nucleated cell dose/kg and CD34+/kg at collection were respectively 8.8 (2.4-37) x107 and 3,3 (0.7-21.83)x105. 8 patients received a TBI containing, and 36 received a chemotherapy based myelo-ablative regimen. All patients received ATG. Probability of neutrophil engraftment was 97% with a median day of engraftment +22 (10-58). Acute-GvHD (grade II-IV) was observed in 27% (4.5% grade III, no grade IV), while chronic-GvHD was seen in 18% (7% extensive) at 2 years. Two years overall survival (OS) and disease/event free survival were 71% and 63%, respectively. Between malignant and non-malignant diseases no difference in OS / EFS (61% vs. 67% / 71% vs. 71%, respectively) was found. NC-dose and CD34+-dose did not influence the endpoints. In conclusion, outcomes following mainly HLA-incompatible UCBT, with relatively high cell doses, for (non)-malignant are encouraging in this dual center experience. (Mismatched)cord blood appears to be a good alternative stem cell source for malignant as well as for non-malignant diseases. Bone marrow transplantation in children affected by malignant osteopetrosis: a single-centre report C. Forino*, A. Razza, D. De Martiis, R. Marzollo, S. Aliprandi, A. Lanfranchi, E. Mazzolari, F. Porta Ospedale dei Bambini (Brescia, IT) Osteopetrosis are a heterogeneous group of bone remodelling disorders characterized by an increase in bone density due to a defect in osteoclastic bone resorption. Malignant osteopetrosis is a lethal disorder which, if untreated, has a fatal outcome. We present a retrospective study on 20 children seen within our institution between January 1991 and October 2007, 11 males and 9 females; the disease was diagnosed at a mean age of 4 months (range 10 d to 9 m). We found that 9 children had mutations of the ATP6i (TCIRG1) gene, 1 patient mutations of the CLCN7 gene, 3 of the OSTM1 gene and one of the RANKL gene. The remaining 6 children have unknown genetic defects. Of the 20 children 14 underwent BMT, 8 of whom are still alive: 5 from a genotypic HLA identical sibling donor, 4 from an haploidentical mismatched related donor and 9 from a matched unrelated donor (MUD). Four children underwent a double BMT while 6 patients didn't underwent BMT: 3 due to a severe neurological impairment; 1 patient didn't find a compatible donor and his parents refused a MUD-BMT. The remaining 2 children died, one at the age of 16 months for sepsis and the other at 10 months for respiratory insufficiency. Only 1 patient (CLCN7 defect) died after an HLA identical BMT from his brother. Despite the fact that he presented a normal donor engraftment and a normal immunological reconstitution, he developed a severe neurological encelophaty that lead him to death 45 months after BMT. All grafted children showed in standard radiographs, a normalization of bony density: at the last follow-up, 3 patients developed a bony porosity. No patient has an improvement or a worsening of visual function after BMT. Two patients developed a moderate delay in psychomotory development with a psychosis with autistic peculiarities that weren't present before BMT and one of the two presented a critic event 10 years after BMT. As concerns GVHD, 8 children developed acute GVHD grade I-II, while 2 children developed chronic GVHD, one extensive, one limited. In our hands disease free survival is 80% for recipient of a genotype HLA identical sibling donor, 33% for patients who received a haploidentical graft and 60% from a MUD. In conclusion bone marrow transplantation from an HLA sibling donor has excellent probability of success, while as concerns MUD BMT largest cohort of patients are needed to understand the real figure of event free survival. By now, there are no reports investigating the effects of natural killer (NK-) cell-mediated immunotherapy of hepatoblastoma (HB). NK cells isolated from healthy donors were tested for cytotoxicity against HB cell lines with and without stimulation through interleukin-2 and -15. NK cell mediated cell lysis in vitro was highly effective down to NKcell/tumor cell ratios of 2.5:1. Anti tumor effects were significantly increased through stimulation with interleukin-2 and -15 in all cell lines (p<.0001). HLA analysis revealed a KIR mismatch in all analysed approaches but HLA class I expression was generally low in all HB cell lines and therefore they are likely to be also susceptible to non alloreactive NK cells. We analysed expression levels of NK cell-interacting molecules on 13 primary HB samples as well as on 3 HB cell lines. All primary samples and cell lines strongly expressed the natural killer cell-activating ligand CD155 (poliovirus receptor). HB cell lines presented either weak or negative surface expression of NKG2D ligands (MICA, MICB, and ULBP) whereas gene expression of MICA and MICB in primary tumor samples was relevant. Expression analyses revealed no differences between the various histological subtypes. Also, expression was not altered through varying chemotherapy regimens. Blockade of CD155 through monoclonal antibodies resulted in decreased lysis rates. This effect could be reversed by IL-2 stimulation. Our findings show that NK cells may serve as promising tool for immunotherapy of human hepatoblastoma. High-dose chemotherapy in treatment of children with high-risk retinoblastoma I. Dolgopolov*, T. Ushakova, R. Pimenov, N. Subbotina, V. Boyarshinov, I. Visochin, G. Mentkevich Institute of Ped. Oncology/Hematology (Moscow, RU) In order to improve DFS we performed a HDCT followed by autoPBSCT as a consolidation phase in pts with high-risk retinoblastoma (RB). High risk(HR) was defined as the involvement of the optic nerve's cut end and/or orbit after enucleation, extraocular spreading, metastatic disease and relapse after previously 1st line therapy. From 2003 to 2006 9 pts (5 female, 4 male) with high-risk retinoblastoma (RB) were treated in our center. The median age was 3 (1.1-9) years and the median body weight was 13.8 (9.8-32) kg. Six pts had unilateral localized primary RB (T4bN0M0-3, T4bN1M0-1, T3cN0M0-1, T3bN0M0-1), 2 had a relapse (metastatic in both pts with lymph nodes, bone marrow and bone involvement), and 1 pt experienced a bilateral RB operated on left eye and relapsed in the right eye 3 months after surgery. Induction phase included 4 courses of chemotherapy including cyclophosphamide, VP-16 and carboplatin, surgery and external beam radiotherapy at a dose of 50-52 Gy. PBSCs were harvested after the 1st course of CT. Consolidation included HDCT with busulfan 16 mg/kg and melphalan 140 mg/m² followed by autologous SCT. Pts received autologous SCT with a median number of 3.5 (2.2-4.8) x 106 CD34+ cells/kg. Toxicity was moderate in all but one pt, who died on d+8 from sepsis caused by Klebsiella pneumonia. Median number of days to WBC>1.0 x 109/l, Plt>20 and 50x109/l was 10 (9-11), 14 (10-20) and 17 (14-35) days, respectively. One pt out of 2 with metastatic disease had a progression of disease in bone, BM, testis and lymph nodes 3 mo. after transplantation and died. Seven pts are alive and well with a median period of follow-up of 52 mo. EFS and DFS were 64% and 76%, respectively. We concluded that HDCT followed by autologous SCT seems to be an effective tool for overcoming adverse prognostic factors in high-risk RB patients and permits to decrease a number of relapse in pts with primary local tumors. Retrospective evaluation of EBV viraemia after paediatric allogeneic haematopoietic stem cell transplantation: single-centre experience V. Bordon*, E. Padalko, E. Vandecruys, J. Verlooy, B. De Moerloose, V. Mondelaers, Y. Benoit, G. Laureys, C. Dhooge Ghent University Hospital (Ghent, BE) Introduction: Epstein Barr virus (EBV) is a frequent human latent infection. The cellular immunity plays a crucial role in the EBV control. The immunosuppression given during and after hematopoietic stem cell transplantation (HSCT) and the development of graft-vesus-host-disease, deplete and alter the function of the T-lymphocytes. As a consequence, EBV can reactivate and eventually cause EBV-lymphoproliferative disorders (EBV-LPD). Objective Evaluation of the frequency and risk factors of EBV viremia en EBV-LPD after allogeneic HSCT. Material and methods: Between January 2002 and June 2007, 51 consecutive pediatric allogeneic HSCT were performed in our institution. EBV load was measured every 2 weeks, by real time PCR until 3 months after transplant and was considered positive above the level of 300 EBV copies/µg leucocyte DNA. A viremia was considered significant if it was positive on 2 consecutive determinations. EBV-LPD was defined by positive detection of viral DNA associated with clinical manifestations of lymphoproliferative syndrome. Fourteen patients (pts) received a graft from an HLA-identical sibling, 28 from a matched unrelated donor, there were 8 haploidentical transplants and 1 familial phenoidentical donor. The diagnosis was a hematological malignant disease in 34 pt (62%), a immunological disorder in12 (23%), a storage disease in 3 pt, 1 pt suffer from SAA and 1 had a thalassemia major. The mean age was 8.5 years (3 months -19 years) . All patients with other donors than identical sibling received ATG during conditioning, with exception of patients with severe combined immunodeficiency. Results: Ten patients developed a significant EBV viremia, the incidence was 7% in the sibling group but 32% for the MUD group. Two patients developed EBV-LPD. The median time to develop EBV viremia was day + 54 (range 24-94). All significant viremias were seen in EBV-seropositive patients and/or donors, and all were MUD group. Only the 2 patients with EBV-LPD received specific therapy with anti-CD20 monoclonal antibodies, with good evolution. Conclusion: Eighty percent of all cases of EBV viremia had a good evolution without any specific intervention other than lowering the immunossuppression if it was possible. The evolution of the EBV-load overtime seems to be more predictive than the EBV-load itself technology) using unified primer/probe systems. Absolute quantification of WT1 expression was based on serial dilutions of a WT1 plasmid; normalisation was done referring to Abelson (ABL) as housekeeping-gene (duplicate measurements). Results were given by the ratio WT1/1E4 ABL copies. Both plasmids were developed and both qPCR systems were standardised in our laboratories. Quality controls (QCs) were performed every six months. Quantification of WT1 and ABL expression is reliably possible from 2.5x1E6 to 2.5x1E1 copies covering 5 logs. QCs comprised 69 AML and 47 healthy bone marrow (hBM) donors' samples that were simultaneously analyzed in the four laboratories. The method revealed a high accuracy and reproducibility: The individual WT1 expression of a respective sample obtained in each of the four laboratories was within the 0.5 log range of the mean in 82% of the samples (AML: 91%, hBM: 68%). The analysis of the pair wise differences of the WT1 expression of a respective sample measured in the participating labs revealed that 95% of the paired values range within < 1 log difference (AML: 98%, hBM: 88%). The following WT1/1E4ABL expression quantiles were defined: (1) Pediatric diagnostic AML samples: 5%: 8.1; 50% 3524.0; 95% 38574.0. (2) hBM donors' samples: 5%: 1.0; 50% 23.2; 95% 139.4. This 95% quantile was defined as the upper limit for WT1 expression in hBM. As much as 86% of the WT1 expressions of the pediatric AML patients at diagnosis ranged above this upper limit of the WT1 expression in hBM. This reveals the potential of WT1 as a MRD marker. Based on this standardized procedure the clinical impact of WT1 as a marker for the risk profile of childhood AML as well as its utility as a MRD marker will be assessed. This prospective multicentre study will be conducted over four years and is the largest one on WT1 expression in childhood AML. The knowledge about glomerular and tubular renal disturbances associated with hematopoietic stem cell transplantation (HSCT) is very limited in children. Objectives: Aim of this study was to assess glomerular and tubular renal function after HSCT in children in a prospective trial. Methods: Renal function assessed prospectively before HSCT (on day -10), on day +30, on day +100 and at least 6 months after transplantation in 34 patients (21 females/13 males) with a mean age of 8.2 years. The following parameters were investigated: glomerular filtration rate (GFR) by creatinin clearance (CrCl), cystatin C (CysC)-based formula and plasma clearance of radiolabeled diethylenetriaminepentaacetic acid (99mTc-DTPA), urinary excretion of beta2-microglobulin (B2M), beta-N-acetylglucosaminidase (B-NAG), fractional excretion of sodium (FENa) and fractional tubular phosphate reabsorption (TP/ClCr). Results: Nine patients (26.4%) suffered from acute renal insufficiency within the first 100 days after transplantation. All patients who developed acute renal insufficiency were treated successfully without renal replacement therapy. None of age, sex, primary diagnosis, sepsis, veno-occlusive disease, acute graft versus host disease and use of vancomycin were significant risk factor for acute renal insufficiency. The medians 99mTc-DTPA-based GFR of patients after HSCT showed a statistically significant decreasing when compared pre-transplant values. The frequencies of pathological findings investigating the renal tubular function before HSCT were as follows: An elevated excretion of B-NAG was found in 35% and of B2M in 63%. The value of FENa was elevated in 15% and TP/ClCr was low only in 4%. After HSCT, the change in the values was not significant in those of TP/ClCr. Compared with pre-transplant results B-NAG excretion was significantly elevated in the first 30 days after HSCT. B2M levels were elevated in early phase, as well. Although the median values of FENa were higher than normal limits sometimes in early and late period, they were either not significant or borderline compared with pre-transplant results. Conclusion: The investigation of both glomerular and tubular renal function should be part of a long-term follow-up in children who underwent hematopoietic stem cell transplantation. Optimised haematopoietic stem cell mobilisation and harvest in children with malignancies by using pegfilgrastim E. Merlin* (1), R. Veyrat-Masson (1) In children, the technical challenges of apheresis require the highest quality of stem cell mobilization. G-CSF remains the reference. The use of pegfilgrastim (pegf) allows the maintenance of an elevated serum level of G-CSF after only one injection. It is therefore a candidate for an optimized mobilization regimen. Objective: to assess the effectiveness of pegf for stem cell mobilization in children with malignancies. End-point: percentage of pts achieving at least 5x106 CD34+ cells/kg in 1 standard apheresis (≤ 3 blood volume processed, BVP) Patients and methods: 29 children (aged 1 to 18) undergoing chemotherapy for malignancy were consecutively included in this prospective study. Mobilization consisted of one subcutaneous injection of 300 µg/kg pegf in haematological steady state. Aphaeresis was begun once the value of 20 CD34+/µl cells was reached in the blood, or at day 4 after pegf injection. Results: 19/29 pts (65%) met the successful criterion (95% CI: 46%-82%). Ultimately, 25/29 pts reached a CD34+ cell target of 5x106/kg. The CD34+cell peak level occurred on day 3 after injection. Serum G-CSF level was undetectable at day 7 in all pts. Pegf was well tolerated without any sever side-effect. Asymptomatic hyperleukocytosis ≥ 100x109/l occurred in 2 pts. After reinfusion, neutrophil recovery (> 500/µl) was achieved on day 11 (9-22) and platelet recovery (>20 G/l) on day 17 (8-49). Conclusion: In children, pegf at 300 µg/kg in haematological steady state is a safe, efficient and well tolerated method of stem cell mobilization. As compared with non-conjugated G-CSF, the combination of predictable mobilisation kinetics, a better acceptance profile together with an efficacy which appears at least as good, is a seductive approach to stem cell mobilisation in children. However, the innocuity of receiving such a high dose of haematopoietic growth factor must be confirmed in larger studies. Stem cell transplantation from matched family donors for children with recurrent acute lymphoblastic leukaemia in second or third complete remission. Long-term results reported by the ALL-REZ BFM Study Group and the PAED AG-KBT E. Saribeyoglu* (1), T. Klingebiel (2) , W. Ebell (3) , A. Borkhardt (4), K. Sykora (5), B. Kremens (6) , W. Friedrich (7) Objective: Aim of this analysis was to evaluate the long-term results of matched family (MFD) stem-cell transplantation (SCT) in patients (pts) with acute lymphoblastic leukemia (ALL) in 2nd or 3rd complete remission (CR) treated according to ALL-REZ BFM protocols in the era before introduction of prospective controlled SCT protocols. Methods: All pts registered in ALL-REZ BFM trials and transplanted from MFD in CR2/3 between 1989-2003 were included and analyzed for clinical, therapeutic, and outcome parameters. Conditioning regimens contained total body irradiation and etoposide in most patients, Graft-versus-host disease (GvHD) prophylaxis was heterogeneous. Results: A total of 224 patients (192 CR2, 32 CR3) were evaluable for the analysis. The 5 years event-free survival probability (pEFS) at a median follow up of 8 years of 192/32 pts transplanted in CR2/CR3 was .52±.04 / 39±.09, the rates of treatment related mortality (TRM) 13%/25% and the rates of subsequent relapse 35%/34%, respectively. In 41% of subsequent relapses, extramedullary sites were involved. Chronic GvHD was associated with a significant lower cumulative incidence of subsequent relapses (extended: .13±.09; limited: .19±.10; none: .45±.05; p=.02). Multivariate analyses revealed age, acute GvHD, and site of relapse as independently associated with TRM, and lineage and time to relapse associated with EFS. Conclusion: Relapse was the most frequent cause of treatment failure with frequent involvement of extramedullary sites. The TRM rate was nearly twice as high in patients transplanted in CR3 as compared to CR2. Whereas acute GvHD was associated with a higher TRM, chronic GvHD contributes to disease control. Prospective controlled studies will warrant a homogeneous methodology allowing for addressing the adequate intensity of conditioning regimens and GvHD prophylaxis. Defibrotide as prophylaxis for veno-occlusive disease in children undergoing allogenic and autologous stem cell transplantation J.C. Gray*, L. Marshall, A. Qureshi, R. Ridwan, S. Sankpal, M. Ethell, M. Potter, D. Lancaster Royal Marsden Hospital (Sutton, UK) Veno-occlusive disease (VOD) is a common (10-50%) and frequently serious complication of myeloablative chemotherapy and haematopoietic stem cell transplantation (HSCT). Severe disease results in multi-organ failure with a high (>50%) mortality. The risk of developing VOD is recognised as being particularly high when conditioning regimes contain busulphan. In recent years defibrotide, a polydeoxyribonucleoside adenosine receptor agonist with antithrombotic, anti-inflammatory and anti-ischaemic properties, has emerged as safe and effective for VOD. Several studies in S299 adult patients undergoing HSCT have suggested that the prophylactic use of defibrotide may be effective in reducing the incidence and severity of VOD. However there is little data regarding the prophylactic use of defibrotide in children. Here, we report a retrospective series of 92 children receiving high dose chemotherapy and autologous (46) or allogeneic (46) HSCT. All children received prophylactic defibrotide at a dose of 20mg/kg/day from the start of conditioning chemotherapy until day +28 post-stem cell infusion. In 12 of the 92 patients the dose of defibrotide was increased to a treatment dose (40 mg/kg/day) at some point in the early transplant period when VOD was suspected, as part of the differential diagnosis of deranged liver function tests. However, in only 6 of these patients was VOD felt to be the most likely diagnosis and the Baltimore diagnostic criteria met. In 5 of these 6 patients, the features of VOD were mild and resolved with a treatment dose of defibrotide and appropriate supportive care. One patient developed severe VOD and subsequently died. 24 patients received chemotherapy regimes containing busulphan and of these, only one patient developed mild VOD. In all patients the defibrotide was well-tolerated without any haemorrhagic complications. This suggests that defibrotide may be used safely to achieve a low incidence of VOD in children undergoing HSCT, even in those receiving high risk, busulphan-containing, conditioning regimes. A larger, randomised controlled study may be useful to address this issue further. (2) Introduction: Lymphocyte recovery constitutes an essential part of immunoreconstitution post stem cell transplantation (SCT). The process is affected by e.g. GVHD and infections encountered. Delayed and/or insufficient recovery leads to an increased risk of post-SCT complications. The aim of this study was to evaluate several key aspects of this immunological reconstitution process and compare the process among recipients of related and unrelated marrow grafts and those of autologous stem cell rescue. The effects of mere immunosuppressive therapy was evaluated using nontransplanted nephrological patients on cyclosporin as a reference group. Material and methods: Between 6/01-3/04 a total of 66 (autologous 15 and allogeneic 51) patients with a mean age of 7,6 yrs with either a malignant (53) or non-malignant (13) disease were transplanted using matched unrelated (31) or sibling donors (20) or given autologous (15) stem cell rescue. T-cell recovery was evaluated by analyzing lymphocyte subpopulations by flow cytometry, determining T-lymphocyte responses to key mitogens and measuring antibody responses to established antigens. B-cell recovery was studied by flow cytometry and functionally by ELISPOT. Also the effects of passive immunization (25 children) using modified primary immunization schedules following SCT are currently being analysed. Blood samples were collected once prior to transplant and at every three months during the first 18 months post-transplant. Results: The regeneration of naive (CD3+CD45RA+CD27+) T lymphocytes in the recipients of allogeneic stem cell grafts was slow and did not reached the pre-SCT level until 6 months post SCT. Lymphocyte proliferation after stimulation with PHA, Con-A and PWM reached that of the immunosuppressed, non-transplant controls at 9 months. IgA and IgM productions in the allotransplanted children reached that of the controls at 9 and 6 months, respectively. However, extensive chronic GVHD associated with delayed T and B lymphocyte recovery (Table 1) Despite high-dose chemotherapy and autografting, the outcome for patients with primary refractory or multiple relapses Hodgkin's disease (HD) remains unsatisfactory. We report the result of reduced intensity conditioning (RIC) allogeneic stem cell transplantation in four paediatrics patients who have failed to standart multilineage curative therapy. They all enter in a prospective national paediatric study of SFGM-TC. The conditioning regimen consisted of fludarabine (30 mg/m² once daily for 6 d), busulfan (4 mg/kg once daily for 2 d) and ATG (2.5 mg/kg, 1 d). They all have matched related donor. Bone marrow were used for three patients and peripheral blood haematopoietic stem cells for one. A median number of 4.03 106 CD34+ cells/kg (2.64-8) and 25.8 10 6 CD3 + cells/kg (17.76 -205) were injected. GVHD prophylaxis was cyclosporine alone. Median age was 16 years. Time from diagnosis to graft was 9 to 24 months. Patients received three or four lines of therapy prior to RIC allogeneic SCT including autograft. At the time of RIC, they were in partial response except one who had progressive chemoresistant disease. The median follow-up was 3 to 8 months post-allograft. All patient's transplant engrafted rapidly and they all alive. The median time of hospitalisation was 35 days (23-69). The median time to neutrophil recovery (≥ 500/µl) was 19 days (15-28). They were all in complete donor chimerism at day 60. Four patients developed skin (grade ≤ II) acute GVHD that need a brief steroid therapy for two patients and maintain of cyclosporine (median time of Csa: 50 days, 36-163). Disease response was assessed monthly by scan-Tc and fluorodeoxyglucose-positron-emission tomography (FDG-PET). All responded. Two patients are in complete remission and two in partial remission. These patients in partial remission benefit from two DLI each (CD3+ cell dose: 1 107/kg and 5 107/kg). FDG-PET show in these patients evidence of disease response with reduction of lymph nodes volumes. These observations, along with the responses after DLI, suggests the presence of a graft-versus-lymphoma effect in patients with advanced active HD. The use of new imaging modality such as FDG-PET and its prognostic significance have to be defined. The optimal dosage and timing of DLI has to be identified. Prospective studies are needed to identify patients who would benefit most from this treatment approach. 5 CD3+cells/kg (range 1.1-3.1). Engraftment was rapid and sustained: WBC > 1.0 G/L, ANC > 0.5 G/L and PLT > 50 G/L on days +10, +11 and +11, respectively. In 4 pts engraftment correlated with aGvHD (gr. II) incl. one most severe case of engraftment syndrome (ES) with pulmonary oedema and liver failure, which required ICU treatment. It was the patient, who had received MMF as the only immunosuppresive drug. On day +9 he recovered with 2793 NK cells/mcl and 399 T cells/mcl in PB, respectively. His clinical status improved after high dose steroid treatment, however on day +34 GvHD grade II reoccurred 2 days after steroid cessation and methylprednisolone 2 mg/kg/d was given again. He required continuous treatment with CsA and steroids for 6 and 3 months posttransplant, respectively. All other pts were given steroids as ES prophylaxis from day +7 and despite this, GvHD grade I-II occurred. However, no ES developed in pts given steroids upfront. All pts remain alive and well with excellent immune reconstitution from 3 to 13 months posttransplant. Our data show, that CD3/CD19 depletion in SCID pts undergoing haploidentical PBSCT permits rapid, sustained engraftment, faster immune reconstitution and excellent survival. However, quality of engraftment may be unpredictible, and there is an increased risk of engraftment syndrome and/or GvHD. For this cohort of pts steroids (± OKT-3) are required not for graft rejection prophylaxis, but definitely for GvHD/ES prophylaxis. Successful allogeneic PBSCT from matched unrelated donor after reduced-intensity conditioning regimen in a patient with Nijmegen breakage syndrome and concomitant secondary T-ALL post B-cell lymphoma J. Owoc-Lempach, K. Kalwak*, M. Ussowicz, E. Gorczynska, A. Dyla, J. Musial, A. Chybicka Wroclaw Medical University (Wroclaw, PL) Nijmegen Breakage Syndrome (NBS) belongs to a group of chromosomal instability syndromes and is clinically characterized by microcephaly, dysmorphic facial features, immunodeficiency, hypersensitivity to ionizing radiation and cancer predisposition. B-cell lymphomas are the most frequent malignancies in NBS, but cases of precursor T-cell, B-cell leukemia, myeloid leukemia and solid tumors also have been reported. In pts with NBS, chemotherapy and radiotherapy must be modified because of potentially serious toxic complications. There have not been any reports on matched unrelated donor (MUD) BMT or PBSCT in a patient with NBS so far. However, two patients in UK have undergone successful allogeneic MSD and phenoidentical related donor BMTs using an FA conditioning regimen, respectively. Here we report on a 14 years old boy with NBS, who underwent MUD PBSCT, because of secondary T-cell ALL. Primarily B-cell NHL in nasopharyngeal area was diagnosed at the age of 5 years, which was successfully treated with B-NHL 93 protocol. Because of his bird-like facial appearance a suspicion of NBS was made and diagnosis of NBS was confirmed by mutation analysis of NBS1 gene. At the age of 13 years the patient developed secondary T-ALL and was treated acc. to high risk arm of ALLIC 2002 protocol. No significant side effects of chemotherapy were observed except for severe renal insufficiency after MTX use. After reduced conditioning regimen incl. BU 2 x 1 mg/kg, Flu 5 x 30 mg/m², ATG 3 x 20 mg/kg (GEFA) the patient received PBSC (5.08 x 10(6) CD34+/kg bw recipient) from 9/10 HLAallele MUD. OKT-3 and CsA were used as graft rejection and GvHD prophylaxis, respectively. A sustained allogeneic engraftment (neutrophils > 0.5 G/L on day +15, platelets > 50 G/L on day +18) was observed. The immunosupressive therapy was discontinued one month later because of the occurrence of 3.3 % autologous content in PB. The posttransplant period was uneventful except for skin aGvHD st. III on day +55, which was successfully treated with steroids and CSA. Skin GvHD recurred on day +235 and was again successfully treated with steroids. The patient remains alive and well in CR 15 months posttransplant, with full allogeneic chimerism and without any symptoms of cGvHD. This report demonstrates that administration of reduced intensity conditioning allows the succesful outcome of UD PBSCT, even in pts with chromosomal instability syndromes like NBS with concomitant primary or secondary malignancies. The Wilms' tumor gene WT1 is expressed at high levels in various hematologic and solid malignancies including lung, breast and colon carcinomas. First evidence for WT1 expression has also been found in rhabdomyosarcoma (RMS). Clinical outcome of RMS is still poor despite improvements of surgical, chemotherapeutic and radiotherapeutic regimens. Allogeneic stem cell transplantation (allo-SCT) as an alternative treatment approach is currently under investigation. In order to improve the outcome of transplantation, WT1-directed adoptive immunotherapy might represent a promising strategy in this clinical setting. In adult patients with WT1-expressing tumors spontaneous antibody and cytotoxic T-lymphocyte (CTL) responses against the WT1 protein have been described. This indicates that WT1 is highly immunogenic and may represent a promising target for cancer immunotherapy. MHC class Irestricted CTL-and class II-restricted helper epitopes of WT1 protein have been identified, and early clinical studies on specific vaccination using these peptide epitopes have been initiated. Here we analyzed WT1 mRNA and protein expression levels in RMS cell lines and primary tumors by RT-PCR and Western blot analysis, and detected WT1 expression in 3 of 7 RMS cell lines and in 4 of 5 primary tumors. To investigate the feasibility of WT1-specific T cells for the treatment of RMS and other WT1-expressing tumors, we analyzed the frequency of T cells specific for different HLA-A2 and HLA-A1-restricted WT1 peptide epitopes in patients and healthy volunteers. WT1 peptide epitopes were used for the in vitro stimulation of WT1specific CTLs. Peptide recognition and in vitro cytotoxicity were analyzed in Interferon-gamma Elispot and standard 51Cr-release assays after expansion of T-cell lines and T-cell cloning. We could show that in vitro activated CD8+ T cells lysed T2 cells pulsed exogenously with the relevant peptide, as well as WT1-expressing tumor cell lines. Our preliminary results demonstrate that WT1-specific CD8+ T cells are found in a large proportion of HLA-A2+ breast cancer patients and healthy volunteers. This may offer the opportunity to utilize ex vivo expanded donor-derived WT1-specific T cells for the adoptive T cell therapy after allo-SCT. Arguments for systematic psychodiagnostic assessment in children and adolescents, before and after haematopoeitic stem cell transplantation, in relation to cognitive aspects and the influence on quality of life P. De Vos*, A. Bomans, N. Nolf, V. Bordon University Hospital Ghent (Ghent, BE) Background: Hematopoeitic Stem Cell Transplantation (HSCT) has become the standard treatment for a variety of hematogical/oncological malignancies and inborn errors of the metabolism. With this intensive treatment, a substantial percentage of patients can be cured. Although some studies in adults report a rather good quality of life (QoL) on a long-term base, a significant percentage of patients however suffers from late complications, such as cognitive deficits, psychological distress and fatigue. Both cranial irradiation and high-dose chemotherapy are known causes of delayed encephalopathies. In HSCT highdose chemotherapy is often combined with total body irradiation (TBI). These are potentially neurotoxic on the central nervous system (CNS). The maturing brain of children/adolescents is more vulnarable for potentially toxic treatments. Impairments in the CNS have an important impact on academic achievement and therefore on choices concerning school and work. Objective: Prospective investigation in children and adolescents of the frequency and severity of delayed cognitive deficits after HSCT and their impact on QoL. Methods: Since 2005 we started with systematic psychodiagnostic assessment of allogeneic HSCT-patients before conditioning therapy and at least 2 and 4 years after transplantation. The age of the patients varies between 2,5 and 16 years. We use the WPPSI-R and the WISC-III-NL (IQ), the BSID-II-NL (development) and the TEA-Ch (attention and concentration). Results: The results we yet obtained, indicate a possible impairment of cognitive skills and attention deficits. Conclusion: Although our population is yet too limited to obtain solid scientific data, we observed the following: decrease in IQ, attention deficits and concentration problems. Our patients report also difficulties in keeping track in the classroom activities. Information processing seems to be decreased compared with results before transplantation. This has strong repercussions on their academic skills and choice of future studies and professional career, and therefore on QoL. Nucleoside analogs are active drugs in leukaemias.Clofarabine(CLO), a novel purine nucleoside analog,inhibits DNA repair following exposure to DNA damaging agents (eg,cyclophosphamide) in leukaemia blasts.CLO is active in both myeloid and lymphoblastic acute leukaemia and several studies have already shown the effectiveness in paediatric patients with refractory disease after standard treatment. Acute Lymphoblastic Leukaemia arising within the first year of life is a rare disease defined as Infant Leukaemia (IL). It is characterised by extremely poor prognosis, due to high resistance to therapy and great incidence of early relapse. We report the case of a 1-year girl affected by IL, MLL gene rearranged, in 2nd complete remission (CR) who was referred to our centre to undergo Unrelated Cord Blood Transplantation (UCBT). The patient has been treated, as first line, by Interfant 99 protocol (October 06) and at the time of the first relapse, occurred during the maintenance therapy (May 07), by AIEOP REC 2003 protocol. The 2nd CR was achieved in June 07. In August 07, during the consolidation therapy before UCBT, she developed high white blood cells count (179.4x10 9 /l) and 2nd relapse was diagnosed. As third line treatment Cyclophosphamide as cytoreduction therapy (200 mg/m 2 iv x 4 days) followed by 2 cycles of CLO (52 mg/m 2 iv daily x 5 days) with ten-day interval was administered; informed consent was obtained from parents. The third CR was obtained after the first cycle of CLO.Transient hepatotoxicity (raised ALT/AST) was observed during the two cycles of CLO. Two weeks after the last CLO, before haematological recovery, a 2-antigen mismatched UCBT was performed with a preparative regimen of Busulfan, Cyclophosphamide, Melphalan and Antithymocyte globulin. Total nucleated cell dose was 11.7x10 7 /kg. Cyclosporine and steroids were used for Graft-versus-host disease prophylaxis. Neutrophil engraftment occurred at day +27 with no Granulocyte Colony-Stimulating Factor therapy. Molecular studies demonstrated 100% donor engraftment on serial bone marrow examinations on day +21 +35 +60. Even if at short follow up we can conclude that a relapsed chemotherapy-refractory patient was successfully transplanted with a mismatched cord blood unit after two cycles of CLO that was well tolerated. Three months after transplantation the child is in continuous CR, has full donor chimerism and no signs of serious adverse events. conventional myeloablative stem cell transplantation (CST) and children received nonmyeloablative HSCT (NST). Methods: Quantitative analysis of chimerism was performed on 51 paediatric patients who underwent allogeneic HSCT in Transplantation Unit in Department of Paediatric Haematology and Oncology in G.Gaslini Institute. 15 and 36 out of 51 children underwent NST and CST, respectively. We used a STR method with polymerase chain reaction (PCR) amplification of 9 genomics polymorphic loci with tandem repeat sequences. We evaluated 10 and 20 Pts receiving NST and CST, respectively, at take and on days +30, +60, +90 and +180 from HSCT. Results: Our results showed mixed chimerism (MC) in 10/10 Pts underwent NST, at take and day +30 with 95% of donor cells. 7/10 Pts reached a complete chimerism (CC) at day +90 post HSCT. The remaining 2 Pts rejected , and 1 Pts maintained a MC with 75% of donor cells. 12/20 Pts who underwent CST reached a CC on day +30 The remaining 8 showed a chimera comparable to Pts subjected to NST, at the same time post HSCT. Conclusions: During the last few years NST has been increasingly used on paediatric patients and mostly in Pts with non neoplastic pathologies. Previous studies on adults indicate that Pts undergoing NST reach a CC later than Pts receiving CST. In our experience CC at day +30 was reached in 60% of patients receiving CST and in none patients who underwent NST. This preliminary data suggest that there is difference between the two groups of patients even in children. The greater availability of NST on paediatric patients and a careful monitoring of the follow-up with STR analysis on a greater number of Pts, could certainly allow us to analyze better this issue. Lymphocyte subsets in children after allogeneic HSCT-Impact of conditioning regimen M. Choma*, K. Drabko, A. Zaucha-Prazmo, B. Wojcik, J. Kowalczyk Medical University of Lublin (Lublin, PL) Introduction: Immunological reconstitution after hematopoetic stem cell transplantation (HSCT) has a major impact on outcome. Conditioning regimen is one of the factors which may influence immunological recovery. Aim: Comparison of immune reconstitution after allogeneic stem cell transplantation using three different myeloablative regimens: based on total body irradiation (TBI), busulfan (Bu) and treosulfan (TREO). Material: 50 consecutive patients, who underwent 52 HSCT between 2000 and October 2007 were included into the study. Children were transplanted due to malignant (n=45) or nonmalignant diseases (n=7) from matched related donors (MRD) n=33 or matched unrelated donors (MUD) n=19. Sexteen children were conditioned with TBI 12 Gy (6x2 Gy) (TBI group) in combination with VP 16 12/16 and other drugs 4/16. Eighteen patients received oral busulfan 16 mg/kg/bw (BU group) in combination with cyclophosphamide in 14/18 patients and other drugs in 4/18 patients. Eighteen children were conditioned with the use of treosulfan i.v 12g/m 2 (TREO group) in combination with cyclophosphamide (7/18), with VP 16-(5/18) or other drug (6/18). Method: Absolute counts of lymphocyte subpopulations were determined in the following time points: 100 days, 6, 9, 12, 18 and 24 months after transplantation. Lymphocyte subsets: CD4+(T-helper), CD8+(T-supressor), CD19 (B-lymphocyte) and CD16, 56+(NK lymphocytes) were analysed by flow cytometry (FACScan, Becton Dickinson) using Cellquest software package. Results: We did not observed statistically significant differences between absolute counts of CD 4, CD 19, CD 16+56 between analyzed groups. There was a trend to lower CD 8 counts in patients who received treosulfan in conditioning regimen, and this difference was statistically significant 12 months after HSCT. CD8 counts in all analyzed time points are shown in figure 1. Acute GvHD grade >II or ccurred in 11 patients: 4 after TBI, 5 after busulfan and 4 after terosulfan. Chronic GvHD was observed in 4 childred: 3 in busulfan group and 1 in treosulfan group. Conclusions: 1. Children treated with TBI, busulfan or treosulfan in conditioning regimen have comparable immune reconstitution in subpopulations of CD19, CD4 and CD16+56 lymphocytes. 2. Lower CD 8 count after conditioning with treosulfan may contribute to decrease immunological complication after allogeneic transplantation HSCT. A peculiar clinical presentation of a condition mimicking macrophage activation syndrome: severe combined immunodeficiency S. Aliprandi*, C. Forino, R. Marzollo, D. De Martiis, D. Dallera, R.F. Schumacher, E. Mazzolari, F. Porta Ospedale dei Bambini (Brescia, IT) A 4 months old female presented with clinical and laboratory evidences that suggested a diagnosis of hemophagocytic lymphohistiocytosis. Infact, first child born at term of a couple of not related parents, despite a regular development in weight and height till the age of 3 months, presented diarrhoea, weight loss, fever not responsive to antibiotic therapies, hepatosplenomegaly and cough. Laboratory evaluation showed pancytopenia (lymphocytes 2031/mm³, platelet count 74.000 mm³, Hb 8.9 g/dl), hypertransaminasemia (AST 216 U/L, ALT 48 U/L), hypogammaglobulinemia, increasing triglycerides (398mg/dl), hypofibrinogenemia (fibrinogen 51dl mg/dl). Overall the clinics and the lab tests suggested Hemophagocytic Lymphohistiocytosis, meanwhile the child got worst. She presented severe pulmonary distress and lethargy, needing intensive care regimen (c-PAP ventilation). At the chest x-ray diffuse intestinal pneumoniae was present while rachicentesis was negative. She didn't responded to steroid therapy as we would imagine. Therefore we decided to go deeper with our investigation and we analysed the lymphocyte subset (CD3 1.1%; CD4 0.2%; CD8 0.2%, CD19 86.1%, CD16 6%), the EBV and CMV serology (CMV PCR > 12.500.000 copies/ml, EBV PCR < 316 copies/ml) and the NK activity (normal). A diagnosis of SCID T-B+NK+ was now evident. Molecular analysis of genomic DNA performed at the IL7RA locus, shown 2 different mutation (IVS1(-2)a>g; T182C. She was immediately treated with gancyclovir, foscarnet, cidofovir, methylprednisolone, broad-spectrum antibiotics, immunoglobulins for 10 days; after 10 days of treatment CPAP ventilation was stopped and CMV PCR decreased (638.640). Since an HLA matched family donor wasn't available, a search of Matched Unrelated Donor (MUD) was started. While in therapy with antiviral drugs, a MUD bone marrow transplantation (BMT) with conditioning (antithymocyte globulin, cyclophosphamide, busulfan) was performed at the age of 8 months, with successful outcome. The child was discharged at day +34 in good clinical conditions, the CMV PCR was decreasing with antiviral therapy (6.600 copies/ml). She is currently 7 months out of BMT. Lineage specific chimerism analysis using immunomagnetic cell separation in children following haematopoietic stem cell transplantation J. Stein* (1), D. Kristt (2) , T. Klein (2) , Y. Kodman (1) , I. Yaniv (1) (1)Schneider Children's Medical Center (Petach Tikva, IL); (2)Rabin Medical Center (Petach Tikva, IL) Lineage specific chimerism analysis (LSCA) permits serial analysis of engraftment performance in the specific lineages (T, B and NK cells) that are important for long term graft function. By contrast, the use of unfractionated blood or marrow specimens for chimerism analysis yields a potentially skewed value that reflects the integration of donor/host contributions of all the various hematopoietic lineages present in the sample. LSCA has traditionally been performed on cells isolated using automated, high-throughput cell sorters, but automated cell sorting is an expensive, labor intensive process that is not available at many centers. Consequently, we have utilized a small scale immunomagnetic lineage purification system (EasySep®, StemCell Technologies) that can be performed rapidly, that costs approximately 10 euro per sample (for selection of T, B, NK and granulocyte lineages), and that routinely provides lineage specific cell populations at high purity (94-99%) for subsequent chimerism analysis using either fluorescence in situ hybridization or short tandem repeat (STR)-based techniques. In this series, chimerism was determined by STR analysis (SGM+, Applied Biosystems).Data were analyzed for percent chimersim of T, B, NK, granulocyte and the unfractionated sample using Chimertrack® software, which provides a multilineage graphic display and facilitates comparison of serial samples. Sequential samples of either whole blood or aspirated bone marrow from 23 patients were analyzed. Mixed chimerism (>5%, <95%)was detected on whole blood or marrow samples in 9 patients, of whom 7 had received transplants for nonmalignant disease, and 1 each had been transplanted for ALL or CML (PCR negative for BCR-ABL transcript at the time of LSCA). LSCA results led to change of clinical management (titration of immune suppression or the application of donor lymphocyte infusions) in 5 patients with non malignant disease, and predicted imminent relapse in the 2 patients with malignancy, expediting specific pre-emptive therapy. LSCA provides indispensable information for clinicians and can be readily performed using small scale immunomagnetic techniques. Patients with myelofibrosis below the age of 45 should be considered candidates for TBI based myeloablative regimes, where RIC transplants should be applied in older patients P. Kottaridis*, A. Bloor, R. Pearce, K. Towlson, K. Thomson, J. Apperley, G. McQuaker, D. Marks, C. Craddock, S. McCann, N. Russell, S. Mackinnon, G Myelofibrosis with myeloid metaplasia (MMM) is a chronic myeloproliferative syndrome characterised by bone marrow fibrosis, cytopenias and extramedullary haematopoiesis. The majority of patients are elderly, however a significant proportion is younger and therefore candidates for allogeneic transplantation. Here we report the UK experience in 51 patients who received either a myeloablative (MA) (n=27) or a reduced intensity conditioning transplant (RIC) (n=24) for MMM. Twenty one were females, 30 males and the median age at diagnosis was 42 years. The median age at transplant was 49 years. Thirty three patients received a sibling allograft while 18 an unrelated donor transplant. Thirty patients at the time of the transplant had an intact spleen, 16 had a splenectomy and splenectomy status was not known for 4 patients. Twenty three recipients of a MA transplant were conditioned with cyclophosphamide and Total body irradiation (TBI) while 4 with cyclophosphamide and busulphan. The conditioning for recipients of RIC was fludarabine/melphalan/Campath for 18 pts and busulphan fludarabine with ATG for 6 patients. For the entire group of patients the NRM at 100 days was 21% (13% for RIC, 26% for MA). The NRM in recipients of RIC below the age of 55 was 7% at 100 days compared to 22% for those above the age of 55, however this was not statistically significant. For the entire group of patients DFS and OS were 39% and 46% respectively at 3 years. DFS and OS for MA transplants at 3 years was 44%, while for RIC transplants 24% and 31% respectively. Recipients of RIC transplant had increased risk of relapse (46% versus 15%, however this was not statistically significant (p=0.06). Acute GVHD more than grade II was developed in 4 recipients of MA transplant and 3 of RIC. Loss of donor chimerism in the RIC group and especially in the MUD transplants was noted in 3/7 patients requiring intervention with DLI (n=2) or a 2nd transplant (n=1). The use of campath-1H in recipients of RIC transplants might have contributed for the inferior outcome in this group of patients as compared to published data using non campath regimes. We conclude that MA allogeneic transplantation is a safe treatment option in selected patients with myelofibrosis. Young patients should be considered candidates for TBI based regimes, where patients above the age of 45 should receive RIC approaches. The optimum regime in RIC setting needs further evaluation in Phase II studies. Background: Indication to allogeneic stem cell transplantation (SCT) in chronic myeloid leukaemia (CML) underwent many prognosis. Furthermore, a mutual transcriptional activation between the nm23 and c-myc genes has been implicated in transformation. We have recently described the identification of Nm23-H2 as a candidate tumor associated antigen in a case of CML and demonstrated the presence of Nm23-H2 reactive T cells 5 years after transplantation. Here, we compare the expression of Nm23-H2 and H1 proteins in leukaemic patients and healthy donors. Materials and Methods: PBMC were collected from 22 CML patients at initial diagnosis, from 12 Glivec-resistant CML patients, from 15 AML patients at diagnosis and from 5 healthy donors. PBMC, CD3+, CD14+, CD33+ and CD34+ populations were stained, fixed and sorted onto microscope slides. Immunocytochemical analysis was performed with Nm23-H1-(Novacastra), Nm23-H2-(Santa Cruz) and ß-Actin-(Sigma) specific antibodies. Evaluation was carried out using a confocal laser scanning microscope LSM 510 (Zeiss). Results: PBMC from 19 out of 22 untreated CML patients (86%) showed a strong Nm23-H2 staining. Fractionation of the PBMC population revealed that CD14+, CD33+ and CD34+ but not CD3+ cells are Nm23-H2 positive in the majority of these patients. Nm23-H2 was also detected in the PBMC of 7 out of 12 Glivec-resistant CML patients (58%). In most cases, Nm23-H2 co-localized with ß-actin. PBMC and sorted subpopulations from 14 of 15 AML patients (93%) and from all healthy donors were Nm23-H2 negative. Nm23-H1 protein was clearly detectable in a number of cell lines (HL-60, U-937, K562 and T47D), but was very weak or absent in primary PBMC from patients with acute and chronic leukaemia or healthy donors. Conclusion: Taken together, our results show that those myeloid populations functionally affected by the bcr/abl mutation specifically over express Nm23-H2 but not Nm23-H1 protein in 19 out of 22 cases of CML. While the molecular basis of this expression pattern and the possible involvement of c-Myc remains to be resolved, these results support the proposal that immune responses to Nm23-H2 may mediate GvL effects and that peptides derived from Nm23-H2 might therefore be attractive targets for specific immunotherapy in CML patients. Imatinib (IM) is now first line therapy for chronic myeloid leukaemia (CML). However 25-30% of patients (pts) respond suboptimally and alternative therapies are important. As IM considerably reduces tumour load even in pts who subsequently lose response, autologous stem cell transplant (SCT) using a largely Philadelphia negative product may be a useful strategy. We have attempted to collect SC from 133 pts with CML in complete cytogenetic response post IM. 125 were diagnosed in CP. 48% received IM due to lack of response or intolerance to alpha-interferon (IFNa), the remainder received IM only. All pts received 10 mcg/kg of G-CSF for 5 days. IM was continued throughout. Leucapheresis was attempted if mobilisation was successful (PB CD34+ cells >10x103/ml) and a successful harvest was defined as >2.0x106 CD34+ve cells/kg. 100 pts underwent leucapheresis at first attempt, 49% achieving a successful harvest in a median of 1 procedure. Median cell dose was 2.890x106/kg. 79% of those achieving a cell dose >2.0x106/kg at first attempt had received IM only prior to mobilisation. 30 second attempts were made following failure to mobilise or harvest adequate SCs, resulting in 12 additional harvests. Third and fourth attempts led to successful harvests in 3 of 8 pts. Overall successful harvest was obtained in 48%, 65% in the IM only group, compared to 29% prior treated with IFNa. Prior IFNa negatively predicted for successful harvest (p=0.005). Factors predictive of a successful harvest included the pre-pheresis WCC (p=0.001), longer time from CCR to mobilisation (p= 0.019) and reduced time from diagnosis to IM, (p=0.003). 7 harvests were RT-PCR negative (median BCR:ABL 0.12%). Engraftment post autografting can occur with lower SC doses. Changing the criteria of successful harvest to >1.5 and >1.0 x106 resulted in 7 and 15 additional successful harvests respectively, an 80% success rate for pts treated with IM from diagnosis and 35% in those given IM following IFNa. Negative and low level BCR-ABL harvests can be obtained in pts previously treated with IM ± IFNa. Prior IFNa significantly reduces the ability to mobilise and harvest adequate numbers of SC. This may simply be due to a reduced SC reserve as IFNa non-responders were diagnosed earlier than those treated with IM alone. However even in those treated with, and responding well to, IM alone, successful harvests cannot be guaranteed. We recommend continuing to collect stem cells from all patients at diagnosis. Late relapses after allogeneic stem cell transplantation for chronic lymphocytic leukaemia C. Moreno*, J. Esteve, P. Abrisqueta, F. Bosch, E. Gine, C. Martinez, M. Rovira, E. Caarreras, E. Montserrat Hospital Clinic (Barcelona, ES) Background: Allogeneic stem cell transplantation is considered to be the only treatment with curative potential for chronic lymphocytic leukemia (CLL), with most series showing disease free survival (DFS) and overall survival (OS) plateaus of around 60% at 4 years after transplantation. Due to the long natural history of the disease, a long follow up is required for a meaningful analysis of the benefits of allotransplantation in patients with CLL. Aim of the Study: To report the long-term results of a series of 32 patients with CLL submitted to allotransplantation in our institution since 1991, with special focus on late relapses. Patients and Methods: Thirty-two patients with poor-risk CLL were included in the study. Median age was 50 years (range, 29 -63). Male/Female ratio: 1.6.The median follow-up for alive patients was 72 m. (range, 11-191) . Twenty-five patients received a transplant from an HLA-matched sibling and 7 from an HLA-matched unrelated donor. Characteristics of the graft, conditioning regimen, graft-versus-host disease (GVHD) prophylaxis and general management of the patients changed over the years as per the protocols employed at our center. Results: Non-relapse mortality was 31% at 1 year after transplantation (CI, 19%-52%). DFS and OS at 5 years were 86% (CI, 77% to 95%) and 56% (CI, 47% to 65%) respectively. Four patients relapsed, one of them within 4 years from transplantation (30 m.) and three more than 4 years after transplantation (62, 68, and 120 m., respectively). The cumulative incidence of relapse was 23% at 10 years. Notably, three out of 12 patients transplanted with a T-cell depleted graft relapsed as compared to one of 20 patients receiving an unmanipulated graft. One of two patients responded to donor lymphocyte infusion. Conclusion: This study shows that albeit allogeneic stem cell transplantation offers the potential for cure in CLL relapses are observed. Importantly, relapses may occur quite late after transplantation and are particularly frequent in T-cell depleted grafts. This calls against the use of T-cell depleted grafts in patients wit CLL and stresses the importance of a careful follow-up even in patients who remain in CR for more than 4 years after transplant. Conditioning with oral busulfan with fludarabine or oral busulfan and cyclosphosphamide in patients with chronic myeloid leukaemia, chronic phase, using sibling donor: experience of coorporative group (Caipira Connection) in Brazil V.A.R. Colturato* (1) In the period between August 1996 and December 2003 we performed 74 hematopoietic stem cell transplantations from an HLA identical sibling (HSCT), in patients with Chronic Myeloid Leukemia (CML), in chronic phase (CP) using the conditioning regimen of Busulfan, 16 mg/kg, and Cyclosphosphamide 120 mg/kg (BUCY). This group stratification for age ≤ to 30 years and > than 30 years, determined survival rate probability significantly better 93% x 62%, p = 0,002, for the smaller age group, due to transplantation relation mortality (TRM) significantly smaller 29,2% x 6,1%, p = 0,009. The donor's sex x recipient's sex factor and the time of diagnosis until the moment of the transplant > or < than 1 year, didn't significantly interfere with the survival rate probability of this group. With the objective of reducing TRM, since December 2003 we have been using the myeloablative conditioning with Busulfan 16mg/kg and Fludarabine 120 mg/kg (BU-FLU), in 45 patients, with CML in CP. After average follow-up of 718 days, the patients above, with a median of age of 43 years, presented survival rate probability of 87% and TRM of 4,4%, against respectively 88% and 9,1% of the BUCY group (N=55), with median of age of 28 years, transplanted in the same period. When we compare the BU-FLU group with the historical BUCY's group, higher than 30 years, N = 41, median of age of 42 years, we verified the statistically superior survival rate probability: 87% x 62%, p = 0,04 due to smaller TRM 4,7 x 29,2 p = 0,04. We have concluded that the BLU-FU myeloablative conditioning, without Busulfan's serum level monitoring, is associated with smaller TRM than the BUCY conditioning, allowing that the patients with higher age (median of 43 years) of BU-FLU's group, have the same survival rate probability than the patients with smaller age (median of 28 years), p = 0,001 in the BUCY group. Curative allogeneic transplant for blast crisis systemic mastocytosis K. Theunissen* (1), K. Buve (2) , G. Bries (1), V. Madoe (1), J. Maertens (2) (1)Limburgs Oncologisch Centrum (Hasselt, BE); (2)UZ Gasthuisberg (Leuven, BE) We present the case report of a 33 year old male. In 2005 he presents with pancytopenia, and the diagnosis of systemic mastocytosis is made. A ckit mutation is present. In May 2006 he undergoes splenectomy after splenic rupture. 3 months later, he presents with massive ascites, with a prominent presence of myelomonocytic leukemic blasts. Bone marrow and peripheral blood show the development of an AML M5. An SM-AHNMD is diagnosed. Consecutive induction treatments with Ara C-Idarubicin, and Etoposide-Mitoxantrone-Imatinib fail, with recovery of peripheral blood blasts and recurrence of ascites. A last induction treatment with high dose AraC yields long lasting pancytopenia, with a fibrotic trephine biopsy with reduced cellularity, still prominent mastocytes but no marked presence of leukemic blasts . Since a related donor is available, and because of the good overall condition of the patient he is refered for a conventional sibling transplant. TBI/Cy conditioning is started with 11% peripheral blood blasts, a serum tryptase of 197 mcg/L, and mild ascites present. The transplant was uneventful with prompt engraftment on day 13. Tryptase levels were still high (172). Mild grade 2 aGVHD of skin and gut was easily controlled with low dose steroids. Calcineurine inhitors were prematurely stopped because of microangiopathic hemolysis. During further follow up, consecutive trephine biopsies showed no evidence of the leukemic component, and a gradual complete resolution of mastocytosis and fibrosis at 6 months post transplantation. Full donor chimerism was observed through-out the post transplantation period. Today, almost 11 months after transplant, the patient has mild cGVHD of the skin and liver. The tryptase has further decreased to an almost normal level of 34 mcg/L. To our knowledge, this is the first report of a successful conventional transplant for an advanced stage of systemic mastocytosis, with at least circumstantial evidence of a lasting graft versus mastocytosis effect. Transplants from unrelated donors may be probably considered comparable to related. MRD mapping identifies distinct GVL response patterns after allogeneic stem cell transplantation for chronic lymphocytic leukaemia: results from the GCLLSG CLL3X trial P. Dreger *, S. Boettcher, S. Stilgenbauer, D. Bunjes, J. Schubert, S. Cohen, M. Hallek, M. Kneba, N. Schmitz, H. Döhner, M The purpose of this study was to comprehensively map distinct minimal residual disease (MRD) patterns and their individual prognostic implications after non-myeloablative allogeneic stem cell transplantation (NST) in high-risk chronic lymphocytic leukemia (CLL). Subjects were the first 30 consecutive patients from a prospective clinical trial (study patients), and 7 pilot patients treated identically. Using quantitative RQ-PCR and/or flow-based MRD monitoring (sensitivity ≥10-4), four distinct patterns of MRD kinetics could be identified: (i) patients who promptly achieved durable MRD negativity without direct evidence of graft-versus-leukemia effects (GVL; n=4; no clinical relapse); (ii) patients with complete and sustained MRD response after GVL (n=18, including one with del 17p-; one relapse); (iii) patients with incomplete and transient MRD response to GVL (n=4; three relapses); and (iv) patients without sufficient MRD response due to lack of GVL (n=2; two relapses). The remaining patients could not be clustered due to insufficient sampling (n=5), graft rejection (n=1), early relapse (n=1), or early death (n=2). Including 2 non-relapse-related deaths, study patients had a 50% probability of being alive and MRD-negative 12 months post transplant. MRD negativity occurring at this time or later was durable in all cases except one. With a median follow-up of 42 (7-70) months, 3-year event-free and overall survival of the 30 study patients was 58% and 71%, respectively. In summary, MRD mapping indicate that in high-risk CLL effective GVL activity can be induced in most patients after Treplete NST. However, in a significant proportion of cases this does not translate into sustained disease control due to development of secondary GVL resistance. This data may help to understand the physiology of GVL activity and resistance to it, and gives a rationale basis for designing MRD-guided interventional studies on NST in CLL Quantitative monitoring of T315I BCR-ABL mutation by the Invader assay M. Yamamoto*, K. Kakihana, K. Ohashi, T. Yamashita, H. Akiyama, H. Sakamaki Tokyo Metropolitan Komagome Hospital (Tokyo, JP) Although imatinib mesylate has a highly inhibitory effect against BCR-ABL kinase, primary and secondary refractoriness have been observed. Point mutation within the kinase domain of BCR-ABL is one of the most important drugresistant mechanisms. Of 25 reported point mutations, T315I BCR-ABL mutation might be dismal in clinical settings because it mediates clinical resistance to imatinib, nilotinib, and dasatinib, and the only established therapeutic option is hematopoietic stem cell transplantation at this moment. However, there is no information about the kinetics of this mutated clone. Here we developed quantitative invader assay to monitor T315I BCR-ABL transcript. By using fluorescent resonance energy transfer system, the amount of released 5' flap is measured as signal intensity, which enables us to calculate the copy number of T315I products with standard curve. Using this assay, we serially monitored T315I BCR-ABL transcript in a CML patient whose BCR-ABL transcript was still detectable (more than 10000 copies/microgram mRNA) 6 months after starting imatinib therapy. Although we have continued to monitor T315I BCR-ABL transcripts in 30 patients (chronic phase) up to 12 months, there was no patients who appear to be apparently resistant to imatinib due to a given T315I mutant, so far. In contrast, in a case of Ph+ ALL being treated with chemotherapy including imatinib, we serially monitored both wild type and T315I BCR-ABL transcripts and observed the increased level of T315I transcript during relapse (0%at the time of diagnosis and 54.8% at relapse). Thus, our new strategy could be a useful tool to study the kinetics of the mutant clone and the pharmacokinetics of the drug resistant to T315I mutation. Comparable long-term results after related or unrelated stem cell transplantation for chronic myeloid leukaemia in first chronic phase, transplanted within one year after diagnosis -a single-centre analysis K. Kolbe*, T. Fischer, W. Herr, R. Meyer, C. Huber Johannes-Gutenberg University (Mainz, DE) We retrospectively analysed the outcome of seventy-two patients with chronic myeloid leukaemia in first chronic phase who got an allogeneic haematopoietic stem cell transplant (HSCT) at our centre between 1993 and 2005. Forty patients with a median age of 38 years (range 19-54) had related donors, thirty-two patients with a median age of 39 years (range 17-55) received stem cells from an unrelated donor. Total body irradiation (TBI, 12 Gy) and cyclophosphamide (2 x 60mg/kg bw) was used as conditioning regimen in 28/40 and 24/32 cases. Before unrelated HSCT patients additionally received anti-thymocyte globulin (ATG, rabbit, Fresenius) 3 x 20-30mg/kg bw as part of the preparative schedule. 50% related and 65% unrelated donors spent peripheral blood stem cells. The graft was HLA-matched in 36/40 pairs in the related and 26/32 pairs in the unrelated cohort (with molecular typing for HLA-A, -B, -C, -DRB1, and -DQB1 since 2000). Prophylaxis against graft-versus-host disease (GvHD) consisted of cyclosporin A and short course of methotrexate. 30/40 patients after related and 28/32 patients after unrelated HSCT are alive. Transplant related mortality (TRM) was 20% in the related and 10% in the unrelated group. Relapse rate (RR) was higher in the unrelated cohort (40% versus 13%, p=0,012). Acute GvHD grades III-IV occurred in 5% and 6%, chronic GvHD in 20% and 10% of evaluable patients after related and unrelated HSCT, respectively. With a median follow up of 4,4 years (range 0,01-10,6) and 3,8 years (0,5-8,5) the 5-year probability of overall survival (OS) and disease free survival (DFS) is 74% and 68% in the related and 85% and 52% in the unrelated group. Considering only patients transplanted within one year after diagnosis TRM and RR show no difference after related and unrelated HSCT. The 5-year probability of OS and DFS is 80% and 73% versus 94% and 67% in the related and unrelated cohort, respectively. Outcome after unrelated stem cell transplantation has improved over years, mainly due to more specific HLA typing methods and better supportive care. Though introduction of imatinib has changed management of CML patients dramatically, there are still indications for allogeneic stem cell transplantation in the course of disease. For those indications transplant with cells from an unrelated donor is an excellent alternative to related HSCT. Adverse effect of cytotoxic pre-treatment for myelofibrosis on survival after allogeneic haematopoietic stem cell transplantation M. Ditschkowski* (1), R. Trenschel (1) , N.K. Steckel (1) , A.H. Elmaagacli (1) , C. Schulte (1) , H. Ottinger (1) , D.W. Beelen (1) ( Primary myelofibrosis (PMF), essential thrombocythemia (ET) and polycythemia vera (PV) can be cured by allogeneic haematopoietic stem cell transplantation (HSCT) but TRM might be substantial. 48 patients (pts) (27 male, 21 female; median age=48 ys) with PMF (n=28), post-PV myelofibrosis (MF) (n=6) and post-ET MF (n=8) or blast phase disease (n=6) underwent allogeneic HSCT between 1994 and 2007. Myeloablative conditioning was based on TBI (n=42), melphalan (n=1) or treosulfan (n=5). Donors were HLAidentical related (n=22), matched unrelated (n=17), mismatched sibling (n=3) or mismatched unrelated (n=6). Transplants consisted of unmanipulated PBSC (n=42), bone marrow (n=5) or purified CD34+ cells (n=1). CSA+MTX (n=29), CSA+MTX+Anti-thymocyte-globulin (ATG) (n=4), Alemtuzumab+CSA (n=13), in vitro T-cell depletion (n=1) or CSA+MMF (n=1) were used as GVHD-prophylaxis. 42 pts were screened for JAK2 pre-transplant. Of 22 pts without cytotoxic treatment (group 1) 15 pts were in non-advanced and 7 pts in advanced disease stages. 26 pts had cytoreductive therapy (group 2) prior to HSCT (10 nonadvanced, 16 advanced). In both groups median Lille-score was 1 and median marrow fibrosis grade 2. 16 pts had splenectomy (group 1: 5; group 2: 11; n.s.), JAK2 gene mutation was found in 46% of all pts (group 1: 41%; group 2: 46%). After a median follow up of 26 (3-154) months (group 1: 20; group 2: 36) survival probability was 36% for all pts. Survival was significantly (p<0.01) higher in group 1 compared with group 2 (73% vs. 40% after 2 years) resulting in a relative mortality risk of 3.2 for group 2 compared with group 1 pts. Considering only non-advanced pts of group 1 and 2 (n= 25) no significant influence of cytotoxic pre-treatment on posttransplant survival was found. Of all advanced pts (n= 23) the pre-treated (n=16) showed a survival probability of only 15% while for advanced pts without cytotoxic pre-treatment (n=7) it was 54 % (p=0.032). One year TRM was 23% in group 1 and 31% in group 2 (n.s.) and correlated with the presence of peripheral blasts prior to HSCT, advanced disease stage and occurrence of chronic GVHD after transplant. Relapse rate was significantly higher in group 2 (p<0.05). Our data indicate that among advanced disease pts cytotoxic pre-treatment seems to be an adverse prognostic factor for transplant outcome after allogeneic myeloablative HSCT for myelofibrosis. Thus, in suitable pts allogeneic HSCT should be performed before cytotoxic treatment becomes necessary. Long-term observation of 99 CML cases transplanted from matched sibling or alternative donors: single-centre experience A. Lange*, M. Sedzimirska, K. Suchnicki, D. Duda, E. Nowak, S. Madej, J. Lange Lower Silesian Center for Cellular Transplantation (Wroclaw, PL) CML is at present a rare indication for allogeneic HSCT. However, in some cases imatinib toxicity or lack of positive effects of tyrosine kinase inhibitors may prompt us to perform allo HSCT. Therefore it is good to know what transplant modality could be advocated in this situation. In this study we present the data of allo HSCT performed in one center in two cohorts composed of patients receiving myeloablative (cohort 1: Busulfan 16 mg/kg b.w., Cyclophosphamide 120 mg/kg b.w. + anti-thymocyte globulin -ATG 20 mg/kg b.w. in alternative donors transplantation) and non myeloablative (cohort 2: Bu 8 mg/kg b.w., Flu 120 mg/kg, ATG usually 10 mg/kg b.w.) preparative regimens. Patients characteristics in cohort 1 and cohort 2 are as follow: n=40 (n=59), 1st CP: 31 (50), > 1st CP: 2 (9), ACCP or BC: 7 (0), sib/alternative donors: 30/10 (24/35), marrow/PBPC: 35/5 (14/45), years of transplant: 1989-2000 (1999-2007) . The results of the transplant procedure when compared cohort 1 vs. cohort 2 differed with respect to the incidence of transplant related toxicity ≥ III grade (10/40 vs. 2/59, p<0,001), but not with respect to aGvHD (19/40 vs. 19/59, p=0 .125), extensive cGvHD (11/40 vs. 15/59, p=0.818) and relapses incidence (5/40 vs. 9/59, p=0,700). Overall survival of RIC patients (cohort 2) was significantly better than myeloablative patients (cohort 1) (Log-rank test, p=0.017). From an univariate analysis it became apparent that the results of the procedure was affected by: time from diagnosis to transplantation (p<0.017), years of transplant (p<0.005), stage of the disease (p<0.025) and intensity of conditioning (p<0.018). There are several bias behind the latter comparisons: myeloablative and RIC transplantations were performed in two different time periods what may affect the outcome due to the improvement in supportive care, on the other hand cohort 2 patients received more frequently VUD then sibling transplantation. Therefore, a multivariate analysis was performed which documented that only time from the diagnosis to alloSCT (p<0.009) and intensity of conditioning (p<0.007) independently affected the outcome. In conclusion patients transplanted under RIC with a low dose of ATG enjoy in 62% long term survival affected by 25% incidence of extensive cGvHD independent whether they received sibling or MUD transplant providing high resolution typing. Haematopoetic stem cell transplantation in CLL: promising results using allogeneic reduced intensity conditioning compared with an autologous setting S. Reitter*, W. Zinke-Cerwenka, A. Zebisch, S. Sormann, H. Sill, W. Linkesch Medical University Graz (Graz, AT) Background: We retrospectively evaluated the role of autologous stem cell transplantation (ASCT) and allogeneic reduced intensity transplantation (allo-RIC) in patients with chronic lymphatic leukaemia (CLL). Methods: Between 1999 and 2006 14 patients (11 male, 3 female) with CLL underwent ASCT, whereas allo-RIC was performed in six patients ( 4 male, 2 female). Four of those allo-RIC were sibling-transplantations, two of them were match-unrelated donor transplantations (MUD-SCT). One patient underwent ASCT and MUD-RIC which was due to PD after ASCT and two allo-sibling RICs were performed in another patient, which was due to primary graft failure after the first stem cell transplantation. For ASCT mean age at time of transplantation was 55 years (range: 42-67) and the mean time from diagnosis of CLL to ASCT was 50 months (range: 5-140). Pre-transplant remission status before ASCT was CR in three patients, VGPR in two patients, PR in five patients, and PD in four patients. For ASCT conditioning regimen consisted of fractionated TBI (8-12 Gy) and cyclophosphamide (120 mg/kg). For allo-RIC mean age at time of transplantation was 51 years (range: 39-59) and mean time from diagnosis of CLL to allo-RIC was 40 months (range: 9-83). Before allo-RIC, one patient was in CR, one patient in VGPR, and four patients in PR. For allo-RIC conditioning therapy consisted of fludarabine (150 mg/m²) and TBI (4 Gy), or fludarabine (90 S310 mg/m²) and cyclophosphamide (900 mg/m²). GVHD prophylaxis consisted of cyclosporine A and mycophenolate mofetil. For MUD transplantation low dose ATG Fresenius (4x5 mg/kg days -4 through -1) was added to this regimen. Results: There was no transplantation related mortality (day 100) neither in the ASCT nor in the allo-RIC group. With a mean follow up of 44 months (range: 14-98) for ASCT the CLL progression free survival is 50%. The overall survival in patients undergoing ASCT is 64.2%. Mean follow up in allo-RICs is 40 months (range: 20-61) and shows progression free survival of 83.3%. Overall survival reaches 100% in the allo-RIC group. Remarkably, no single patient treated with allo-RIC experienced acute or chronic GVHD. Conclusion: Our data suggest that allo-RIC carefully applied in selected patients with CLL displays a promising therapeutic option. Primary chemotherapy (PC) is a standard approach for the treatment of patients with breast cancer to achieve a tumor shrinkage and to allow a breast-conserving surgery. Moreover locally advanced disease can become operable after PC. Pathological complete remission after PC has been showed to identify a good prognosis group with a 5-year DFS of 80%. Five-year DFS is about 40 % for pts who do not achieve more then a partial response and with axillary positive lymph nodes (LN) > 4. For these high-risk pts the optimal management after surgery is still unclear. We have performed a retrospective analysis on pts with locally advanced breast cancer treated with high-dose chemotherapy (HDC) and autologous hemopoietic support after primary PC between Jan 1990 and Dec 2005 at 22 italian GITMO centers. Objective of the study was to analyse the outcome of the pts according to prognostic factors including age, clinical stage, hormonal receptor (HR) and Her-2 expression, menopausal status, axillary positive LN after PC, HDC procedure. Data on 234 pts were collected from the GITMO registry and additional data from the investigators. HR status was available in 200 pts, Her-2 status in 138 pts, menopausal status in 185 pts, number of axillary positive LN in 205 pts.. Median age was 45 yrs (26-63); postmenopausal pts were 62.7 %, HR+ 67 %, Her-2+ 42.7 %, multiple transplants were performed in 36 % of the pts. Thirty pts (13 %) had received HDC as part of a primary treatment. Median number of axillary positive LN after PC was 12 (range 4-53). Pts with HR+ status were given adjuvant hormone therapy. At a median FU of 72 months, 5years DFS and OS were 60 % and 75 % respectively. Treatment-related mortality was 0.4 %. There was no difference in outcome between the pts receiving HDC before or after surgery and between patients with HR+ vs. HR-. Seven-years DFS and OS for the group 4-9 LN, 10-19 LN, > 20 LN were 74 %, 57 %, 47% (p: 0.002) and 75 %, 70 %, 45 % (p: 0.007) respectively. DFS was significantly prolonged in Her-2+ pts vs. Her-2-(p: 0.001) but the difference in OS was not statistical significant (p: 0.07). DFS and OS were significantly prolonged for pts receiving multiple vs. single transplant, and Her-2+ vs. HR+Her-2-vs. HR-Her-2-tumors. In conclusion HDC is a feasible procedure as a part of a multidisciplinary approach for pts with locally advanced primary breast cancer. Optimal treatment for high-risk pts after PC remains to be evaluated in a randomized study. Allogeneic stem cell transplantation in ovarian cancer: the EBMT experience J.O. Bay*, R. Tabrizi, D. Blaise, P. Viens, G. Ehninger, M. Bornhauser , S. Slavin, G. Rosti, T. Demirer, M Objectives: Preliminary results suggest that AHSCT (Allogeneic Hematopoietic Stem Cell Transplantation) in ovarian cancer is a feasible procedure and might be of potential therapeutic benefit when associated with GvHD (Graft versus Host Disease). It is still difficult to evaluate the potential benefit of AHSCT in ovarian cancer. Objective of this study was to assess the potential correlation between GvHD and patient overall survival (OS). Methods: From November 1995 to November 2005 AHSCT in ovarian cancer reported to the EBMT database were retrospectively studied. Results: The median age at transplantation was 50.5 years [range 31-63] and median time from diagnosis to transplant was 41.5 months [range 7-117]. Before transplants, 13 diseases were progressive, 11 stable, 7 in partial and 1 in complete response. Median number of prior chemotherapy line was of 3 [2] [3] [4] [5] [6] [7] . All donors were genoidentical with a median age of 46 years . Graft source was PBSC in 84% of grafts. Two AHSCT have been performed with myeloablative regimen, all the others were non myeloablative. Hematopoietic engraftment was available only for 30 allografts because of early patient deaths. The median time to reach an absolute granulocyte count of 1.0 x 109/l was 14 days [range 11-24] and a platelet count of 20 x 109/l was 12 days [range 9-42]. Platelet count did not decrease below 20 x 109/l in 5 allografts. Thirty grafts were evaluable for tumor response. Objective response was observed in 50% (95% CI, 32.1-67.9) of patients which was significantly higher in patients who had received DLI (86%, 95% CI, 67-96 versus 37.5%, 95% CI, 20.7-54.3; p=0.041). Although chronic GvH and acute GvH grade 2-4 seemed to be more frequent in patients with objective response than in those with no disease regression, difference failed to reach statistical significance (p = 0.16, p=0.072 respectively). With a median follow-up of 9.8 months , the median overall survival was of 9.8 months with a 12 months OS of 32% (95% CI, 14.1%-49.9%). The patients group with cGvH, presented significant improvement of OS (23.4 months versus 5.2 months, p= 0.0089). Conclusion: Our study results suggest a graft-versus ovarian effect which is correlated with OS and potentially to tumor response. Long-term outcome in adult Ewing's sarcoma patients treated with high-dose chemotherapy: results of a retrospective analysis from the EBMT A. Bertuzzi* (1), A. Tienghi (2) , F. Peccatori (3) Objectives: To retrospectively evaluate the impact of highdose chemotherapy (HDCT) on outcome of Ewing's sarcoma patients (pts). Methods: One-hundred-seventy-four EBMT transplant Centres enrolled 604 pts on a period of 27 yrs (from 1977 to 2003) . To minimize the impact of missing data, only complete data of pts enrolled from 1995 (n=181) were considered for this analysis. Results: The characteristics of 180 pts were: 59 females and 121 males, with a median age at transplantation of 26 yrs (range 18-64). Metastatic disease was diagnosed in 45% of pts. Transplantation was offered at diagnosis after induction CT to 124 pts (HDCT-D), whereas 56 pts received transplantation at relapse (HDCT-R). Considering the whole population, no statistically significant difference in terms of overall survival (OS) was observed for stage at diagnosis and age. With a median follow-up of 32 months (range 6-233), 2-yr OS was 60% vs 50% (p 0.047) in HDCT-D group and HDCT-R group, respectively. We analysed the treatment response in the two groups (HDCT-D/HDCT-R) according to the status of disease at transplantation (CR 57%/52%, PR 36%/36%, SD 6%/7% and PD 1%/4%). Considering the best response at 100 days from transplantation the overall response rate (ORR) was 67% (55% CR, 10% PR, 2% SD) vs 64% (46% CR, 16% PR, 2% SD) for HDCT-D and HDCT-R, respectively. In univariate analysis the status of disease at transplantation, busulphan as conditioning regimen and the best response at 100 days were statistically significant for OS for HDCT-D group. In HDCT-R group, age and busulphan were statistically significant for OS. Conclusion: Although in this series the ORR at 100 days were similar, pts who received trasplantation at diagnosis had a statistically significant better outcome. Noteworthy, busulphan in the conditioning regimen showed a significant impact on OS in both subgroups of patients. Further analysis from prospective studies are warranted to confirm these retrospective data. Intensive induction chemotherapy in poor-prognosis adult pPNET patients: a single-centre experience A. Bertuzzi*, E. Stroppa, V. Quagliuolo, G. Gullo, L. Siracusano, L. Castagna, L. Giordano, A. Santoro Istituto Clinico Humanitas (Rozzano (Milan) , IT) Objectives and methods: To evaluate retrospectively the impact of intensive versus conventional chemotherapy (CT), as induction treatment before high dose chemotherapy (HDCT), in the treatment of poor-prognosis (local advanced, metastatic or relapsed) pPNET patients (pts). Conventionaldose induction CT (CDICT) consisted of 3-5 cycles of ifosfamide/vincristine/epirubicin (IVE) followed by high dose cyclophosphamide or vepesid to mobilize peripheral blood stem cells (PBSC). Intensive-dose induction CT (IDICT) consisted of 2-5 cycles of IVE alternating 1-3 cycles of carboplatin/cyclophosphamide/vepesid (mCAPEC) with reinfusion of PBSC mobilized after the first cycle of IVE. Only responsive pts (CR, PR, SD) after induction CT proceeded to HDCT. Conditioning regimens included mitoxantrone 60 mg/mq (or thiotepa 600 mg/mq) plus melphalan 160 mg/mq or melphalan 200 mg/mq or busulphan 12mg/kg plus cyclophosphamide 120mg/kg. In case of local advanced disease, surgery and radiotherapy were performed after induction CT or at the end of all treatments. Results: Thirty-two consecutive pts (16 CDICT, 16 IDICT) from 1997 to 2006 were treated at our Institution out of clinical trial. Characteristics of all pts were: median age 30 yrs (range 13-69), local advanced disease 44%, metastastic disease 44%, relapsed disease 12%. Overall response rates (ORR) after CDICT and IDICT were respectively 56% (CR 6%, PR 44%, SD 6%) vs 69% (CR 25%, PR 31%, SD 13%). Five pts are not evaluable for response due to radical surgery at diagnosis. HDCT was performed in 23 responsive pts (72%). At the end of treatment (induction CT plus HDCT) ORR was 69% (CR 63%, PR 6%) and 57% (CR 37%, PR 14%) in CDICT vs IDICT groups, respectively. With a median follow-up of 18 months (range 2-117), median time to progression (TTP) and median OS were 16 vs 11 months and 18 vs 13 months for CDICT vs IDICT groups, respectively. Only one transplantrelated death was observed in IDICT group. Conclusions: In our experience, IDICT achieves a higher ORR than CDICT but also an unexpectedly higher rate of early relapses was observed. The causes of this phenomenon are now under investigation by our group. These poor results do not support the routine use of this myelosuppressive CT with PBSC support as part of induction phase of treatment. Alloreactivity and antitumour activity segregate within two distinct subsets of cytokine-induced killer cells: implications for their infusion across major HLA-barriers D. Sangiolo* (1), E. Martinuzzi (2) , M. Todorovic (2) , K. Vitaggio (1) , A. Vallario (2) , N. Jordaney (2) , F. Carnevale-Schianca (2) Cytokine induced killer (CIK) cells are ex-vivo expanded and activated lymphocytes, expressing both T and NK surface markers and endowed with a broad non-MHC restricted antitumor capacity. Donor-derived Cytokine-Induced Killer (CIK) can be infused as adoptive immunotherapy after hematopoietic cell transplant (HCT). Promising results were recently reported in HLA-identical HCT, where mild graft versus host (GVH) events were observed. To extend this strategy across major HLA-barriers (e.g. HLA-haploidentical HCT), further studies on CIK cells alloreactivity are needed. We hypothesized that alloreactivity and antitumor activity of CIK cells segregates within two different cell subsets and could consequently be separated according to CD56 and CD3 expression. We tested CIK cells expanded from 7 patients who underwent HCT as treatment of metastatic colorectal cancer. CIK cells were expanded in vitro from peripheral blood mononuclear cells (PBMC) cultured with timed addition IL2, IFN-a and anti-CD3 antibody. The alloreactive proliferation of CIK cells against normal HLA-mismatched PBMC was measured by a 3H-thymidine incorporation assay. Cytotoxicity of CIK cells against colon carcinoma cell lines and normal allogeneic PBMCs was determined by a standard Cr51 release reaction. We found that CIK cells maintained their alloreactivity across major HLA-barriers when tested as bulk population; after CD56 positive selection, antitumor activity was restricted to the CD3+CD56+ cell fraction (Fig.1) , and alloreactivity versus HLA-mismatched PBMC was restricted to the CD3+CD56-cell fraction (Fig.2) . Bulk CIK cells from engrafted patients did not exhibit alloreactivity in response to host-or donor-derived PBMC, confirming their low potential for GVH across minor HLA-barriers. Moreover we tested if CIK cells expanded from engrafted patients after HCT were as effective as donor derived ones and could be considered as an alternative option. The expansion rate and tumor cell killing was comparable to that observed in sibling donors. In conclusion, depletion of CD3+CD56-cells might reduce the risk of GVH without affecting the tumor killing capacity, and could help extending CIK infusions across major HLA-barriers. Engrafted patients after HCT could also be considered as an effective alternative option to donor-derived CIK cells. Reduced-intensity conditioning for allograft after cytoreductive autograft in metastatic breast cancer A.M. Carella, S. Nati, A. Congiu Division of Hematology (Genoa, IT) The benefits of allografting noted in some malignant diseases might be safely extended to metastatic breast cancer by a combination of cytoreduction with high-dose chemotherapy (HDT) and autologous stem-cell transplant (ASCT) with graftversus-tumour effect mediated by transplanted donor immune cells with nonmyeloablative allografting (reduced intensity conditioning transplantation, RICT). Twenty patients with heavily pretreated disease were given tandem transplants. Sixteen patients sustained donor engraftment. Four had partial remission after HDT and ASCT and complete remission after RICT; they achieved full chimerism and all developed graft-versus-host disease (GVHD) before regression of cancer. Another patient did not respond to HDT and ASCT but had partial remission after RICT, giving an overall response rate of 24%. Seven patients had grade II or higher acute GVHD and nine had extensive chronic GVHD. No nonrelapse-related deaths occurred during the first 100 days. Four patients died of extensive cGVHD/infections. Six patients died early from disease progression (<4 months after RICT). At October 2007 six patients (31%) are alive at a median of 2035 days (range, 270-3420): 3 patients achieved complete remission and are now alive CR at 270, 2580 and 3420 day; one patient in CR (2330) developed colon cancer and 2 patients are in stable disease at 1350 and 1740 days. The incorporation of high-dose paclitaxel as a part of sequential high-dose chemotherapy and autologous stem cell transplantation in patients with germ cell tumours, a phase II study G. Koumakis, N. Tsoukalas*, D. Tryfonopoulos, K. Papadimitriou, V. Barbounis, I. Filis, S. Demiri, G. Lipas, N. Pistamaltzian, M. Vassilomanolakis, S. Droufakou, C. Panopoulos, M. Moraki, A. Efremidis "Saint Savvas" Anticancer Hospital (Athens, GR) Background: To determine the activity of sequential high dose chemotherapy incorporating paclitaxel, in patients with resistant germ cell tumors. Materials and Methods: Eighteen patients (17M/1F), median age 33 years (range 16 to 50) with resistant germ cell tumors were enrolled. Following standard premedication Paclitaxel (400mg/m 2 to 560mg/m 2 ) was infused over 24 hours. About 1/3 of previously collected and cryopreserved peripheral blood stem cells (PBSC's) were transfused after 72 hours. Following hematologic reconstitution second high dose chemotherapy was infused, consisting of Carboplatin 1200mg/m 2 , Etoposide 1200mg/m 2 and Melphalan 120mg/m 2 over 72 hours. Remaining 2/3 of cryopreserved PBSC's were transfused 72 hours after megatherapy. Results: Overall response rate was 77,78% (7 CR, 7PR) whereas 3 SD and 1 PD were observed. Of 8 non-responding to conventional treatment patients 2 converted to CR, 5 to PR and 1 showed PD. Two patients who were in CR at transplantation remained in CR and of 8 patients in PR after conventional treatment 3 showed CR, 2 PR, and 3 SD. Main non-hematologic grade III-IV toxicities consisted of neuropathy (12.5%), mucositis (6.3%) and febrile neutropenia (12.5%) after paclitaxel and mucositis (31.3%), nausea-vomiting (12.5%) and febrile neutropenia (50%) after carboplatin, etoposide and melphalan. Mean TTP was 20,38 months (SE 4,46) and mean OS was 29,93 months (SE 4, 40) . Incorporation of high-dose paclitaxel chemotherapy in sequential double graft programs appears to benefit patients with resistant germ cell tumors and bears manageable toxicity. Fludarabine/total body irradiation (TBI) with allogeneic peripheral blood haematopoietic stem cell transplantation is ineffective in metastatic renal cell cancer and has a high rate of graft failure F. Gigli*, R. Pastano, C. Rabascio, F. Bertolini, L. Calabrese, G. Martinelli, F. Peccatori European Institute of Oncology (Milan, IT) Metastatic renal cancer (MRC) is a fatal disease with a median overall survival of only 11 months. In the last decade, allogeneic peripheral blood haematopoietic transplantion (AlloPBSCT) with reduced intensity conditioning (RIC) has been used with the aim of eliciting a potentiated host anti-tumor response and increase disease control. Here we report our experience from April 2003 to November 2005 with 11 patients (pts), 7 males and 4 females, median age 51 yrs (34-67) with Interferon/Interleukin-2 resistant MRC treated with RIC followed by fully matched sibling AlloPBSCT. Conditioning regimen consisted of Fludarabine (30 mg/mq d -4 to-2) and single fraction 200 cGy total body irradiation (TBI) in 8/11 pts. In the remaing 3 pts, Endoxan (60mg/Kg from day -7 to -6) substituted low dose TBI. Fully matched sibling AlloPBSCT (median cell dose 5.64 x106/Kg) was given after conditioning. Graft vs host disease (GVHD prophylaxis consisted of Mycophenolate Mofetil 15mg/Kgx2/daily from day 0 to +28 and Cyclosporin targeted to a serum level of 300-400 ng/dL from day -3 to day +36, with subsequent taper. At transplant, all pts were in progression after cytokine containing regimens, with a median time from diagnosis of 15.8 months. Six out of 11 pts had bone metastases, calcium was normal in all pts, 1 had elevated LDH, 9/11 had anemia. The average Motzer score was 2, with 2 pts with an ECOG performance status >2. At day +28, 9/11 pts had a full donor engraftment, while 2/11 pts had <20% engraftment. In these pts, mixed chimerism did not improve after Cyclosporin withdrawal. Two pts who fully engrafted at day +28, eventually lost engraftment, with an overall graft failure of 36% (4/11). In the engrafted pts there was a trend for late donor T cell engraftment compared with granulocytes engraftment. Grade 3-4 acute GVHD occurred in 1 patient, who eventually died due to progressive disease (PD). No fatal infections were observed with a +100 TRM=0%. All pts progressed after transplant and died because of PD. Median time from transplant to progression was 41 days and median time from transplant to death was 97 days (24-777). In 1/11 pts DLI was performed, without any GVHD or tumour response. These data suggest the limited role of Fludarabine/TBI regimen in pts with advanced/resistant MRC. A possible explanation can be found in the high rate of graft rejection, in the low incidence of GVHD and subsequent graft vs tumour effect and in poor patient selection. Allogeneic haematopoietic stem cell transplantation for metastatic breast cancer: the GITMO experience A. Ballestrero*, A.M. Carella, M. Musso, M. Bregni, L. Castagna, S. Siena, M. Martino, F. Blandino, G. Da Prada, B. Bruno, D. Boy, P In the field of allogeneic stem cell transplantation (ASCT), small series of patients (pts) with metastatic breast cancer (MBC) have been reported over the last decade. Clinical responses have been documented suggesting that a graftversus tumor effect exists against BC. Aim of the present study is to evaluate toxicity and efficacy of allogeneic haematopoietic stem cell transplantation (ASCT) in the cohort of pts who underwent this procedure in Italy and were registered at the national database. Between 1995 and 2006, 62 women with heavily pre-treated MBC received ASCT from an HLA-identical sibling in 17 Italian centers. The median age of patients at transplantation was 43 years (range 27-58). Nearly half of pts underwent high dose chemotherapy with autologous stem cell rescue before ASCT in order to achieve maximum tumour cytoreduction before immunotherapy. The median time from BC diagnosis and ASCT was 51 months. The majority of pts received reduced intensity fludarabine-based conditioning regimen. Detailed information regarding these pts was obtained from the GITMO Registry and all Centers reporting activity in this setting are cutrently being contacted for additional detailed data on toxicity, including GVHD, and outcome. The study is ongoing and results will be presented at the meeting. The study is conducted on behalf of Gruppo Italiano per il Trapianto di Midollo, di Cellule Staminali Emopoietiche e di Terapia Cellulare (GITMO) S314 Cellular and gene therapies P1007 Allogeneic haematopoietic stem cell transplantation from unrelated donors for chronic lymphocytic leukaemia: a population-matched analysis from the EBMT registry M. Michallet (1) , Q.H. Le (1) , P. Dreger (2) , A. Van Biezen (2), D. Milligan (2) , D. Niederwieser (2) , L. Sutton (2) , A. Buzyn (2) , R. Tapani (2) , A. Nagler (2), J. Schetelig (2) , V. Koza (2) , T. De Witte (2) (1) Hôpital Edouard Herriot (LYON, FR) ; (2) EBMT CLL Subcommittee CLWP (Leiden, NL) We performed from the EBMT registry a population-matched analysis of allogeneic for chronic lymphocytic leukaemia (CLL) after either myelo-ablative or non myelo-ablative conditioning. Two hundred ninety four patients were studied, 161 from HLA identical siblings and 133 from unrelated donors. We matched the centre, the type of conditioning (standard or reduced intensity), recipient gender and age, haematopoietic stem cell (HSC) source, year of transplantation and we defined almost 3 common factors. We then obtained 120 pairs matched allotransplantations, 60 in each group (Table 1) . In group 1, on 59 evaluable patients, 28(47%) developed acute GVHD with 15(25%) ≥grade II [12(20%) grade II and 3(5%) grade IV], on 58 evaluable patients, 21(36%) patients developed cGVHD [10limited and 11(19%) extensive]. At the last follow-up, 21(35%) patients relapsed and, 23 patients died and 37 are alive. In group 2, on 57 evaluable patients, 37(65%) patients developed acute GVHD with 24(42%) ≥grade II [15(26%) grade II, 7(12%) grade III and 2(4%) grade IV], on 50 evaluable patients 30(60%) developed cGVHD (15 limited and 15(30%) extensive). At the last follow-up 10 patients (17%) relapsed, 27died and 33 are alive. We found a very significant difference concerning the non relapse mortality with 12% (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) for group1 and 39% for group2 (p<0.001). In addition, we observed no significant difference concerning the probability of overall survival and event-free survival at 3 years: for group 1, 66% (54-81) and 56%(44-72) respectively with a median follow-up of 45 months and for group2, 52% (39-68) and 46% (34-62) respectively with a median follow-up of 35 months (6-76) (p=0.11 and p=0.24). In conclusion, this study showed no difference between group 1 and group 2 due to higher non relapse mortality after unrelated transplantation and higher relapse rate after related transplantation for CLL. Minimum requirements for fully humanised clinical scale propagation of multipotent mesenchymal stromal cells K. Schallmoser, E. Rohde, A. Reinisch, A. Obenauf, M. Speicher, C. Bartmann, G. Lanzer, W. Linkesch, D. Strunk Medical University Graz (Graz, AT) Ex vivo propagation of human multipotent mesenchymal stromal cells (MSC) is currently considered as a prerequisite for MSC therapy. This study was performed to define the minimum requirements for producing sufficient MSC numbers for therapeutic application in a completely animal serum-free standard system from small bone marrow aspiration volumes within clinically acceptable short time. In compliance to good manufacturing practice we established a time and resource saving efficient procedure for MSC propagation. Bone marrow was seeded without manipulation directly in pooled human platelet lysate (pHPL) and Lglutamine supplemented minimum essential medium without antibiotics. Clinical scale expanded MSCs were harvested already after primary culture. MSC quality, identity, purity and function were assessed according to a defined panel of release criteria. Complete genomic hybridization was used to determine genetic stability of the expanded cells. Starting from 790 to 1130 million nucleated cells in aspiration volumes of 14 to 17mL (3 male donors; age: 30, 36, and 47 years; 1 female donor; age 13 years), four clinical scale expansions resulted in 906, 963, 256 and 982 million MSCs, respectively. This yield was achieved within a single culture period of 11 to 16 days. MSC quantity represents one to four application doses of >200 million MSCs each. MSC viability was 95-97.7% and flow cytometry revealed a CD73+/CD90+/CD105+ phenotype with less than 2% hematopoietic cell contamination. Bacterial, fungal and mycoplasma contamination was excluded by repeated negative testing. Endotoxin levels remained below 0.05 EU/mL. Initial plating density inversely correlated with the fibroblast colony-forming unit (CFU-F) frequency. The differentiation potential of MSC into adipo-, chondro-and osteogenic lineages was verified. Complete genomic hybridization showed balanced profiles for all four MSC samples after expansion. The opportunity to achieve up to four application doses of MSCs without animal serum in a standardized single culture phase procedure within 2 weeks with a minimum of manipulation and without antibiotics supports therapeutic approaches that depend on the fast and safe availability of sufficient MSC doses in the clinical setting. R.M. Gonzalo-Daganzo*, T. Martin-Donaire, C. Regidor, R. Sánchez, N. Panadero, Y. Gutiérrez, M. García-berciano, M.A. Rico, G. Bautista, I. Krsnik, E. Ruiz, R. Forés, E. Ojeda, B. Navarro, J.A. Garcia-Marco, I. Sanjuán, I. Millán, J.R. Cabrera, M.N. Fernández Hospital Universitario Puerta De Hierro (Madrid, ES) Our group has pioneered co-infusion of a low number of T-cell highly depleted mobilized haematopoietic stem cells (MHSC) from a third party donor (TPD) as a tool to support cord blood transplant (CBT) engraftment and full chimerism in adults with high risk haematologic malignancies ("dual transplants", Haematologica 2006; 9:640-8). The conditioning regimens used are myeloablative although of reduced extrahaematological toxicity. With this strategy we have obtained very favourable results regarding both CBT engraftment and full chimerism. We are now evaluating the addition of other TPD cells in this setting, aiming to optimizing CBT engraftment and immune reconstitution. We report results of a pilot study to evaluate the use of TPD Multipotent Mesenchymal Stromal cells (MSC), designed to evaluate tolerance, effect on CB engraftment kinetics, development of GVHD and immune reconstitution, as well as therapeutic effect of refractory GVHD. For this purpose we have ex-vivo expanded MSC from bone marrow samples from the same TPD under GMP conditions according, to the EBMT consortium protocol. Median total expansion time was 30 days (23-38) and the final number of MSC ranged from 80-335 x 10 6 (median 248). Nine patients have received the MSC immediately after the CB and the TPD-MHSC. The number of co-infused MSC ranged from 2.15 to 1.04 x 10 6 cells/kg (median 1.20) . No immediate adverse effects occurred. No beneficial effect has been observed on CB engraftment kinetics, both in terms of granulocyte and platelet recovery. Acute GVHD grade I-IV was developed by 5/9 patients and grade II/IV by 3/9, respectively compared to 29/46 and 11/46 in patients not receiving MSC. In 2 cases aGVHD proved refractory to conventional treatment and therapeutic infusions of MSC were given. Both patients achieved complete remission: one after a single infusion of 1.2 x 10 6 cells/kg; the other after 3 doses of 2.22, 1.88 and 1.15 x 10 6 cells/kg given along 1 month. Conclusion: We have observed beneficial effect of TPD MSC as a therapeutic tool for treatment of a-GVHD in the setting of "dual CB transplant", but not derived from its prophylactic use. No data are yet available about possible effects on immune reconstitution. Supported by Grants from the EC-6thFP (AlloStem IP), the Government of Spain (FIS) and CAM, the Gabrielle Rich Foundation and Vidacord. Inhibitory and activating killer immunoglobulin-like receptors (KIRs) regulate function of NK cells. KIR expressing T cells have been documented to play a role in several autoimmune conditions whereas expression of KIRs on NKT cells has not been studied so far. The goal of this study was to evaluate the KIR expression pattern in both T and NKT cells in the context of clinical outcome after alloHSCT. 40 adult patients treated with alloHSCT from either sibling (n=17) or unrelated (n=23) donors were included. Conditioning was myeloablative. The GVHD prophylaxis consisted of CsA, Mtx ± ATG. KIR expression on T cells (CD3+CD56-) and NKT cells (CD3+CD56+) was studied in all recipients on days +28,+56,+100,+180, and +365, using antibodies specific for KIR2DL1/DS1, KIR2DL2-3/DS2, and KIR3DL1/DS1. In addition the donors were tested KIR genotype. At all time-points T and NKT cells expressing KIRs, for which the donors had respective genes, could be detected in peripheral blood in a majority of alloHSCT recipients. The proportion of T and NKT cells bearing particular receptors varied strongly ( Table 1 ). The expression of KIR2DL1/DS1 on T and NKT cells was increased for patients with grade II-IV acute GVHD on day +100 (p=.008 and .002, respectively). On day +56, patients with acute GVHD had increased expression of KIR2DL2-3/DS2 on T and NKT cells (p=0.008 and 0.05, respectively) and on day +100 (p=.05 and .008, respectively). The increased expression was associated with skin and liver, but not intestinal manifestation of GVHD. On day +365 the presence of chronic GVHD correlated with increased proportion of T cells bearing KIR2DL1/DS1 (p=.02), which was particularly marked in case of extensive form of this complication (p=.006). We conclude that T cells and NKT cells expressing KIR2DL1/DS1 and KIR2DL2-3/DS2 may be involved in the pathogenesis of GVHD. They may play a role as either effector or regulatory cells. Elucidation of this issue requires further investigation, in particular discrimination between activating and inhibitory KIR phenotype. Genetic polymorphism of NQO1 is associated with an increased treatment-related mortality in patients undergoing allogeneic transplantation M. Koldehoff*, A.H. Elmaagacli, N.K. Steckel, D.W. Beelen University Hospital (Essen, DE) Background: The widely expressed detoxification enzyme NAD(P)H:quinine oxidoreductase 1 (NQO1) is involved in the cellular response to oxidative stress and irradiation and protects cells against the mutagenicity from free radicals and toxic oxygen metabolities. NQO1 is subject to a genetic polymorphism (C609T) leading to a change in its amino acid sequence. Heterozygous individuals C/T have intermediate activity and homozygotes T/T are NQO1 deficient. The influence of genetic polymorphisms of NQO1 on patients in allogeneic stem cell transplantation (HSCT) recipients has so far not been evaluated. Methods: Here we genotyped in a retrospective study 198 patients (and their donors) for NQO1 genotype expression who underwent allogeneic transplantation for various diseases and analyzed their outcome. Genotyping of NQO1 was performed by real-time PCR using subsequent melting curve analysis. Results: 145 patients (73.2%) were genotyped as homozygous wild-type gene C/C, 48 patients (24.2%) were genotyped as heterozygous genotype C/T and five patients (2.5%) were genotype as homozygous gene mutation T/T. From the donors 147 donors (74.2%) were C/C, 50 donors (25.3%) were C/T, and one donor (0.5%) had a homozygous gene mutation T/T. Calculated genotype frequencies did not differ from those reported earlier by other studies in Caucasians. Five-year estimate of treatment-related mortality (TRM) was highest in genotype C/T-and T/T-patients with 39% +11% compared to homozygous wild-type gene C/Cpatients (21% + 3.9% [p<0.045]), whereas the five-year estimate for relapse or overall survival were not statistically different between the groups. No differences for five-year estimates of TRM, relapse rate, or OS were seen in patients with either genotype C/C-, C/T-or T/T-donors. No statistic differences were found in the incidence of acute GVHD grade 2-4 within the study groups. Conclusions: These results suggest that patients with genetic polymorphism of NQO1 do have an increased TRM after P1016 Amplifying graft-versus-tumour effect by donor regulatory T-cell depletion before donor lymphocytes infusion: a phase I/II clinical study S. Maury* (1), F. Lemoine (2) , M. Rosenzwajg (2) , Y. Hicheri (1) , J. Beaumont (1) , N. Azar (2) , M. Cherai (2) , H. Trebeden-Nègre (2) , C. Giverne (2) , A. Buzyn (3), J. Vernant (2) , C. Cordonnier (1) , D. Klatzmann (2) , J. Cohen (2) (1)Hopital Henri Mondor (Créteil, FR); (2) Background: We have previously shown that depletion of CD4+CD25+FoxP3+ regulatory T cells (Treg) enhances the alloreactivity of T lymphocytes, as attested by an accelerated GVHD after allogeneic hematopoietic stem cell transplantation (HSCT) in mice. We thus designed a clinical trial to test whether Treg-depleted donor lymphocytes infusion (dDLI) could induce an improved graft-versus-tumor (GVT) effect in patients (pts) refractory to standard DLI (stdDLI) for treatment of relapse after HSCT. Patients and Methods: Eleven pts (21-54 years) with relapse of non-CML malignancies (5HD, 4 AML, 1 RAEB, 1 ALL) after HSCT from sibling (n=9) or matched unrelated donors (n=2) were included between Feb 2006 and Sept 2007. dDLI was administered after failure of 1 to 4 previous stdDLI of at least 107 CD3+ cells/kg, defined after a minimal follow-up of 2 months after the last injection. The absence of previous clinical manifestations of GVHD was required to be included. To prepare dDLI, CD25+ Treg were depleted from donor leukaphereses using anti-CD25 magnetic microbeads and a CliniMACS device (Myltenyi). In order to evidence the potential effect of dDLI, the dDLI cell dose was adjusted to be below or equal to the maximal cell dose previously received in stdDLI. Results: dDLI was administered after a mean interval of 9 (2-26) months from the last stdDLI. The CD25 depletion led to a CD3+CD25+ as well as a CD4+FoxP3+ cell-depletion constantly higher than 90% as assessed by flow cytometry and RQ-PCR. A mean dDLI cell dose of 4.5 (1-10)x107 CD3+ cells/kg was infused without any acute toxicity. Four pts had stable disease after dDLI (3 in PR and 1 CR) and 3 of them developed acute or chronic GVHD after 20 to 256 days following injection. Seven pts had progressive disease (PD) within the 3 months following injection and did not develop GVHD. Two of them received a second dDLI preceded by a lymphodepleting chemotherapy associating cyclophosphamide (Cy) and fludarabine (Flu) and developed acute GVHD after this second injection. Overall, out of 11 pts with no previous history of GVHD after HSCT or previous stdDLI, 5 developed clinical manifestations of alloreactivity after dDLI or Cy/Flu/dDLI. At a median follow-up of 13 months after dDLI, 6 of 11 pts are alive in PD (n=2), PR (n=3) or CR (n=1). Conclusion: Treg-depleted DLI may induce clinical manifestations of alloreactivity in pts with no past history of GVHD and might thus amplify the GVT effect in patients in relapse after HSCT. Background: mesenchymal stromal cells (MSC) are promising candidates for clinical cell therapy approaches because of their intriguing properties, i.e. high proliferation and differentiation capacity, potent immune-modulatory capability, stroma function, etc. MSC might be used to reconstitute the hematopoietic stroma in vivo (for example after high-dose therapy). However, in vivo homing and engraftment of transplanted human MSC has thus far not been conclusively demonstrated. Therefore, we studied whether MSC are capable of transendothelial migration, which is a prerequisite for homing. Materials and methods: Bone marrow (BM) mononuclear cells (MNC) were isolated by density gradient centrifugation. BM preparations enriched for primary MSC were obtained by antibody depletion (CD3,CD14,CD9,CD19,CD38b,CD66b) using the RosetteSep technology. CD271+ bone marrow cells were isolated by MACS. Culture-derived MSC were generated from MNC using standard methods. SDF1-induced transendothelial migration across an endothelial cell layer was assessed in vitro. Results: Bone marrow MNC and RosetteSep enriched cells (R-MNC) migrated across endothelium as indicated by SDF1induced migration rates of 17.6 ± 4.6% and 1.87 ± 0.9% for MNC and R-MNC, respectively, compared with spontaneous migration rates of 6.7 ± 5.6% (MNC) and 1.17 ± 0.9% (R-MSC). Flow cytometric analysis of transmigrated cells revealed that CD271 cells had migrated in both populations. In contrast, no transendothelial migration was observed for standard culture-derived MSC (0.03 ± 0.03% spontaneous versus 0.04 ± 0.04% SDF1-stimulated migration). Primary bone marrow CD271+ cells (positively selected by MACS, purity 95-97%) on the other hand clearly showed a response to SDF1 with SDF1-induced migration rates of 7.85 ± 2.9% compared to a spontaneous migration rate of 2.85 ± 0.94%. CD184 expression (CXCR4) could be demonstrated on the majority of transmigrated CD271+ cells. Finally, blocking experiments using anti-CXCR4 antibody reduced SDF1induced migration of CD271+ cells by approximately 50% indicating that SDF1 is an important chemotactic activity mediating its activity through the chemokine receptor CXCR4. Conclusion: Taken together, our data demonstrate that primary human bone marrow CD271+ cells but not culturederived MSC transmigrate across endothelium in response to SDF1. These results are of importance when aiming at designing strategies to reconstitute/repair the hematopoietic environment by intravenous cell transplantation. Phase II human trial of autologous mesenchymal stem cell transplantation for the treatment of decompensated cirrhosis K. Alimoghaddam*, M. Mohamadnejad, M. Bagheri, M. Bashtar, H. Ghanati, M. Mohyedin Bonab, A. Ghavamzadeh, R. Malekzadeh University of Tehran (Tehran, IR) Introduction: Liver transplantation is the standard treatment in decompensated cirrhosis; however, it has several limitations including high cost, and several complications. Recent animal works have shown that mesenchymal stem cell (MSC) infusion through the rat tail vein can lead to regression of liver fibrosis. But there has been no human trial of autologous MSC transplantation (Tx) in cirrhosis. The aim of this study was to evaluate safety and feasibility of MSC transplantation in decompensated cirrhosis. Aims & methods: After protocol approval by the ethic committee, 21 patients with decompensated cirrhosis were included. 50-100 ml of their bone marrow was aspirated and MSCs were cultured. After two months culture, 10 to 300 million MSCs obtained which 95% of the cells were viable (as assessed by trypan blue method) and 95% of the cells were morphologically and immunophenotypically MSCs. The MSCs were infused through a peripheral vein. Outcomes were changes in liver function tests, MELD score, and liver volume (as measured by CT volumetry) 5 months after Tx. During follow up, patients medications (e.g duretics, or propranolol) remained the same as before MSCs Tx. Results: For first four patients (1 male, 3 female) aged 30 to 56 were included. The etiology of cirrhosis was autoimmune hepatitis or cryptogenic. The follow up for the first patient has been completed, and other 3 patients have 2.5 to 4 months of follow up. In first patient, PT (18.5 to 13 seconds), and albumin (2.9 to 3.8 g/dL) normalized, and MELD score was decreased from 16 to 11. Her liver volume increased from 495 to 815 ml. In patient 2, PT decreased from 20.1 to 17.5. In patient 3, PT decreased from 19.1 to 15.7 seconds, and MELD score decreased from 17 to 14. Patient 4 has not yet been shown any significant improvement in liver function tests. The procedure was safe, and there was no any adverse effect. For other 17 patients we will present updated data in ebmt.2008. Conclusion: MSCs Tx is a novel approach to treat cirrhosis. For the first time, we showed that this procedure is safe and feasible, and signs of improvement found in some of our patients are promising From 1998 to 2002 8 patients with chronic or acute myeloid leukemia in first complete remission (2 CML, 4 AML, 2 sAML) have been transplanted according to myeloablative conditioning regimens from HLA-identical family donors with CD34-enriched hematopoietic stem cells without further immunosupression. Two of these patients received prophylactic, untransduced donor T-cells, 2 had leukemic relapse prior transfusion of donor leukocyte transfusion (day +193 and day +881 after HSCT), 1 of these died due to septic complications after aGvHD and extensive chronic GvHD 2 years after HSCT. Three patients of 4 receiving DLI developed aGvHD grade II or more and 2 developed chronic GvHD. Thus, in 2002 the standard transplantation protocol was adapted to include a prophylactic transfusion of gene-modified donor T-cells after HSCT, in order to induce graft-versusleukemia (GVL) and to investigate the possibility of controlling graft-versus-host disease (GvHD). Seven patients with AML and 2 patients with chronic myelogenous leukemia (CML) were included in this gene therapy trial. Donor T-cells were transduced with the replication-deficient retrovirus SFCMM-3 which expresses the herpes simplex thymidine-kinase (HSV-Tk) as a suicide gene and the truncated low affinity nerve growth factor receptor (Delta-LNGFR) for selection purposes (HSV-Tk DLI). SFCMM-3 transduced T-cells were detectable in all patients by PCR and FACS-analyses immediately after transfusion and during the follow up period for over 5 years (range: 1.2-5.8 years). Only 1/9 patients developed acute GvHD grade 1 of the skin at 56 days after the HSV-Tk DLI. To date 2 AML patients had leukemic relapse. Both were retransplanted with non-depleted PBSC, 1 died due to septic complications. Loss of bcr-abl gene expression was observed in patient UPN914, after an expansion of transduced cells. Eight of 9 patients are alive, well and 7 are in complete hematological or molecular remission. Dendritic leukaemia cell hybrids generate specific anti-leukaemia CTLs in vitro M. Shpringer*, R. Eshel*, B. Tartakpvsky, B. Katz, R. Ben-Yosef , A. Vexler , E. Rom, I. Fabian , E. Naparstek Tel-Aviv Sourasky Medical Center (Tel Aviv, IL) Background: Allogeneic stem cell transplantation (alloSCT) contributes significantly to better disease control in patients diagnosed with high risk AML. Nevertheless, a significant proportion of patients suffer from recurrence of the disease. For those high risk patients, novel therapeutic strategies based on cellular immunotherapy have been explored to improve the clinical outcome of alloSCT. However, in most cases the leukemia specific antigens are unknown, and hence the cellular or non-antigen specific immunotherapy is based preliminary on the administration of allogeneic, donor derived T lymphocytes (DLI), aiming to induce a clinically significant graft-versus-leukemia responses. Aim: The aim of our study was to induce a potent and specific anti-leukemia cytotoxic T lymphocyte (CTL) response, utilizing dendritic-leukemia cell hybrids, to treat leukemic relapse in patients after alloSCT. Such fusion cell vaccine has the advantage of presenting both known and unidentified leukemic antigens, in the context of co-stimulatory signals. Methods and results: Purified human monocyte-derived dendritic cells (DCs) were isolated from peripheral blood mononuclear cells of 5 healthy HLA-identical stem cell donors. Immature DC were successfully fused with recipients irradiated leukemic cells utilizing polyethylene glycol (PEG), and underwent further maturation in the presence of TNFalpha , IL-6 , IL-1beta , and PGE2. Fused population of DCleukemia was estimated by flow-cytometry using two membrane incorporated fluorescent dyes, and DAPI stain was utilized to confirm the true presence of DC -leukemia cell hybrids. Generation of leukemia specific donor CTLs was performed by co-culture of donor mononuclear cells with irradiated DCs-leukemia hybrids under IL-2 deprivation. T cells were then further expanded in culture, and tested for their specific in-vitro cytotoxic activity against the leukemia cells that was utilized as fusion partner by LDH cytotoxicity colorimetric assay. In 4 out of 5 cases we were able to demonstrate a significant and specific cytotoxic activity of the hybrids-primed CTLs against the patients leukemic cells. Conclusions: Our results clearly demonstrate that the hybrid vaccination approach in AML is technically feasible. Such specific anti-leukemic donor CTLs may be utilized to maximize the anti-tumor effects of DLI in patients relapsing after allogeneic transplantation. Dendritic leukaemia cell hybrids generate specific anti-leukaemia CTLs in vitro M. Shpringer*, R. Eshel*, B. Tartakpvsky, B. Katz, R. Ben-Yosef , A. Vexler , E. Rom, I. Fabian , E. Naparstek Tel-Aviv Sourasky Medical Center (Tel Aviv, IL) Background: Allogeneic stem cell transplantation (alloSCT) contributes significantly to better disease control in patients diagnosed with high risk AML. Nevertheless, a significant proportion of patients suffer from recurrence of the disease. For those high risk patients, novel therapeutic strategies based on cellular immunotherapy have been explored to improve the clinical outcome of alloSCT. However, in most cases the leukemia specific antigens are unknown, and hence the cellular or non-antigen specific immunotherapy is based preliminary on the administration of allogeneic, donor derived T lymphocytes (DLI), aiming to induce a clinically significant graft-versus-leukemia responses. Aim: The aim of our study was to induce a potent and specific anti-leukemia cytotoxic T lymphocyte (CTL) response, utilizing S320 dendritic-leukemia cell hybrids, to treat leukemic relapse in patients after alloSCT. Such fusion cell vaccine has the advantage of presenting both known and unidentified leukemic antigens, in the context of co-stimulatory signals. Methods and results: Purified human monocyte-derived dendritic cells (DCs) were isolated from peripheral blood mononuclear cells of 5 healthy HLA-identical stem cell donors. Immature DC were successfully fused with recipients irradiated leukemic cells utilizing polyethylene glycol (PEG), and underwent further maturation in the presence of TNFalpha , IL-6 , IL-1beta , and PGE2. Fused population of DCleukemia was estimated by flow-cytometry using two membrane incorporated fluorescent dyes, and DAPI stain was utilized to confirm the true presence of DC -leukemia cell hybrids. Generation of leukemia specific donor CTLs was performed by co-culture of donor mononuclear cells with irradiated DCs-leukemia hybrids under IL-2 deprivation. T cells were then further expanded in culture, and tested for their specific in-vitro cytotoxic activity against the leukemia cells that was utilized as fusion partner by LDH cytotoxicity colorimetric assay. In 4 out of 5 cases we were able to demonstrate a significant and specific cytotoxic activity of the hybrids-primed CTLs against the patients leukemic cells. Conclusions: Our results clearly demonstrate that the hybrid vaccination approach in AML is technically feasible. Such specific anti-leukemic donor CTLs may be utilized to maximize the anti-tumor effects of DLI in patients relapsing after allogeneic transplantation. The development of chronic GVHD (cGVHD) and its associated morbidity is one of the major limitations of allogeneic hematopoietic stem cell transplantation. Progress in treatment of cGVHD requires better understanding of the underlying pathophysiology including the development of animal models. We describe here two approaches for induction of cGVHD in a rat model. Methods: In the first experiment female LEW/BN rats (n=9) received 3x10 7 bone marrow (BM) cells depleted with MicroBeads against MHC II, OX52 and CD45R from female LEW-rats one day after total body irradiation (TBI) with 9.2 Gy. On day 10 animals received enriched 1x10 7 naive LEW spleen T-cells (CD45RC+CD4+). A second group (n=4) received the naive T-cells on day 14 instead. The second approach consisted of a double transplantation. First LEW/BN-rats received 4x10 7 BM cells from either LEW-Rats (n=14) or LEW/BN rats (n=14) one day after TBI with 5 Gy. Two months after the first transplantation all rats received a TBI with 7.5 Gy followed by an injection of 4x10 7 LEW BM cells and 1.5x10 7 LEW T-spleen cells (n=16) or 4x10 7 LEW BM cells and enriched naive spleen T-cells (CD45RC+CD4+) (n=12) one day after TBI. Enrichment was conducted by depletion of CD8a, CD45RA and gamma delta TCR positive cells with MicroBeads. Results: First approach: 6 out of 9 rats receiving spleen cells on day 10 developed cGVHD. The remaining 3 rats developed acute GVHD (aGVHD). All rats receiving naive T-cells on day 14 showed no or only mild signs of cGVHD and no signs of aGVHD. Second approach: All rats receiving LEW/BN BM cells at the first transplantation developed lethal aGVHD after the second transplantation in the double transplantation experiment regardless of the T-cell source (enriched naive or spleen Tcells) used for the second transplantation. 5 out of 8 rats receiving LEW BM cells during the first transplantation and whole spleen T-cell suspension in the second developed cGVHD, the 2 remaining rats developed mixed aGVHD and cGVHD. One animal died due to anaemia. 2 out of 6 animals receiving LEW BM cells during the first transplantation and enriched naive T-cells in the second developed cGVHD, the remaining 4 rats showed signs of mixed aGVHD and cGVHD. Conclusion: The two described models provide a tool to study the pathophysiology of cGVHD. While the first approach allows studies concerning the composition of donor lymphocyte grafts, the second focuses on studies on the role of antigen presenting cells. Infusion of IL-2 activated donor NK cells after haploidentical SCT reduces the incidence of high-grade GvHD L. Uharek* (1), C. Gentilini (1) , U. Hilbers (2) , V. Huppert (3) NK cell alloreactivity can mediate strong GvL effects following haploidentical stem cell transplantation (HSCT). In an attempt to further improve the antileukemic effectiveness of this approach, we have adoptively transferred donor NK cells during the early phase after transplantation. In addition, we activated the transferred NK-cell population ex vivo in short term cultures with high doses of IL-2 in order to enhance the antileukemic activity and inhibit the occurrence of severe GvHD. In a phase-II study, 19 patients (10 AML, 1 MDS, 1 HD, 2 CML, 3 ALL, 2 AL, median age 37 yrs, range 17-50 yrs) were transplanted in late phases of their disease and received purified NK cells from their haploidentical donors at day +2 after HSCT. Conditioning consisted of fTBI, Thiotepa, Fludarabine and OKT3. NK cells were isolated from the CD34fraction using an automated two-step procedure of CD3+ depletion and subsequent CD56+ selection. Eight patients received activated NK cells and 11 patients received unstimulated NK cells. Cells were activated by 16h incubation with IL-2. After selection and subsequent overnight activation, a mean number of 6.7 x 10 6 /kg NK cells was transferred at day 2 after transplantation. The mean number of contaminating CD3+ cells in the transfused NK product was 0.8 x 10 4 /kg. No differences in yield or number of contaminating T cells were observed between IL-2 activated and not activated NK cells. No severe acute toxicity attributable to NK cell infusion was observed in both groups of patients. Comparing the rate of high-grade GvHD revealed an interesting result. Whereas only one patient developed GvHD ≥ grade II after treatment with IL-2 activated NK cells, seven out of ten patients showed GvHD ≥ grade II after transfer of non-activated NK cells (p<0.05). Moreover, immunocytometric analysis of lymphocyte subpopulations at different time points after transplant revealed a long-lasting cellular immunodeficiency in all patients. As for the incidence of GvHD, there was also a striking difference in immune recovery between the two groups of patients. Patients receiving activated NK cells showed significantly lower numbers of NK-and T cells during the first months post transplant, whereas no differences in the number of granulocytes were present between the two groups. Based on these findings we can assume that the use of IL-2 for NK cell activation could play a role in reducing the incidence of severe GvHD after haploidentical HSCT. Bone marrow stem cell transplant into intra-bone cavity prevent type 2 diabetes: role of heme oxygenase and CO N. Abraham* (1), L. Vanella (1), S. Ikehara (2) (1)New York Medical College (Valhalla, US); (2)Kansai Medical University (Osaka, JP) Increase in endothelial cell sloughing and diminished function of endothelial stem cell progenitors in diabetic subjects are well known phenomena. We hypothesized that transplantation of bone marrow stem cells (BMSC) including mesenchymal stem cells but not limited to CD34+ stem cells into Type 2 diabetic ob mice would restore insulin sensitivity and glucose tolerance. This approach, when combined with induction of HO-1 (a cytoprotective antioxidant system) in the recipient, would further improve bone marrow function. Sublethally irradiated ob mice received BMSC or CD34+ stem cells from B129SF2/J mice (genetically related) via i.v. or to the bone marrow cavity (IBM-BMT) at a dose of 5X10 6 cells. CD34+ i.v. administration to ob mice modestly improved glucose tolerance, whereas BMSC administered by the IBM-BMT significantly increased BMSC function, serum adiponectin and glucose tolerance. Induction of HO-1 in the recipients greatly enhanced the ability of BMSC to prevent diabetes. These findings suggest that transplantation of BMSC-mesenchymal stem cells via IBM-BMT in conjunction with induction of HO-1 can eradicate Type 2 diabetes. The beneficial effect of HO-1 induction further suggest that the abnormality in endothelial progenitor cells is due to mesenchymal stem cell-stromal cell disorder exacerbated by oxidative stress. Thus, transplantation of BMSC using IBM-BMT strategy in conjunction with HO-1 induction offers a novel approach for the treatment of Type 2 diabetes. A. Poggi (1) , M. Zocchi (2) (1)National Institute for Cancer Research (Genoa, IT); (2)San Raffaele Scientific Institute (Milan, IT) Bone marrow stromal cells (BMSC) can exert an immunosuppressive effect by inhibiting proliferation of T lymphocytes in vitro. Transforming growth factor-beta, interleukin (IL) 10, prostaglandin E2 and indoleamine dioxigenase as well as BMSC to lymphocyte contact have been reported to have a role in mediating immunosuppression. Noteworthy, it has been proposed to infuse BMSC to improve bone marrow engraftment and to treat graft versus host disease. It is relevant to determine whether a) stromal cells derived from other tissue may play this immunosuppressive effect and b) this is a unique feature of cells of mesodermal origin. Indeed, to obtain BMSC, bone agobiopsy is needed, furthermore, only the inhibition of in vitro proliferation exerted by BMSC may give some information about their expected effect in vivo. Thus, we have analyzed whether skin fibroblasts (SF) and BMSC from healthy donors may inhibit lymphocyte proliferation. We found that both BMSC and SF can inhibit lymphocyte proliferation to phytohemagglutinin (PHA) or anti-CD3 monoclonal antibodies (mAbs). This effect was contact and dose dependent, furthermore the effectiveness of SF was comparable to that of BMSC (up to 90-100% of inhibition at 1:1 stromal:lymphocyte ratio) or even higher (up to 80% of inhibition for SF vs 35-50% for BMSC at 1:10 stromal:lymphocyte ratio). Then, we analyzed whether thymic stromal cells (TSC) and/or thymic epithelial cells (TEC) could inhibit proliferation to polyclonal stimuli including PHA or anti-CD2 or anti-CD28 mAbs of autologous thymocytes. TSC were characterized by the expression of marker similar to that expressed by BMSC and SF as they expressed SH2, SH3, SH4, ICAM1, LFA3 and prolyl-4-hydroxylase (P4H) while TEC were negative for the expression of P4H but positive for epithelial specific cytokeratins. Surprisingly, we found that both TSC and TEC can strongly inhibit lymphocyte proliferation to the above mentioned stimuli. Interestingly, the inhibiting effect of BMSC, SF, TSC and TEC was abolished by the addition to co-culture with lymphocytes of IL2-activated NK cells. Indeed, the interaction between MICA and ULBP3 expressed by BMSC, SF, TSC and TEC with NKG2D activating receptor on NK cells led to killing of stromal or epithelial cells blocking immunosuppression. These findings would suggest that stromal cells from different tissues and ectodermal cells may possess immunosuppressive properties and that NK cells can regulate this phenomenon. Toll-like-receptors (TLRs) are part of the innate immune system, which are able to recognize and bind to the so-called pathogen-associated molecular patterns (PAMPs) from invading pathogens. TLR9 is activated by DNA containing unmethylated CpG motifs and produces potent Th1-type innate and adaptive immune responses. Recently, it was reported that synthetic agonists of TLR9 containing CpG motifs have shown antitumor activity in a number of preclinical trials. In this study we wanted to evaluate if gene variants of TLR2, -4 or -9 influence the outcome of transplant. Here we evaluated the genotype of 405 patients with their donors for the occurrence of the TLR2 (G2408A), TLR4 (A12874G) and TLR9 (T1237C) and (T1486C) by real-time PCR who underwent transplant. First, we retrospectively analyzed the TLR gene variants in a cohort of 290 transplant recipients and their donors, then prospectively in a 2nd cohort of 115 transplant recipients and donors. An analysis of the first cohort showed that the homozygous CC gene variant of TLR9 at position 1486 compared to TC/TT gene variants was significantly associated with an markedly improved 5-year TRM (14.1% + 5.4% versus 37.7% + 0.4%, p<0.01) and 5-year OS (83.7% + 4.5% versus 52.3% + 0.4%, p<0.001) and a significant lower rate rate of leukemic relapse (16.7% + 0.7% versus 36.1% + 0.45%, p<0.012), whereas the incidence of acute GVHD grade 2-4 or severe acute GVHD grade 3-4 was not different. An analysis of the second cohort confirmed a lower 1-year TRM (9.5% +6.4% versus 39.3% + 0.79%, p<0.047) and 1 year OS (90.5% + 0.46% versus 52.2% and a higher relapse risk of 5% + 4.9% versus 34.1% + 7.5% P<0.03). The homozygous CC gene variant of TLR9 was found in 17.9% of patients in the 1st cohort and in 19.1% in the 2nd cohort. A multivariate analysis including factors which influence the OS, TRM, and relapse rate confirmed that the CC gene variant of TLR9 is a strong independent factor for OS, TRM and lower relapse rate. TLR2 (G2408A), TLR4 (A12874G) and TLR9 (T1237C) gene variants had no significant influence on any endpoints in this study. The results presented here suggest that the CC gene variant of TLR at T1483C is an independent and strong marker for outcome of transplant. The gene variant of TLR9 might be helpful in patients planning a transplant as a prognostic positive factor. TLR9 could be a rational therapeutic target for preventing leukemic relapse or improving outcome of transplant. neurological symptoms, herpetic rushes, oral and gut mucositis and hemorrhagic cystitis were registered. Results: Frequency of HSV reactivation was revealed in 23% of the cases studied, and no differences have been found for related and unrelated HSCTs. HSV reactivation is associated with development of severe mucositis. A highly active 4G allele of thrombogenic gene PAI-1 proved to be associated with more mild initial course of mucositis. Moreover, persistence of HSV and CMV in peripheral blood was found to be connected with delayed recovery of mucositis lesions. Generally, reactivation of HSV was associated with more active 2G/2G genotype of matrix metalloproteinase-1 (MMP-1), thus being suggested to favour matrix penetration of virusinfected cells. Meanwhile, EBV reactivation proved to be associated with intestinal mucositis. and posttransplantation cystitis. The study suggests a potential clinical value of regular HSV and EBV monitoring after allogeneic HSCT. Role of amniotic fluid mesenchymal stem cells in gene and cell therapy F. Bolda (1) Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into diverse lineages including osteocytes, chondrocytes, adipocytes, neuronal cells and cardiomyocytes. MSCs were initially identified in adult bone marrow (BM), but cells resemblings BM-MSCs have also been found in many other tissues, including adult and foetal peripheral blood, foetal liver, foetal spleen, placenta, umbilical cord, amniotic membrane and synovial fluid (J. Kim et al. 2007 ). BM-MSCs have been widely used in clinical applications, for example, for cell-based therapy of Osteogenesis Imperfecta and some metabolic diseases like Hurler syndrome and metachromatic leukodystrophy. Therefore, finding alternative sources of MSCs that are useful in gene and cell-based therapy, is an important goal. Human amniotic fluid (HAF) obtained during the process of amniocentesis is a valid source of MSCs and they could be a valid vehicles for gene therapy. The aim of this study was to isolate MSCs from amniotic fluid and to test their transduction efficiency. HAF specimens (n = 11) were obtained between 15 and 18 weeks' gestation by amniocentesis, previous written consent. Amniotic-fluid MSCs (AFMSCs) were cultured under specific conditions for 7 weeks and analysed by flow cytometry and quantitative real time PCR to asses the presence and the expression levels of specific markers. Mesenchymal markers (CD73, CD106, CD54, CD90, CD29, CD44, CD105) present a peak of expression between 3rd and 4Th week of colture. The replication potential in vitro was evaluated to be 30 times after 4 weeks. To asses the transducibility of AFMSC we used a SIN HIV-1based lentiviral vector. This vector encloses SFFV-U3 promoter and eGFP. It also encodes a mutant WPRE and cPPT. Transduction efficiency was 60%. We demonstrate with the immunoistochemestry and molecular techniques the presence of human multi-potent MSCs in the second-trimester amniotic fluid. We suggest that AFMSCs may be good target for prenatal gene therapy because of their ready expandability, their ability to differentiate into multiple lineages and their high transduction efficiency. Further studies are now indicated to evaluate the effect of transduction on self-renewal and differentiation in long term coltures. The model under investigation is severe combined immunodeficiency (SCID) due to adenosine deaminase deficiency (ADA). Background: Cyclosporine-A (CsA) is frequently used in the prophylaxis of graft-versus-host-disease (GvHD) after haemapoetic stem cell transplantation (HSCT). The target range for CsA blood concentration levels depends on the suspected susceptibility for GvHD and the balance with graftversus-malignancy in case of malignant diseases. We retrospectively analyzed the relation between the CsA dose (iv and po), the trough blood concentration at steady state (Ctrough) and clinical outcome. Methods: All pediatric patients receiving an allogeneic HSCT between 2004 and 2006 receiving CsA were included. The association with primary endpoints acute-GvHD (grade II-IV), Event Free Survival (EFS defined as death, graft-failure or relapse) and secondary endpoints nephrotoxicity (GFR <50 ml/min/1,73m2) and veno-occlusive disease (VOD) were analyzed using uni-and multivariate Cox regression analysis. The Ctrough targets were divided in 3 groups. The dose of CsA (iv or po) was adjusted according to the Ctrough levels and was measured weekly. When the patient was able to eat, intravenous CsA was switched to its oral form. Results: 75 patients were included: median age 5.7 (range 0.1-18) years. All patients received iv CsA after transplantation, 63 patients switched to oral CsA after a median of 28 days. A mean starting dose of 4mg/kg was used. In 75% of patients the dose was increased, in 4% the dose decreased to reach the target Ctrough. Patients who reached the target Ctrough after a longer period of time (median time 6 days, range 1-27 days) showed worse EFS (OR=0.9 per day, P=0.045). AGvHD (grade II-IV) occurred in 23 patients (31%). A target concentration of CsA of 0.2-0.25 mg/L, was associated with lower incidence of aGvHD compared to a target of 0.1-0.15mg/l, (OR=0.09, p=0.027). 5% of patients developed nephrotoxicity and 21% developed VOD, neither correlated with CsA dose or Ctrough. Based on the Ctrough 0.2-0.25 mg/L, children <5 years of age were found to require a mean dose of 10mg/kg, while older children needed a mean dose of 7mg/kg (range 1.5-19mg/kg). In order to reach a similar Ctrough, a converting factor of 1.5 for switching to oral CsA was found, independent of age. Conclusions: CsA target blood concentration of 0.2-0.25 mg/l prevented the occurrence of aGvHD. CsA dose nor concentration were associated with nephrotoxicity or VOD. This Ctrough requires a dose of 7mg/kg in children <5 and 10mg/kg in children >5 years of age and a converting factor of 1.5. Bone marrow stromal cells (BMSC) can exert an immunosuppressive effect by inhibiting proliferation and function of effector T lymphocytes. This phenomenon is patients after RIC SCT. We observed a trend to a higher level of chimerism on days +28; +56, and +100 in patients after standard conditioning versus RIC. It is necessary to note that the relapse rate was significant above in RIC patients (39%) than in patients with standard conditioning (19%) (log rank t<0,05). Quantitative chimerism analysis in CD34+ cells was more sensitive to detect relapse in the early terms. MRD assessment from days +28 till day +100 after SCT allowed identification of patients at risk of relapse. After a median follow-up of 556 days, 62% of patients remain alive; 90% from them keep complete remission and CC. In conclusion, simultaneous studies of both chimerism and MRD are a useful tool in order to predict risk of relapse in patients undergoing SCT and so can be helpful for individualizing treatment strategies after transplant. Reduced intensity allogeneic stem cell transplantation (RIC alloSCT) is a therapeutic option for relapsed chronic lymphocytic leukemia (CLL) and can lead to 50% of progression-free survivors. The aims of the study were to assess the rate of molecular remissions (MR) after RIC alloSCT, the correlation between MR and relapse risk and the role of molecular monitoring in post-transplant immunotherapy. Twenty-nine patients with a molecular marker (heavy chain gene immunoglobulin rearrangement, IgH) were monitored for minimal residual disease (MRD). All patients had a relapsed disease; 75% had an unmutated IgH; 6 out of 16 patients (38%) showed a 17p deletion. Median age was 60 years (range, 44-69). Median number of previous chemotherapy was 3 (range, 1-6) . Eleven patients (38%) were chemorefractory before transplant. The conditioning regimen included an in vivo T-cell depletion in 13 patients (45%). Molecular monitoring was performed by nested-PCR on bone marrow using CDR-2 and CDR-3-derived patient-specific primers. For real-time PCR relative quantification was estimated by using a FR3-derived probe and a pre-transplant sample as positive control. Nine patients (31%) were PCRnegative: 5 of them (17%) have been always PCR-negative while 4 patients (14%) experienced a delayed clearance of MRD. All these patients are in complete remission (CR) at the median follow-up of 20 months (range, 6-71). Seven patients (24%) showed a mixed pattern of PCR positivity and negativity: one patient died of TRM, one patient had a nodal relapse, the others are in CR at a median follow-up of 30 months (range, 7-61). Thirteen patients (45%) were always PCR-positive: 7 of them relapsed after a median time of 9 months; 2 patients died of TRM; 4 patients are in CR after a median follow-up of 17 months (range, 3-24). Relapse rate was higher for PCR-positive patients compared to PCRmixed/negative patients (p=0.009). T-cell depletion was associated with MRD positivity (p=0.027). In 3 PCR-positive patients that did not relapse a decreasing tumor load was detected by real-time PCR; the patients were affected by extensive chronic GVHD. In 2 PCR-positive patients the tumor load increased on day +300 and +270: both patients relapsed on days +420. In conclusion, in relapsed CLL MR can be achieved in a sizeable fraction of patients after RIC alloSCT. Persistent PCR positivity correlates with a high incidence of relapse, while a mixed-PCR pattern can be observed without clinical relapse. Quantitative monitoring of NPM1 mutations versus chimerism for detection of impending relapse after allogeneic stem cell transplantation in AML U. Bacher* (1), A. Badbaran (1) , B. Fehse (2) , T. Zabelina (1), F. Lehmann (1), S. Zeschke (1) , A.R. Zander (1) , N. Kröger (1) (1)University of Hamburg (Hamburg, DE) ; (2) Johann-Wolfgang-Goethe University (Frankfurt, DE) Background: In chronic myeloid leukemia increase of BCR-ABL as assessed by quantitative PCR is more sensitive for the detection of impending relapse than chimerism after allogeneic stem cell transplantation (SCT). In other myeloid malignancies, the question which parameters are most suitable for detection of relapse after SCT still needs to be clarified, especially in Acute Myeloid Leukemia (AML), where the rapid dynamics of relapse would require early intervention. We compared retrospectively molecular chimerism as assessed by quantitative PCR and the minimal residual disease load as indicated by the Nucleophosmin (NPM1) mutations in mutated AML patients after SCT. Patients and methods: We included 15 pts (21-69 years) at diagnosis/relapse of AML. Quantitative chimerism was performed on the basis of 11 single nucleotid polymorphisms (SNP) as previously described (Alizadeh et al., 2002) . In 3 patients, quantitative Y-PCR was performed (Fehse et al., 2001) . Other genetic markers being followed were 1b (1pt), 7a (2 pts), 7b (4 pts), 8a (1pt), and 8b (2 pts) according to Alizadeh. In 2/15 pts chimerism could not be assessed. Significant decrease of chimerism was defined as <99% in cases having previously achieved full donor chimerism or as decrease of more than 2.5% in cases with mixed chimerism. PCR screening for NPM1 mutations was performed by realtime (Taqman) PCR from peripheral blood/bone marrow samples taken before and after SCT. Quantification of the NPM1-mutational levels of the most frequent subtype A was performed by normalization with the HCK gene. Results: Relapse was detected in 11/15 pts (73%) with the NPM1-mutational subtype A after SCT. This was preceded by increase of the NPM1-mutational level in 10/11 cases (91%). When compared to molecular chimerism, the NM1-mutational level was increasing in 7/10 cases (70%), before a significant decrease of chimerism was observed with a mean interval of 15 days ranging up to 36 days. Conclusions: Also when performing chimerism with the so far most sensitive method (quantitative real-time PCR for genetic polymorphisms), certain molecular mutations seem to precede significant changes in chimerism in impending relapse in AML. In NPM1-mutated cases, this marker provides an appropriate approach after SCT. More studies should compare other recurrent mutations -e.g. MLL-PTD, NRAS, or CEBPA -with chimerism in AML patients after SCT. Minimal residual disease and chimerism in CML patients receiving allogeneic transplants after myeloablative or reduced-intensity conditioning M. Uzunel* (1), J. Mattsson (2) , D. Hauzenberger (3) , O. Ringden (2) (1)Karolinska Institutet (Stockholm, SE); (2) It is not known whether reduced conditioning is associated with prolonged period of detectable disease after SCT in patients with CML. We have therefore compared BCR-ABL S336 transcript levels in patients receiving a conventional myeloablative conditioning with those receiving reduced conditioning. We also made chimerism studies in both patient groups. Myeloablative Conditioning (MYC): 31 patients, 19 matched unrelated donors (MUD) and 12 sibling donors (Sib). Median age was 42 years (10-61). Conditioning with Bu+Cy (n=31). Twenty of the MUD patients were also given ATG. Seven patients have died (6 GVHD, 1 Relapse) . Median follow-up time: 56 months (10-99). Reduced Intensity Conditioning (RIC): 24 patients, 13 MUD and 11 Sib. Median age was 55 years (11-64). Conditioning with Flu+Bu+ATG. Seven patients have died (3 Relapses, 1 GVHD, 3 other reasons). Median follow-up time: 43 months (18-100). Results: There was no statistical significant difference in overall survival and relapse free survival between both groups. 5/29 patients in the MYC group relapsed as compared to 7/23 patients in the RIC group. Only one patient in the RIC group rejected the graft. The incidence of severe acute GVHD was significantly higher in the MYC group (48%) as compared to the RIC group (13%), p=0.004. Interestingly, the incidence of chronic GVHD was slightly higher in the RIC group, 51% vs. 37% (p= 0.11). During the first 3 months, MRD levels in the RIC group was in median one log of magnitude higher than in the MYC group (p=0,01). After that, no significant differences in the MRD incidence and MRD level were found between the groups. Median time to complete donor T-cell chimerism was 34 days (14-285) in the MYC group as compared to 60 days in the RIC group (p=0.04). Also, complete myeloid cell engraftment was delayed in the RIC group, median 44 days (14-165) as compared to 25 days (14-270) in the MYC group (p=0.02) Conclusion: Despite higher BCR-ABL levels during the early post-transplant period and higher incidence of mixed chimerism, nonmyeloablative transplantation for CML patients may induce molecular remission in the majority of the patients. Less chronic graft-versus-host disease with reducedintensity conditioning than myeloablative conditioning in HLA-matched sibling donor transplant E.P. Alessandrino, A.A. Colombo, P. Bernasconi, D. Caldera, M. Gotti, F. Ripamonti, A. Zaniboni, C. Pascutto, M. Lazzarino Fondazione IRCCS Policlinico S.Matteo (Pavia, IT) Chronic GVHD (c-GvHD) incidence, requirements for immunosuppressive therapy and survival were retrospectively analyzed in 40 patients who underwent reduced intensity conditioning (RIC) allogeneic peripheral stem cell transplantation (HSCT)and in 46 patients who underwent standard myeloablative ( STA) HSCT from HLA identical sibling. The RIC transplantation regimen consisted of Thiotepa 10 mg/kg recipient body weight over two days and fludarabine 125 mg/m² ev. over five days followed by immunosuppression with cyclosporine (CSP) continous infusion ( 1,5 mg/kg recipient body weight) and methotrexate ( MTX) 10 mg/m² at day +1 and 8 mg/m² at day +3, +6, +11; the myeloablative conditioning consisted of Busulfan and Cyclophosphamide followed by immunosuppression with CSP 1,5 mg/ Kg recipient body weight twice day ev and methotrexate 10 mg/m² at day +1 and 8 mg/m² at day +3, +6, +11. The median age of patients was 46 years ( range: 19-62 years), 55 were male, 31 female. RIC and STD groups were similar for sex ( Male/Female ratio) prevalence of previous infective episodes, status of disease at transplant. Forty nine patients 57%) developed c-GvHD; On multivariate Cox regression analysis significant variables associated with a higher risk of c-GvHD were: previous acute c-GvHD (HR 3.9 P 0.002) and standard conditioning ( HR 2.6 P 0.017). Relapse was not higher after RIC than after standard transplantation ( P=NS). Survival was similar in the two groups. The prevalence of grades II-IV acute GVHD was significantly lower after RIC transplantation, but there were no differences in the cumulative incidence of chronic GVHD. The occurrence of extensive c-GvHD requiring immunosuppressive treatment was lower in the RIC group ( P 0.03). These data indicate that RIC was associated with a lower incidence of extensive c-GVHD and with the use of fewer systemic immunosuppressive agents. Correlation between MRD and chimerism kinetics in CLL patients after myeloablative and non-myeloablative allogeneic haematopoïetic stem cell transplantation A. Plesa (1) We studied the correlation between chimerism and minimal residual disease (MRD) in 12 patients (pts) (9M, 3F, 51 years) who underwent allogeneic hematopoietic stem cell transplantation (HSCT) for CLL. Chimerism was assessed from total peripheral blood (PB), bone marrow (BM) and CD3+ cells by STR-PCR (sensitivity[S]:5%)and by SNP-PCR (S:0.2%); the MRD was tested from PB and BM by an international standardized multicolour flow cytometric approach (S:0.01%). At diagnosis, there were 4 Binet stages A, 7 stages B and 1 stage C. Four pts were in CR, 7 in PR and 1 in PD pretransplant. 11 pts underwent 1 HSCT and 1, 2 HSCT from HLA identical sibling donors (10 PBSC, 3 BM); 2 pts received a myeloablative regimen and 10 a reduced intensity conditioning. After transplantation, 6 pts developed an aGVHD ≥ grade 2 and 8 a cGVHD. The chimerism kinetics showed a conversion from mixed chimerism (MC) into full donor chimerism (FDC) at day 30 post transplant in 6 pts, between day 30 and day 120 in 4 pts, 2 pts never converted to FDC and remained either in stable MC or progressed to recipient profile. The median follow-up was 39 months. Among the 11 pts who received 1 HSCT, 2 pts died from infection and they showed FDC and MRD negativity, 9 pts are alive (7 in CR and 2 were too early). Among the 7 CR pts, 6 were in FDC and MRD negative and 1 in MC and MRD negative. We studied 246 samples for chimerism and 88 for MRD and to correlate quantitative MRD levels with chimerism data only 56 samples were comparable. Considering kinetics, we observed a concordance in 50/56 (89%) with 16/50 (32%) both positive early after transplant and 34/50 (68%) both negative later. Among the 6 non concordant samples which belonged to 2 patients, 5/6 (83%) were MC and MRD negative explained by the MC profile of CD3+ cells and 1/6 (relapsed patient) was MRD positive and FDC explained by the better sensitivity of MRD. Relation between MRD and chimerism for PB and BM was tested using a mixed effect linear regression and this analysis showed a significant correlation between PB MRD and chimerism (p<0.001) and between BM MRD and chimerism (p<0.001). In conclusion, because of the better sensitivity, specificity and advantageous cost of MRD flow we could recommend in the future to combine chimerism and MRD data during the first 90 days after allogeneic HSCT for CLL pts and to limit to the flow cytometry MRD approach the further follow-up to guide therapy after transplant. and risk category by the underlying disease (standard risk vs high risk), and sex mismatch (male recipient/female donor vs other combinations) as categorical variables a higher TREG number and lower CD34 and CD3 cell numbers were associated with improved survival. Regarding relapse standard-risk disease and a higher NK, NKT, and TREG cell number were associated with a significantly lower risk for relapse. A higher NKT cell number was also associated with a lower risk for acute GVHD II-IV (RR 0.54) although this did nor reach statistical significance due to low patient number. Discussion: These preliminary results suggest that donorderived regulatory NK T cells might have a clinical impact on relapse and graft-versus-host disease following allogeneic peripheral blood stem cell transplantation. Differences in the use of allogeneic haematopoietic stem cell transplants in the Nordic countries L Brinch* (1), P. Ljungman (2) , H. Sengelöv (3) Several surveys have shown significant differences between the number of haematopoietic stem cell transplants (HSCT) per number of inhabitants (transplant rates) performed in the European countries. As a main factor more transplants are performed in countries with a high, than in countries with a low Gross National Income (GNI/cap). The Nordic countries all belong to the group of high income by World Bank definitions, they have a common cultural background, and well developed health care systems. However, surveys have shown marked differences between Norway and the other Nordic countries. In 2005 Norway had the highest GNI/cap of all European countries. Still between 2000 to 2005 about half the number of HSCT were performed in Norway compared to Sweden and Finland, and about 20% fewer than in Denmark. The aim of this analysis is to learn more about the reasons behind these differences with focus on allogeneic HSCT. Methods: The analysis is based on the annual of EBMT activity surveys 2000-2005. Total of transplants performed and transplant rates for the most common indications were compared. Results: The table shows the total number of first transplants, and the transplant rates for allotransplant for selected diagnoses, per 10 million inhabitants in the Nordic countries 2000-2005. The percentages of RIC allotransplants were 34% in Sweden, 28% in Denmark, 20% in Finland, and 13% in Norway. The main indication for transplantations seemed in general to be about the same, with some exceptions. Allogeneic transplant for solid tumors were only performed in Sweden, and more allotransplants for myeloma were performed in Finland than in the other countries (1.1 vs 0.2-0.4/10 mill.). Interpretation: There is a clear difference in the transplant rates between especially Finland and Sweden, compared to Norway.The low percentage of RIC SCT performed in Norway compared to especially Sweden explains some of the differences. The selection of patients for transplants is centralized in Norway and Denmark with only one centre each, while Finland has two and Sweden six allogeneic centres. There may be a difference in the attitude to resources allocated to performing SCT in general perhaps especially on new and developmental indications. This investigation raises more questions than answers. It illustrates the need to learn more about allocation mechanisms and decision making. Decision on individual patients as well as on health care planning for the infrastructure in the future could then be based on rational elements. Introduction: Stem cell transplantation is a curative approach using chemo-, radio-, and immunotherapy for malignant and non-malignant hematological disorders. EBMT is collecting yearly data on survey basis since 1990. The variables within the survey are limited to detailed indications, number of the patients, transplant type, stem cell source, type of conditioning regimen and donor type. The transplant rates in certain indications, patterns of stem cell source selection and donor availability and alternative donor use were analyzed in detail. Patients and Methods: The TTR data within EBMT-EAS was delivered by the EBMT Activity Survey Office. We compared national data with international EBMT-EAS data pool. Results: In 2005 and 2006 24 national centers responded to EBMT-EAS. In 2005 the total activity in Turkey reached 600 and in 2006 exceeded 800. The ratio of auto-to allo-HCT is 1:1 which was quite different in EBMT-EAS (2-2.5:1). Our population based transplant rates for both allo-and auto-HCT were 1-50/10 million inhabitants. The major indications for allo-HCT are acute leukemia (28%), chronic leukemia (22%) and non-malignant hematological problems (20%) (Fig 1) . There is a sharp drop in allo-transplants for CML (66%). In autologous setting the major indications were lymphoproliferative disorders (69%), solid tumors (16%) and leukemias (Fig 2) . The graphical outline for indications and their growing patency were nearly identical for both allo-and auto-HCT. The use of peripheral blood stem cells reached a ratio of 71%, which is the same in 2006 EBMT-EAS. For autologous setting the use of PBSC is a standard approach (98%). The number of alternative donor transplants is rising in last 2 years. The ratio of MUS and allo-cord blood HCT is 7% in 2006, which is quite far from the general 41% use of alternative donors in Europe. Conclusion: The TTR activity is in concordance with the indications in EBMT-EAS. The drop in allo-HCT for CML, use of more PBSC in allo-HCT and tendency in rise of allo-RIC were concordant with the published EBMT-EAS. The only exception is the nearly equal number of allo-and autoHCTs. The transplant rates for allo-and autoHCT are quite low and still at a low survey segment. The number of alternative donor transplants has risen within 3 years. Report from two centres K. Kalwak* (1) Tuebingen/ show excellent engraftment, faster immune recovery /incl. NK cells/, low rate of GvHD and EBV-LPD after haploidentical PBSCT using CD3/CD19 negative selection. On the other hand, adult data show, however, a significant proportion of pts developing aGvHD CD 34 positive selections used before led to significant rate of autologous recovery or graft rejection (44%), therefore the protocol was changed for CD3/CD19 depletion. Pts (4 male, 1 female; median age 7 months, range 1-11 months) were given either: Bu, Cy, MMF (n=2) or TREO, CY, MMF (n=3). Due to SCID phenotype and thus decreased risk of graft rejection CD34+cells/kg This retrospective multicenter analysis provides an overview on the transplantation activities of the centers participating in the German Registry of Stem Cell Transplantation (DRST) within the period 1998 to 2004. Methods: 30 centers contributed data of 1716 consecutive pts (1594 adults, 122 children) with CML who received an allograft within this period. Multivariate analysis was performed by Cox-Regression Median age was 40 years (r: 1-68). 66 pts were >59 years of age (3.8%), whereas the majority was between 20-59 years old Peripheral blood stem cells (PBSC) played a major role (1069/1709; 62.6%) when compared to bone marrow (BM) (640/1709; 37.4%). Five years after SCT 1066/1716 pts (62.1%) were still alive. Between 1998 and 2004 annual transplantations were decreasing from 346 to 94 (27.2%) with a marked reduction in 1. chronic phase (CP) (1998: 223 annual SCTs 1998 to 50/94 (53.2%) in 2004. RIC was performed in 156/903 cases (17.3%), whereas 747 pts (82.7%) had standard conditioning. Conditioning included total body irradiation in 842/1688 pts (49.9%) Four (17%) pts had previous autologous SCT. Median interval from diagnosis to allo-SCT was 46 months. Results: All pts engrafted. Median intervals to neutrophil and platelet recovery were 17 days (range, 12-28) and 13,5 days (range, 10-30) resp. Acute graft-versus-host disease (aGVHD) occurred in 15 (65%) pts (5x grade I, 9x grade II and 1x grade III). Of the 20 evaluable pts 6 (30%) developed chronic GVHD (3x extensive, 3x limited). Overall 100-day and 3-year treatment related mortality (TRM) rates were 9% and 17% resp. Relapse incidence was 13%. With a median folow-up of 19 months, estimated overall survival (OS) at 3 years is 78%, progression free survival (PFS) 73% and relapse risk 27%. From 8 pts with chemoresistant CLL at allo-SCT, five (63%) achieved remission (3xCR, 2xPR), 3 pts died (2 for relapse, 1 for TRM). Overall 6 (26%) pts died, 4 for TRM and 2 for relapse. At time of last follow-up, 17 pts (74%) are alive, 15 in CR, 2 in PR. Overall 12 pts were evaluable for MRD, from 10 in CR 5 are MRD-and 5 MRD+, both pts in PR are MRD+. We didn´t found any significant difference (measured by OS or PFS) according to donor, conditioning, pretreatment, age or chemosensitivity. Conclusion: Allogeneic SCT represents an effective therapeutic option for patients with high risk CLL P1026 Treosulfan and cyclophosphamide conditioning for paediatric allogeneic transplantation R. Cutting, A. Vora* Sheffield Childrens Hospital (Sheffield, UK) P1064 Conditioning and graft-versus-host disease decrease CBFß/MYH11 fusion transcript monitored by quantitative PCR in patients with inv(16) AML after allogeneic haematopoietic stem cell transplantation A. Ghavamzadeh, A.A. Hamidieh*, M. Jahani, K. Alimoghaddam, A. Mousavi, M. Iravani, B. Bahar, A. Khodabandeh, L. Nedaiefard, A. Ashouri, S. Basirpanah, A. Mousavi, Z. Shahriari, S. Khalilvand Hematology-Oncology and SCT Research Center (Tehran, IR) Analysis of CMV-specific T-cell immunity by intracellular cytokine staining in patients undergoing allogeneic haematopoietic stem-cell transplantation early after transplant identifies a high-risk group for viremic CMV reactivation I. Benet (1) , M. Clari (1) , C. Solano* (1) , J. Nieto (2) , R. de la Cámara (3) , , M.J. Remigia (1) , J. López (4) , A. , J. Alberola (1) , A. Tamarit (1) , C. Gimeno (1) , D. Navarro (1) (1) University of Valencia (Valencia, ES) ; (2) Hospital Morales Meseguer (Murcia, ES) ; (3) Hospital de La Princesa (Madrid, ES) ; (4)Hospital Ramón y Cajal (Madrid, ES) Cytomegalovirus (CMV) infection is a significant cause of morbidity and mortality in pts undergoing allogeneic stem-cell transplant (Allo-SCT). Pre-emptive therapy reduces the CMV end-organ disease. This strategy, however, represents a considerable workload and results in treatment of a significant number of pts who would not develop CMV disease. Aims: To evaluate whether assessment of CMV-specific CD8+ and CD4+ T-cells by intracellular cytokine staining (ICS) early after Allo-SCT could predict viremic CMV reactivation (CMV-react+) within 100 days after SCT. Patients and Methods: Thirty-two pts [Sex(M/F): 12/20;Age:48 years(22-66)] undergoing Allo-SCT for hematological malignancies (1/3/07 to 15/9/07). CMV-serostatus: D+/R+ (n=25), D-/R+ (n=6), D+/R-(n=1). Monitoring for CMV-react+:1-2 times/week by plasma RT-CMV PCR and/or pp65-Ag. Pre-emptive antiviral treatment was initiated upon either a positive pp65-Ag or 2 consecutive plasma DNAemias. Enumeration of CMV-specific CD8+ and CD4+ T-lymphocytes was performed by ICS (BD Biosciences Fastimmune tests) using a FACSCalibur in heparinized blood samples at days: +30 and +60 post-SCT. Whole blood was stimulated with 15mer peptides spanning the pp65 and IE-1 CMV proteins during 6 h at 37ºC plus co-stimulatory CD28 and CD45. Results: Fourteen pts developed viremic CMV-react+ (7 before and 7 after day +30) and were pre-emptively treated. Pts who developed CMV-react+ after day +30 had significantly lower both CMV-specific CD8+ and CMV-specific CD4+ T cell counts at day +30 [median:0.32 cells/mcl (range:0-0.68) and 0.24 cells/mcl (range:0-1.02), respectively(p≤0.001)] than those who did not [median 1.31 cells/mcl (range: 0-23.6) and 1.37 cells/mcl (range 0-7), respectively(P≤0.001)]. CMVspecific T-cell counts at day +60 in pts without CMV-react+ were comparable to those of day+30 but significantly (P<0.001) lower than those found in pts with CMV-react+ at the same time (+60). Expansion of CMV-specific T CD8+ cells was associated with rapid CMV viremia clearance in preemptively treated pts. A preliminary safe cut-off cell level could be established (1 cell/mcl for T-CD8+ cells and 1.2 cells/mcl for T-CD4 cells) to discriminate between pts at risk for CMV-react+ (who would need virological monitoring) and those who are not. Conclusion: These results suggest that assessment of the CMV-specific T-cell immune response by ICS early after Allo-SCT may provide useful information for clinical management of CMV infection P915 Immunologic reconstitution after HSCT and its clinical relevance: a single-centre experience M. Serban (1) , S. Arghirescu (1) , C. Jinca (1) , R. Firescu (2) , A. Oprisoni (1) , L. Stana (2) , M. Baica (2) , H. Ionita (1) , M. Lesovici (2) , J. John (1) (1)University of Medicine V.Babes Timisoara (Timisoara, RO); (2) Children Hospital L. Turcanu Timisoara (Timisoara, RO) Introduction: Reconstitution of the immune system after haematopoietic stem cell transplantation (HSCT) is a critical determinant of the outcome of patients with malignancies, who even prior to transplant have a deteriorated immune reactivity. Objectives.Taking into account the characteristics of our patients (multiple relapses, partial remissions with prolonged chemo-and radiotherapy) we aimed at analysing the immune restoration and its clinical impact. Material and methods: The retrospective observational study was conducted on 50 patients: 44 with autologous and 6 with allogeneic HSCT, strikingly, out of which 56.36% with frequent relapses or resistant malignancy. The conditioning regimen was polychemotherapic ( BEAM, BU/CY, BU/CY/ETO, MEL ) in all cases. Median CD34 cell dose was 5.814 x 106 / kg b.w. The immune status has been evaluated two times / year at 6 months interval by flowcytometry with a panel for CD3, CD4, CD8, CD19, CD16, CD56,HLA-DR + . Results: The most delayed reconstitution characterized CD 3+ CD4+ lymphocytes with a recovery of 55% at 1 year for allogeneic HSCT and 35% recovery at 1 year for autologous HSCT. In this last situation at 3 years the recovery was 80%. Regarding CD19+ lymphocytes a 100% reconstitution was achieved for autologous HSCT at day +100 and for allogeneic HSCT at day +180. The recovery of CD16+CD 56+ lymphocytes was for allogeneic HSCT at 1 year 80% and for autologous HSCT arround 40%. More severe in autologous setting, this immunodeficiency did not influence in a significant modality the global rate of infection, but the severity of posttransplant infections was life-threatening: one of the patients developed severe pneumocystis pneumonia, threereactivation with clinical disease of varicella zoster virus and one -aspergillosis. Conclusions: Long-lasting and severe immunodeficiency is noticed in 88% of our transplanted patients. Even if they don't influence significantly the global rate of infections, they are responsible for very severy nosocomial infections: pneumocystosis, reactivation of varicella zoster virus and aspergillosis. Posaconazole and cyclosporine in allogeneic stem cell transplantation, is it a good combination? C. Montes*, L. Yáñez, A. Bermúdez, A. Iriondo Hospital Marques de Valdecilla (Santander, ES) Introduction: The risk of invasive fungal infection is increased among recipients of hematopoietic stem cell transplantation because of neutropenia, immunosupression and graft versus host disease. Posaconazol is a new triazole that shows efficiency in the prevention of invasive aspergillosis and has activity against other fungal species. Objectives: To determine the safety of the cyclosporine when it is co-administered with oral posaconazole. Patients and Methods: We analyzed eight patients who underwent allogeneic stem cell transplantation in our center. Median age was 53 years (18-65) and five were women. In five patients, donors were matched unrelated and in three patients matched related. All patients received posaconazol 200 mg oral suspension 3 times/day and neoral cyclosporine. Indications for use of posaconazol were primary prophylaxis (6) and secondary prophylaxis (2) . The beginning time and prophylaxis duration were variable. Results: In all patients co-administration of posaconazole increased cyclosporine levels (p=0,0005). Dosage reductions of 16-72% for cyclosporine were necessary for all of them (median: 50%) and 4 patients had to stop 1 dose of the immunossupresant treatment. Females needed a major reductions (p=0,03). Two patients showed mild renal impairment and one patient incomplete thrombotic microangiopathy. We did not observe increased levels of alanine aminotransferase, aspartate aminotransferase, or bilirubin during posaconazole treatment. Despite secondary effects of high levels of cyclosporine, all patients are alive and Obective: Hematopoietic stem cell transplantation (HSCT) is curative treatment in many hematologic and nonhematologic diseases. This is follow-up report of pediatric patients whom transplanted in Shariati Hospital Tehran, Iran. Methods: 522 pediatric patients (median=7.75 years) received HSCT between 1991 and 2007. Most common disease were : 307 (58.8%) beta-thalassemic patients, 85 (16.3%) patients with AML, 37(7.1%) patients with ALL, 26 (5%) patients with severe aplastic anemia, 21(4%) patients with fanconi anemia, 12(2.3%)patients with lymphoma and 7(1.3%)with CML and others were congenital immunodeficiency syndrome , storage disease and solid tumors. 386( 82%) patients received stem cell from healthy full human leukocyte antigen (HLA) matched siblings, 12 (2.5%) patient from their parents , 3(0.6%) patients from unrelated full match donor and two (0.2%) patients from other relative.One patient received stem cell from healthy monozygotic twin. The graft source was bone marrow in 213 patients, peripheral blood stem cells (PBSC) in 294 patients, cord blood in 14 patients, and in one patient PBSC and bone marrow. Results: 411 patients are alive, 111 patients dead. Most common causes of death were relapse of disease 34.2% and graft-versus-host disease (GVHD) 21.6%. Acute GVHD occurred 23 % and chronic GVHD occurred 16.8%.Two years overall survival and disease free survival of thalassemic patients were 86% and 76% respectively. Two years overall survival and disease free survival of AML patients were 65% and 64% respectively. Two years overall survival and disease free survival of ALL patients were 73% and 59% respectively. Two years overall survival and disease free survival of sever aplastic anemic patients were 100%and 80% respectively. Both two years overall survival and disease free survival of fanconic anemia patients were 51%. Conclusions: Our results of overall survival and disease free survival of disease are compatible with literature. Neurologic complications in paediatric patients following haematopoietic stem cell transplantation A. Zaucha-Prazmo* (1), K. Drabko (1) , M. Choma (1) , B. Wojcik (1) , , K. Kalwak (2) , M. Leda (3) , G. Grund (3) , J. Styczynski (4) , J.R. Kowalczyk (1) (1)Medical University (Lublin, PL) ; (2) Medical University (Wroclaw, PL) ; (3) Medical University (Poznan, PL); (4)Medical University (Bydgoszcz, PL) Aim: The aim of the study was to evaluate the frequency and type of neurologic complications (NC) in children undergoing haematopoietic stem cell transplantation (HSCT). Patients and methods: We performed a retrospective analysis of the incidence and outcome of NC in 912 children transplanted in 4 paediatric BMT units (6,3%) , 5/73 MMFD (6,8%) , while among autoHSCT patients was 0,94% (3/316). The median age of 21 girls and 27 boys with NC was 13 years (range from 3,2 to 20, 3) . Diagnoses among this group of children were malignant diseases in 40 pts ( ALL -16, AML -8, JMML -1, CML -7, MDS -5, Ewing Sa -2, CNS tumour -1) and non-malignant in 8 children ( SAA -3, Fanconi anaemia -2, BD anaemia -1 and ADL-X in 2). According to Saiz and Graus classification: drug related toxicities occurred in 15 cases; toxicities related to bone marrow depletion (metabolic, septic, hemorrhages) were observed in 12 pts; related to chronic immunosupression (opportunistic infections) were observed in 7 pts. and late events (CNS relapses, second neoplasms) in 14 pts. NC occurred after a median follow-up of 3 months (range from 14 days to 84 months). 54% of NC (26/48) happened within first 3 months postransplant: infections in 10 pts., hemorrhages in 2 pts and drug related toxicities in 14 pts. 46% of NC (22/48) were of late onset: infections in 7 pts., hemorrhages in 7 pts, drug related toxicities in 1 pt., second neoplasms in 3 pts and other events in 4 pts. (hallucination syndrome, loss of consciousness, vision disorders, spastic paralysis). 37 out of 48 patients (77%) died with NC being the cause in 28, 4 children died because of non-CNS relapse, 5 due to severe acute GvHD. Conclusions: Among the analyzed material NC were observed mainly in children undergoing allogeneic transplantation. Half of analyzed neurologic events occurred in first 3 months post transplantation, thus contribute to transplant related morbidity. In our patients infections and drug-related toxicities were the most frequent neurologic complications. JACIE and ISO 9001/2000: to assure holistic care of paediatric patient on HSCT P. Halle*, D. Roudeix, C. Sozeau, V. Souquière, M. Ravel, C. Paillard, E. Merlin, A. David, A. Rieutord, C. Palasse, B. Sobrier, F. Deméocq, J. Kanold University Clermont 1 (Clermont Ferrand, FR) Objective: to assure holistic care of children during pre-, onand post HSCT periods. Methods: Our paediatric HSCT department accredited JACIE in 2006, expanded its quality management program based on ISO 9001/2000 standards, to all care to children suffering from S295 cancer or leukaemias since the diagnosis up to the long-term follow-up. Like the JACIE implementation project, the ISO 9001/2000 program was conducted with the support of Management Team and all members of the patient's health care team were involved. The same process of quality system than in the collection and processing facilities (certificated ISO since 2004) was applied. It complies with JACIE standards, French and EU legislation. Results: We singled out ten physical processes or "job specializations process" (therapeutic and associated) such as "diagnosis", "long term follow-up", "project for the child" and "transitional care planning network" whereas only four processes were singled for JACIE. Particular attention was paid to the needs of families and children especially in education to identify and manage all the problems during the different phases of cancer treatment. The follow cartography which is the systematic representation of the processes and the interactions between them made it possible to draw up a global vision of our ''care'' activity including the HSCT. Whatever the standard JACIE or ISO, the difficulties linked with the implementation of a quality system are identical: 1) help by a quality professional 2) need to involve, motivate the people and support of the management staff.3) required a significant investment of time and resources, 4) Cost, always difficult to estimate. The benefits are undeniable for children, families, health care specialists and other caregivers: improvements of communication (between members of the team, patients, families) and organization, harmonization of professional practices, recognition for the work of individuals, true group dynamics. Conclusion: JACIE has proven to be a strategic tool indisputable: the difficulties of any establishment of a quality program were lifted and the state of play allowed us to consider without huge additional investment to adapt our quality approach the ISO standards. Altogether ISO and JACIE helped us to strengthen cohesion among medicotechnical teams, and between all internal and external partners necessary for the holistic care of the patient. Treosulfan-based conditioning regimen for allogeneic transplantation in children congenital non-malignant disorders -retrospective study J. Wachowiak* (1), G. Grund (1) , K-W. Sykora (2) , R.F. Wynn (3) , J. Kowalczyk (4) , K. Drabko (4), F. Smiers (5), R.G.M. Bredius (5) , P. Veys (6) , A. Chybicka (7), K. Kalwak (7) (Bristol, UK) Children with congenital disorders eligible for allogeneic hematopoietic stem cell transplantation (allo-HSCT) often demonstrate high risk of regimen related toxicity (RRT), and high risk of transplant failure. Therefore, they need preparative regimen with significant myeloablative and immunosuppressive effects, but with low toxicity. According to previous experience in adults and in children with hematological malignancies, such attributes are demonstrated by regimens based on treosulfan (TREO), structural analog of busulfan, Retrospective study was performed in 30 children and adolescents (0.7-20 years; median 8) with metabolic disorders (n=14), immunodeficiences (n=6), beta-thalassemia (n=6), heredidary bone marrow failures (n=4) transplanted between 2000-2005 from MSD (n=13), UD (n=13) and 4 from haploidentical donor (HD) after conditioning based on TREO given i.v. at total dose of 30 g/m² (n=8), 36 g/m2 (n=17) or 42 g/m² (n=5) in various combination with Fludarabine, Cyclophosphamide, and/or Melphalan plus Campath or ATG according to diagnosis, pre-transplant risk factors, conditioning for 1st HSCT, and donor type. Standard prevention of GvHD was used. Early RRT was graded using Bearman criteria (1988). Early RRT I° was observed in 2 (7%), II° in 4 (13%), and III° in 7 (23%) pts. RRT IV° did not occure. Primary engraftment was achieved in 29 (97%) pts (day 10-35, med. 15), except 20 years old patient transplanted 2nd time with from haploidentical donor for thalassemia with low CD34 cell dose (5.3x106/kg). In 5 (17%) pts (3 x Hurler syndrome; 1 x thalassemia; 1 x dyskeratosis congenita) secondary graft failure was diagnosed (day 89-110; med. 109) . Stable complete chimerism was demonstrated in 16 (53%) pts (day 13-73; med. 37), whilst stable mixed one in 8 (27%). Acute GvHD III-IV° and extensive chronic GvHD occurred in only 2 (7%) pts. each. Three (10%) pts. died (SCID after haplo-HSCT on day 51 due to CMV pneumonia; osteopetrosis with pre-transplant injury to CNS due to neurodegeneration on day 160 after UD-HSCT; Hurler syndrome on day 285 due to chronic GvHD after 2nd UD-HSCT for graft failure). For whole cohort 5-year EFS and OS was 79% and 89%, respectively. In children with congenital disorders undergoing allo-HSCT conditioning based on TREO given at total dose of 30-42 g/m² i.v. is safe, and except children with Hurler syndrome, demonstrates myeloablative and immunosuppressive effects sufficient for sustained engraftment. Treosulfan-based conditioning in 28 children with recurrent/refractory leukaemia or non-malignant disease: a follow-up K.W. Sykora, M. Sauer, B. Maecker, B. Meissner, K. Welte Hannover Medical University (Hannover, DE) Background:Some children with recurrent/refractory leukemia or non-malignant conditions are at high risk for TRM because of their underlying disease, previous dose-intensive chemotherapy or persistent infections. They would benefit from a conditioning regimen with high myeloablative activity and decreased toxicity. Treosulfan is an alkylating agent with good ablative activity and a favourable toxicity profile. Low toxicity has been reported in transplantation (TX) of high-risk adult patients. Methods: In the years 2003 to 2007, 11 children with recurrent/refractory leukaemia and 18 with non-malignant conditions were transplanted: Patients qualified for this conditioning regimen if they had recurrent/refractory leukaemia, and/or if pre-existing pulmonary or hepatic disease and repeated or ongoing viral reactivations were present. Conditioning included fludarabine (30 mg/m²/day x 6) and treosulfan (12 or 14 g/m²/d x 3) in all patients, with the addition of melphalan (140 mg/m²/d x 1) or Thio-TEPA (2 x 5 mg/kg) in some. Donors were MFD, 1 CORD, haploidentical MMFD, or MUDs. Among the malignancies, 8 patients had ALL and 3 AML. Of the non-malignant patients 2 had thalassemia, 1 sickle cell anemia, 1 Kostmann's disease, 1 Mannosidosis, 3 SDS, 3 CAMT, and 7 immunodeficiencies (2 Hyper-IgM, 2 CVID, 2 HLH, 1 MHC Class II deficiency). Results: All patients engrafted rapidly and achieved full donor chimerism after the first TX, except two early (thalassemia and ALL) and one late graft failures (CVID). Three of the leukemia patients survived, two of them longer than 2 years. All other leukemia patients died: 1 from extensive cGVHD, 1 from multiorgan-failure and the others from relapse. One infant with SDS died from IPS, and one patient with Hyper-IgM syndrome from late adenoviral septicaemia. One patient had grade IV skin toxicity and one grade IV GVHD. Toxicity and GVHD grades in all other patients were mostly I and II. Conclusion: This treosulfan-based conditioning regimen is feasible, and has low toxicity and good myeloablative activity in children with high-risk malignant and non-malignant disease and pre-existing organ damage or infection. Survival was poor (20%) in recurrent/refractory leukemia due to recurrent disease after BMT, similar to other transplant modalities. In non-malignant disease, survival with low toxicity was excellent (90%, Fig. 1 ). A common European multicentre non-commercial approach for quantitative MRD detection by WT1 gene expression PCR A.M. Willasch* (1), T. Coliva (2) , M. Kalinova (3) The European Study Group on WT1 Expression in Childhood AML has been established in 2003 and encloses centres from Germany, Czech Republic, and Italy. We present for the first time a quality-controlled non-commercial uniform multicentre approach for the standardized quantitative detection of WT1 gene expression applying qPCR for the investigation of WT1 expression on a large cohort of childhood AML patients. The group agreed on a common approach: RNA was extracted, according to local procedures, from BM-MNC at presentation of patients diagnosed in the participating centres. The analyses were performed by qPCR (TaqMan® The role of HLA class I and II mismatch (MM) was assessed in pediatric patients who underwent hematopoietic stem cell transplantation (HSCT) from a non related donor in a single center. Donor selection was performed prospectively on the basis of HLA A, B occasionally HLA C low resolution and DRB1 and DQB1 high resolution testing. HLA A,B,C was performed retrospectively for all donors mostly on high resolution level. 60 patients aged 0-18 years between 1996 and 2004 were studied. They were transplanted with an unrelated donor and had a typical pediatric disease distribution (ALL, AML, MDS, CML, HLH and metabolic diseases). The conditioning regimens were mostly myeloablative and consisted of mainly of Bu/Cy ±VP16 or Mel (n=41), TBI/Cy/VP16 (n=17), TBI/VP16 (n=2). The stem cell source was mostly bone marrow (49/60). The graft versus host disease (GvHD) prophylaxis consisted of ATG, Cyclosporin and Methotrexat. Based on high resolution typing 19/59 were completely matched (M) for HLA A,B,C and DRB1 and DQB1 (10/10 match). 22/59 had one MM in HLA class I or II. 12/59 had 2 MM in class I/II. 6/59 had more than 2 MM. Upon the evaluation of the influence of HLA matching on survival we can see that there is no difference in survival between the fully matched (75%) and the MM 1 (77%) or MM 2 (83,0%) group. In the group of more than 2 MM the survival is 42% but the latter group includes only 6 patients. There was no correlation between survival and class I MM or class II MM. Only when comparing match (10/10) survival (75%) with DQB1 MM survival (58%) a lower survival rate of the latter could be seen. Follow up in M group 63 months, follow up in MM group 41 months. A trend to acute GVHD grade II-IV is seen in the HLA (A,B,C,DR, DQ) MM entity. In the first 100 days patients with MM grafts have an incidence of 53% of aGVHD in contrast to the patients with a matched graft 33% (p=0,25), but without an impact on survival (79% vs 69%; p=0,2). In conclusion our analysis shows that a conventional approach of MUD transplantation procedures in respect to conditioning or GVHD prophylaxe is also rational in a 1-3 MM Situation before to turn to more experimental treatment strategies. Intravenous busulfan before autologous stem cell transplantation in children with cancer N. Le Guyader, T. Landre, S. Haouy, J. Landman-Parker, S. Fasola, A. Auvrignon, G. Leverger* Hôpital Armand Trousseau (Paris, FR) Objective: Autologous stem cell transplantation (ASCT) is a standard treatment for children with different solid tumors and acute myeloblastic leukemia (AML) in second complete remission (2nd RC). We report here our experience with intravenous busulfan (IVBu, Busilvex®, Pierre Fabre Medicament, Boulogne) for conditioning treatment in 18 children. Method: Observational prospective study Results: 13 children were treated for solid tumor: neuroblastoma (NB, 10), Ewing sarcoma (EWS, 2), and rhabdomyosarcoma (1) . Tumors were metastatic at the diagnosis except for 1 NB, initially unresectable and 1 EWS initially localized. Five children presented AML in 2nd RC. Median age was 7.5 years at the time of ASCT (2-13 y) . IVBu was associated with melphalan for 15 children, with cyclophosphamide for 1 and with cyclophosphamide and melphalan for 2 other patients. IVBu dosage was as recommended: 0.8 to 1.2 mg per kilogramme according to child's weight. Two patients treated for NB benefited of a reduced dosage of IVBu because of a renal failure antecedent. Prevention of seizures (with clonazepam) and of veno-occlusive disease (VOD) was realized (heparin and ursodesoxycholic acid). All children except one received peripheral blood stem cells (failure of peripheral stem cell mobilization). All patients achieved engraftment but 3 children never obtained platelets over 50.109/L and relapsed thereafter. Stomatitis was observed in all patients but two. Up to 80% of children presented liver toxicity with increased serum transaminases activities (grade I to III). Two patients, treated for AML, presented VOD and were treated with defibrotide ; one of them received before gemtuzumab-ozogamicin. All the patients with liver toxicity recovered. No major infection occurred during this study. With a median follow-up of 18 months, nine children are still alive. Nine children relapsed with a median delay of 5 months after ASCT and died. Conclusion: Although our monocentric study concerns a limited number of patients, our analysis shows the favorable safety profile of IVBu-based conditioning regimens, and our results are consistent with recent reviews on this topic, reporting a reduced incidence and severity of hepatic toxicity, compared to oral busulfan historical experiences. The avaibility of an IV formulation of busulfan certainly improves the strategy of ASCT in children. Evaluation of the improvement of quality of life will be of interest, particularly for this population. Objective: After allogeneic haematopoietic stem cell transplantation (HSCT), donor and recipient haematopoiesis could coexist in a dynamic process of chimerism. A careful analysis of chimerism on peripheral blood (PB) and bone marrow (BM) post HSCT provides basic information on take of transplant and can predict rejection and relapse. The aim of our study was to analyze possible differences of post transplant chimerism in children received allogeneic P982 A predictive score for patients with idiopathic myelofibrosis undergoing an allogeneic haemopoietic stem transplant A. Bacigalupo, G. Barosi, A. Dominietto, G. Piaggio, M. Van Lint, C. Di Grazia, A. Raiola, F. Frassoni S.Martino's Hospital (Genoa, IT) Background: Allogeneic transplantation is a therapeutic option for patients with idiopathic myelofibrosis (IMF). The risk category of IMF patients may vary from diagnosis the time the patients are referred for transplant. Aim of the study: To identify risk factors for outcome after an allogeneic HSCT. Patients: Forty patients with IMF underwent an allogeneic HSCT in our Unit. The conditioning was a thiotepacyclophosphamide based reduced intensity regimen in 37, and graft versus host disease (GvHD) prophylaxis was cyclosporin and methotrexate. The donor was an HLA identical sibling (ISD) (n=23) or an alternative related or unrelatd donor (UD) (n=17). The median age was 51 , the median interval diagnosis HSCT was 1015 days (225-7178). Predictive Score: The following data were used to compile the risk score: peripheral blood counts >1% (0/1), bone marrow blasts >5% (0/1), a course of AML like chemotherapy (0/1), peripheral blood CD34+ cells over the median (0/1), interval diagnosis HSCT >365, >2390 days (0/1/2), transfusions 22 cm (0/1), splenectomy no/yes (0/1) Results: The median score of the 40 patients was 4. (range 1-8). The outcome for patients with a score 1-4 vs a score 5-8 was as follows: transplant mortality 14% vs 26% (p=0.1); relapse related death (RRD) 0% vs 37% (p=0.002), overall survival 81% vs 31% (p=0.001). Conclusions: We conclude that patients with IMF can classified before allogeneic HSCT, using clinical data related to the disease before transplant. The risk score identifies patients at high risk of relapse related death. This scoring system , if validated prospectively, may be used to identify IMFD patients at high risk of relapse post-transplant. Introduction: Imatinib has profoundly changed the indication of allogeneic bone marrow transplantation (alloBMT) for CML worldwide and is now the first-line of treatment in most countries. However, in Brazil, Imatinib for CML in first chronic phase is provided by Federal Health Agency only for patients refractory/intolerant to interferon (IFN). Objetives: In order to study results of treatment with imatinib and alloBMT in this particular scenario we retrospectively analyzed 264 patients treated for CML in first chronic phase (less than 60 years-old) in three different institutions in Brazil. Methods: End points were event free survival (EFS = absence of hematological response, loss of hematological/cytogenetic response, relapse in accelerated phase/blast crisis or death) and overall survival (OS). From jan/2001 to dec/2006, 174 patients received imatinib 400 mg after failure or intolerance to IFN. Median time from diagnosis to imatinib treatment was 19 months (range: 2 to 205). At the same period of time, 90 patients received an allo-BMT from an HLA-matched sibling (n=83) or an unrelated donor (n=7). Patients receiving peripheral blood stem cell or umbilical cord blood were excluded. Median time from diagnosis to allo-BMT was 16 months (range: 5 to 104). Gender distribution was similar between groups. Imatinib group had a higher median age ( 40.7 vs. 32.8 years, P<0.001) . Results: With a median follow up of 3 years, 5-year estimated EFS was 61.9% for patients receiving imatinib and 51.8% for patients receiving an allo-BMT (P=0.0002). Estimated overall survival at 5 years was 92.7% for patients treated with imatinib and 58.8% for patients receiving an allo-BMT (P<0.0001). Conclusion: Allo-BMT should be no longer recommended as a first-line alternative treatment, even if imatinib is only available for patients refractory/intolerant to IFN. NM23-H2 protein is overexpressed in CML S. Tschiedel*, A. Jilo, D. Niederwieser University of Leipzig (Leipzig, DE) Introduction: The human Nm23 family consists of 8 genes, termed Nm23-H1 through -H8. H1 and H2 are 88% identical and localized to chromosome 17. A variety of studies imply a role for Nm23 proteins in a range of cancers, in which altered levels of expression rather than mutation correlate to Objective: To evaluate disease free survival (DFS) and overall survival (OS) of breast cancer (BC) patients (pts) with more than 20 metastatic axillary lymphnodes who received adjuvant high-dose chemotherapy (HDCT) with peripheral blood stem cell support (PBSC) after conventional dose chemotherapy (CDCT) with anthacycline plus taxane. Methods: Adjuvant program consisted of 3-4 cycles of epi/doxorubicin plus paclitaxel/docetaxel at conventional doses followed by cyclophosfamide 4-7 g/mq plus GCSF to mobilize PBSC. Conditioning regimes used were Thiotepa 400-600 mg/mq plus Melphalan 160 mg/mq (or Mitoxantrone 35 mg/mq). Adjuvant radiotherapy was given in case of breast conservative surgery. All endocrine-sensitive pts were prescrived hormonal therapy for 5 years at completion of adjuvant CT. Results: Twenty pts with more than 20 metastatic axillary lymphnodes were treated from 1998 to 2003 at our Institutions out of a clinical trial with this sequential approach. Characteristics of pts were: median age 47 yrs (range 32-64), ductal carcinoma 13 pts (65%), lobular carcinoma 6 pts (30%), other histology 1 pt, median number of metastatic axillary lymphnodes: 27 (range 20-45), ER and/or PgR positive: 11 pts (55%). All pts completed the adjuvant program with no treatment-related death. Most common adverse events were neutropenic fever (60%) including 3 documented staphylococcus aureus infections, G3-G4 mucositis (50%), G2-G3 diarrhea (20%). One patient experienced central nervous system haemorrage with long-term sequaele. At a median FU of 102 months (range 53-121), median DFS and OS were 34 (8-121) and 61 (13-121) months, respectively. Twelve pts (60%) had sistemic recurrence including 4 bone disease only (34%). No local relapse was observed. Seven out 12 pts (58%) were ER/PgR positive. Median OS from recurrence was 7 months. Conclusions: Median DFS and OS observed in our casistic of very poor prognosis BC pts treated with adjuvant sequential program integrating CDCT with anthracycline plus taxane and HDCT are encouraging and support the further investigation of this approach in randomized clinical trials.transplantation. Genotyping for NQO1 (C609T) might help to identify patients with an increased higher risk of TRM. Methylenetetrahydrofolate reductase gene polymorphisms in allogeneic haematopoietic stem cell transplantation needs further clarification M. Koldehoff*, D.W. Beelen, A.H. Elmaagacli University Hospital (Essen, DE) Background: Methotrexate (MTX) is commonly administered to allogeneic hematopoietic stem cell transplant (HSCT) recipients as graft-versus-host disease (GVHD) prophylaxis usually in association with other immunosuppressive agents. Moreover, the metabolites of MTX are known to suppress folate enzymes, such as 5,10-methylenetetrahydrofolate reductase (MTHFR), which converts 5,10methylenetetrahydrofolate (5,10-methylene-THF) into 5methylterahydrofolate (5-methyl-THF), a major circulating form of folate. Two common polymorphisms in the MTHFR (C677T, A1298C) gene are associated with a 50-60% decrease in catalytic activity. Methods: Here we genotyped in a retrospective study 157 patients (and their donors) for both MTHFR genotypes who underwent allogeneic HSCT for various diseases and analyzed their outcome. Genotyping of MTHFR was performed by real-time PCR using subsequent melting curve analysis. Results: Wild-type of MTHFR C677T and A1298C were found in 50 of 157 analyzed recipient/donor pairs (31.8%), a mutation of 677TT and 1298CC on the recipient side in 18 and 16 of 157 (11.5% and 10.2%) recipient/donor pairs, mutation of 677TT and 1298CC on the donor side in 21 and 18 of 157 (13.4% and 11.5%) donor/recipient pairs, while a mutation of 677TT and 1298CC in both the recipient and donor was found in only 8 and 7 of 157 (5.1% and 4.5%) cases. As of December 2006, 50 of the 157 recipiens (31.8%) had relapsed and 68 (43.3%) had died. Analysis of each MTHFR genotype showed that the five-year estimates for relapse were lowest in recipients with 677TT genotype [677CC/CT, relapse risk=0.37±0.04 versus 677TT, relapse risk=0.13±0.1; p<0.05] and 1298AA genotype [1298AA, relapse risk=0.27±0.07 versus 1298AC/CC, relapse risk= 0.39±0.07; p=n.s.] on the recipient and donor side, whereas the one-year estimate of treatment-related mortality (TRM) and the five-year estimate of overall survival (OS) were not statistically different between the various MTHFR genotypes. No statistically significantly association with the risk of acute GVHD was observed in recipients or donors with MTHFR C677T. Multivariable logistic regression revealed no significant association between MTHFR genotypes and OS. Conclusions: In conclusion, our results illustrate the need to evaluate both polymorphisms concurrently to fully understand the impact of genetic variability in the MTHFR gene. In vivo immune modulating effects of the human chorionic gonadotropine hormone A.H. Elmaagacli*, M. Koldehoff, N. Steckel, D.W. Beelen University Hospital of Essen (Essen, DE) HCG is a naturally occurring immune modulating agent, which is highly expressed during pregnancy. It is often observed that during pregnancy some autoimmune diseases such as multiple sclerosis and Crohns disease improves in women.Here we hypothesized that the improvement might be associated with HCG expression during pregnancy and evaluated its possible immunemodulating effects. Blood samples obtained from 34 women (median age 37, range 18-47) who underwent in-vitro fertilization (IVF) and received therefore HCG s.c at three different time points (10.000 IE first, after 24 hours 5000IE , after 48 hours 5000IE) were examined prior and after HCG application. Thereafter skin transplantation was performed with HCG and without in mice. We noticed a significant increase of the number of Tr cells (CD4+CD25+) to up to 40% (p<0.04) after the first and second hCG application in the blood samples of women. A twofold increased of CTLA-4 expression after the first hCG application (p<0.049) was observed, whereas at the same time FoxP3 expression fell rapidly (p<0.006). Our analysis of Th1 (CD3+CD4+IFNgamma) and Th2 (CD3+CD4+IL4+) type T cells revealed a slight shift after hCG application: the number of Th1 cells did not alter significantly, whereas the number of Th2 cells increased significantly (p<0.048). Further analyses showed that other subpopulations including B cells (CD19+), NK cells (CD16+CD56+), T helper cells (CD3+CD4+; CD3+CD4+CD45RA; CD3+CD4+CD45RO+), cytotoxic T cells (CD3+CD4+CD8+), TCRalphabeta and TCRgammadelta positive T cells were not influenced by hCG. Also, we analyzed cytokine serum levels in women before and after each hCG application by ELISA. Hereby we found that two applications of hCG increased serum levels of both IL-8 and IL-10 significantly (p<0.013 and p<0.03, respectively) measured by ELISA assays (p<0.013), whereas the serum levels for IL-2, IL-4, TNF-alpha, IFN-gamma, TGF-beta and IL-1beta (data not shown) were not significantly altered by hCG. HCG application increased the expression of antiinflammatory interleukin-27 (IL-27) and reduced that of the inflammatory IL-17. In addition, we found increased IL-10 serum levels and increased numbers of regulatory T cells CD4+CD in peripheral blood of women after hCG application. Rejection of BALB/c skin grafts transplanted in B6 mice was significantly delayed when mice received hCG after transplantion. We conclude that hCG may be useful for the induction of immune tolerance in transplantation. We conducted a prospective multi-center trial concerning 33 patients (pts) who underwent haematopoietic stem cell transplantations (HSCT) from unrelated 10/10 antigen HLAidentical donors (high-resolution level). Twenty-seven patients were assessable (13 F, 14 M -56 years , 24 pts received PBSC and 3 received bone marrow from unrelated donors. All pts received fludarabine 30 mg/m²/dx5d, treosulfan 12 g/m²/dx3d and antithymocyte globulins (ATG) 2,5 mg/kg/dx3d. After transplantation, there were 2 graft failures, 7 pts developed aGVHD ≥ grade II (30%). After 3 months, 23 pts were assessable, 7 pts developed a cGVHD (30%) (5 limited, 2 extensive), 12 pts died (7 from TRM causes, 5 from relapse) and 12 pts are alive in CR with a median follow-up of 14 S317 months. Among the global population we defined a low risk subgroup (CR1, CR2, PR2): 17 pts (12 AML, 1 ALL, 3 MM and 1 NHL), 2 pts presented an aGVHD ≥grade II (13%). With a median follow-up of 18 months, 2 pts had a follow-up of less than 3 months, 3 pts developed a cGVHD (20%), 6 pts died (40%) and 9 pts are alive in CR. At one year, the probability of overall survival (OS) and event-free survival (EFS) were 50.4% , 40% [23-70] for the total population and 60% , 51.5% for the low risk subgroup respectively. To try to demonstrate if Treosulfan allowed a better transplant outcome we performed a paired match-analysis [center and 4 out of 5 other parameters (age, gender, HSC, pre-transplant status and diagnosis)] comparing our Treosulfan series from unrelated donors and HLA identical sibling allogeneic transplants receiving fludarabine, busulfan and ATG from the registry of the société française de greffe de moelle et de thérapie cellulaire (SFGM-TC). This paired match-analysis showed no difference in term of OS and EFS between our series and SFGM-TC series [fig1] [ fig 2] . In conclusion, these results demonstrate that Treosulfan appears to be a very promising drug that could be included in the conditioning regimen before HSCT from unrelated donors. New method for autologous bone marrow mononuclear cell processing and cell-based therapies: preliminary experience in 9 patients with diffuse coronary disease and laser transmyocardial revascularisation A. Alegre*, B. Aguado, C. Cámara, M. J. Fernández-Villata, B. Iñigo, M.E. Moreno, R. Rodríguez-Notario, F. Lara, G. Reyes, J. Duarte Hospital Universitario de la Princesa (Madrid, ES) Background: Cell-based therapies for cardiovascular diseases require the availability of a rapid and efficient stem cell separation technique to be combined with surgical procedures. Concentrated mononuclear cells obtained from autologous bone marrow harvest (ABMMC) may be an ideal source for a mixed population of stem, precursor and accessory cells to optimize the autocrine and paracrine effect of cell-based therapies. Prior stem cell separation techniques only isolated specific cell lines, required large bone marrow volumes (500-750cc) and utilized time consuming chemical,selection devices or expansion and cell culture methodologies. Pilot studies suggest that intracoronary transplantation of unselected ABMMC cells may improve Left Ventricular Ejection Fraction(LVEF)in heart failure(HF). We evaluated a point of care device which utilizes density gradient centrifugation to concentrate bone marrow mononuclear cells for transmyocardial laser injection. Patients and Methods: Nine patients with diffuse coronary disease and medically refractory class III/IVangina(7male-2female,mean age 66years old)were prospectively evaluated for cell-based therapy combined with transmyocardial revascularization (TMR). At the time of surgery, 120cc of autologous bone marrow were aspirated from the posterior iliac crest and anticoagulated with citrate.Using a density gradient centrifugal system(HARVEST™,USA),bone marrow harvested was separated into its components, which included 20 cc of concentrated mononuclear cells inclusive of the buffy coat which was immediately available for direct transmural myocardial laser channels injection.Cell counts and flow cytometry were used to determine the total number of mononuclear cells in addition to specific somatic stem cell populations such as CD34+ and CD133+ cells. Results: Time for bone marrow aspiration and concentration averaged 30 minutes.The complete processing was performed closed to the surgery room,in sterile ambient.There were no complications related to the bone marrow aspiration.Average cell counts pre and post concentration were significatively(p<0.001)increased.No correlation between cell counts and patient demographics was detected. Conclusions and comments: These preliminary data shows that density gradient centrifugation with the HARVEST™device allows a safe,fast and efficient point of care concentration of ABMMC for cell-based therapies.Efficacy endpoints regarding cardiovascular applications await further functional evaluation We report our experience of Treosulfan-based conditioning for allogeneic transplantation of 23 children. All patients were treated at a single UK paediatric transplant centre between 2004 and 2007. Indications included; 13 ALL, 3 AML, 2 thalassaemia, 1 Diamond-Blackfan anaemia, 1 osteopetrosis, 2 haemophagocytic lymphohistocytosis and 1 monosomy 7 MDS. All patients received Treosulfan 36g/m² (12g/m²/day x 3 days) and Cyclophosphamide (50mg/kg x 4 days for nonmalignant and 60mg/kg x 2 days for malignant disease); ATG (Genzyme, 2.5mg/kg, days -8 to -4) was added for nonmalignant and unrelated donor grafts. Additionally, the 2 HLH patients received Etoposide (60 mg/kg x 1), 1 bi-lineage leukaemia patient received Melphalan (140mg/m² day -1) and the osteopetrosis patient fludarabine (30mg/m² x 5 days). Nine patients had identical sibling (cord n=1, bone marrow n=8), 4 unrelated marrow/PBSC and 12 unrelated cord blood (UCB) donors. All patients engrafted successfully and median discharge was at day +24.5 (15-35) with median platelet recovery >20x10 9 at day +21 (11-66), >50x10 9 at day +29 (range 14-73) and neutrophils >1x10 9 at day +21 (12-54). Acute morbidity was minimal with no veno-occlusive disease of the liver (including one patient with a history of the condition). Mucositis developed in only 8 patients, five grade I and three >II (national cancer institute common toxicity criteria). The patients with mucositis >II had received either methotrexate (as graft vs host disease prophylaxis) or melphalan (conditioning). After a median follow-up of 23 months (range 6-48 months), 17/23 (73%) patients are alive in complete remission (1 ALL -autologous reconstitution but remains MRD negative 9 months post-transplant, 1 thalassaemia -stable mixed chimaerism 18 mths post transplant, transfusion independent, the remaining 15 > 95% donor chimaerism). One patient (osteopetrosis) died of grade III GVHD, Evans syndrome and adenovirus pneumonitis; 5 have relapsed (ALL), of whom 3 have died of progressive disease and 2 are in CR 5 and 6 months post second allograft with TBI-based conditioning. Our experience suggests that treosulfan-based conditioning achieves high engraftment rates after sibling and unrelated donor transplants for malignant and non-malignant conditions in children. The risk of VOD and mucositis associated with the regimen is much less compared with that reported in the literature for busulfan or TBI based regimens. Multipotent mesenchymal stromal cells (MSC) have been used to improve the outcome of hematopoietic stem cell transplantation (HSCT). However there is a risk that MSC harbor latent viruses. Human parvovirus B19 (B19) is a common pathogen known to persist in the bone marrow and primary infection can lead to aplastic crises in individuals with high red cell turnover. HSCT recipients may be naïve to B19 and are immunocompromised, hence they are under risk of B19 infection. The aim of this study was to investigate possible transmission of B19 by MSC.One of 20 MSC, derived from healthy donors, screened were positive for B19 DNA by PCR. Infected and uninfected cells cultured under standard conditions had similar cell doubling time, phenotype (CD73+, CD90+, CD105+, CD34-, CD14and CD45-) and ability to differentiate. We found that in addition to their characteristic markers, MSC express the B19 receptor (P antigen/globoside) and have a low expression of the erythroid marker glycophorin A. In in vitro co-culture experiments, the infected MSC transmitted B19 to hematopoietic progenitors and to other MSC, suggesting that the virus can persist in the marrow of healthy individuals. Two HSCT recipients received infected MSC (1×10 6 /kg) as treatment for graft-versus-host disease, corresponding to a viral load of 7×10 5 genomic equivalents. One of the patients was IgG seropositive for B19 before HSCT. Both were heavily immunocompromised at the time of MSC infusion, as indicated by reduced proliferation in 3rd party mixed lymphocyte reactions. Neither patient developed viremia or symptomatic B19 infection. Our results suggest that because MSC express P antigen, they are the only cell type, other than erythrocytes permissive for B19 infection in man. They may thus be the natural reservoir for B19 persistence in the bone marrow of healthy individuals. Fortunately, transplantation of B19 contaminated MSC did not develop into clinical infection. However our results indicate that MSC destined for immunosuppressed recipients should be screened for B19. We have used rituximab both prophylactically and therapeutically in 14 patients (9 males and 6 females) suffering from B cell non-Hodgkin's lymphoma following a nonmyeloablative allogeneic stem cell transplantation (NST). Donors were matched sibling allo-SCT (11) or matched unrelated (3). The median patients' age was 51 years (range 23-68). Most patients had diffuse large B cell lymphoma (10), whereas 4 had low-grade lymphoma (3 -follicular, 1 lymphoplasmacytic). Most patients were heavily pre-treated and transplanted while not in remission (relapsed/persistent disease -9, partial remission -3) while 2 were in remission. Additionally, some patients were pre-transplant rituximab unresponsive. Methods: Rituximab dose used was 375 mg/m 2 in all patients. A range of 1-2 cycles given and a median of 4 doses per cycle (range 1-8) with an interval ranging from every week to every month. Treatment was therapeutic in 13 cycles and prophylactic in only 3 cycles. Rituximab treatment was accompanied by donor lymphocyte infusion in 9 patients (1) (2) (3) (4) and interferon alpha treatment in one patient. Results: The 1 year OS and PFS are 50% and 35% respectively (figure 1). With a median follow up of 13.75 months (2-88) from the rituximab treatment, 5 patients are alive and are disease free, including patients previously unresponsive to rituximab. Six patients died with progressive disease, other causes were relapse (1), GVHD (1) and CVA (1) . The only significant complication of treatment was prolonged neutropenia (one patient). Conclusion: rituximab contributes to the GVL effect and its activity following allogeneic SCT may differ to that seen pretransplant. Background: mesenchymal stem cells (MSC) are BM populating cells, different from HSC, which can be used to improve the rate and quality of haematopoietic engraftment by regenerating the marrow microenvironment. MSC can suppress immune response by multiple mechanisms. The usage of MSC for prophylaxis and treatment of severe a and cGVHD seems to be reasonable. Co-transplantation(co-T) of alloMSC with alloHSC can enhance the engraftment, accelerate the immune reconstitution and suppress the GVHD in HSCT. Patients and methods From 10.05 till 11.07 8 pts received co-T of HSC and MSC for prophylaxis of GVHD and 9 pts received isolated infusions of MSC for treatment of steroidresistant aGVHD and cGVHD. Diagnosis:AML-6, ALL-6,MDS-1,CML-BC-2,NHL-2. Related alloHSC was performed in 6 pts,MUD-8,haplo-3 pts. 9 pts received BM and 7 pts PBSC as the HSC source. The sources of HSC for haploHSCT pts were combination of primed BM and PBSC after separation by CliniMacs. MSCs processed by "Trans-Technology" company (lic ¹ 99-01-002224) include selection and expansion for a dose of 2.0x10 6 MSC/kg of recipient. On D-1, pts are given MSC (2.0 x 10 6 /kg) 24 hours before infusion of donor's HSC. Isolated infusion of MSC is provided in a case of developing steroid-resistant a or cGVHD, 6 pts received 1 MSC dose, 1pt-2 doses and 1pt-3 doses. Consent to participate in a research study was signed in all cases. Results No toxicities were observed related to the infusion of MSC. Among the 17 pts, 10 pts remain alive 1-25 months after alloHSCT, 4 pts died-1 pt non-engraftment, 3 pts progression or relapse. In group with co-T of MCS-HSC engraftment was signed at D+13-29, no pts had severe aGVHD (0-I stage-6 pts, II-IV-0%). Add.infusion of MSC for treatment GVHD doesn't require. Overall 2-y survival without complications related with MSC and without increase in relapse is 62%. After isolated infusion of MSC a PR was observed in 3 pts, CR in 3 pts with GVHD, III stage including 2 pts with haploHSCT, PR in 1 pt with mild cGVHD. Overall response was 85%. Conclusion. MSC infusion is well-tolerated, safe, without immediate infusion-related or late MSC-associated toxicities. Infusion of MSC for co-T of HSC during conditioning regimen may prevent severe aGVHD and cGVHD. Infusion of MSC for treatment resistant GVHD lead to reduction of GVHD stage in some patients. Infusion of MSC before HSCT don't influence to engraftment. Randomized clinical trials are necessary for the estimation of therapeutic effect of MSC in alloHSCT. Donor lymphocyte infusion to enhance remission status post allogeneic stem cell transplantation in patients with multiple myeloma who did not achieve complete remission L. Petersen*, F. Ayuk, U. Bacher, C. Wolschke, A.R. Zander, N. Kröger University Hospital Eppendorf (Hamburg, DE) Complete remission (CR) is a condition for long time survival for patients with Multiple Myeloma (MM). 50-60% of patients achieve CR after allogeneic hematopoeitic stem cell transplantation (HSCT). To enhance remission status by graft versus myeloma effect we evaluated donor lymphocyte infusion (DLI) to achieve CR in patients who were only in partial remission (PR) or very good partial remission (VGPR) after HSCT. We treated 20 patients with a median age of 50 years (35-69) who had underwent allogeneic HSCT from related (n=7) and unrelated (n=13) donors. Median time to first DLI after HSCT was 248 (range 61-1365) days (second DLI: median 526, third DLI median 936). Median number of DLIs were 2 (range 1-3) with a median of 1 x 106 (range 0,26-200 x 106) CD 3+ cells /kg in first dose. Before DLI, remission status was PR (n=6) or VGPR (n=14). Four patients received thalidomide in combination with DLI, one patient received bortezomib with DLI. After DLI CR was achieved in 65% of patients, 25% achieved or remained in VGPR, 10% with PR before DLI had stable disease. Of those patients who did not achieve CR (35%, n=7), one patient (5%) achieved a further reduction of the paraprotein. No patient progressed within 5 months after DLI. After a median follow up of 5.5 years, 45% of patients (n=9) are still in remission, 35% in CR (n=7). 35% of patients had acute graft versus host disease (aGVHD) (43% grade I-II, 14% grade II, 43% grade III). Chronic graft versus host disease (cGVHD) was seen in 40% (n=8) (limited disease 25% (n=5), extensive disease 15% (n=3)). For patients achieving CR after DLI, the median TTP was 13 months and those not achieving CR 9.5 months. DLI as post transplant strategy is a feasible and safety approach with acceptable rate of GvHD. Patients who achieved CR had a longer time to progression than those who had not achieved CR. DLI can induce more CR resulting in a prolonged time to progression. A. Chukhlovin*, D. Ostasevich, O. Pankratova, E. Klyuchnikov, I. Kazantsev , L. Zubarovskaya, B. Afanasyev St. Petersburg State Medical University (St. Petersburg, RU) Patients and methods: We have observed one-hundredtwenty-one patients with oncohematological disorders who underwent intensive chemotherapy followed by HLA-matched allogeneic HSCT. Eighty patients received peripheral HSCs, whereas bone marrow was transplanted in forty-one cases. Transplants from unrelated and related donors were carried out in, respectively, seventy-eight and forty-three cases. Laboratory studies included PCR detection of cytomegalovirus (CMV), Herpes Simplex virus (HSV) and Epstein-Barr viruses (EBV) DNA in peripheral blood on weekly basis before and within 100 days after transplant. Virus reactivation was registered in case of >2 positive findings post-transplant. When analyzing clinical features, the cases of pneumonia, mediated by transforming growth factor-beta, interleukin 10, prostaglandin E2 and indoleamine dioxygenase, but also BMSC to lymphocyte contact is needed. It has been proposed to infuse BMSC to improve bone marrow engraftment and to treat graft versus host disease. Thus, to use these cells, it is relevant to know how to regulate their immunosuppressive effect. Herein, we have analyzed whether statins (i.e. atorvastatin, fluvastatin, mevastatin and simvastatin), reported inhibitors of the enzyme 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase involved in cholesterol synthesis, may affect morphologic, phenotypic and functional features of BMSC. Upon exposure to any kind of statins tested, we observed striking morphological changes in cultured BMSC, with a decrease in adhesiveness and a complete detachment of cells from the substrate, that ultimately led to BMSC cell death. This was related to alterations in the assembly of actin microfilaments and microtubules, as indicated by confocal microscopy. Indeed, in statin-treated BMSC actin was localized at the periphery of the cell, close to the cell membrane while tubulin alpha and beta were not assembled into microtubule organizing centre. The S-phase of cell cycle was abrogated by statins and consequently BMSC division and proliferation was blocked. Statins could reduce the intensity of cell surface expression and distribution of ICAM1 and LFA3 adhesion molecules. As these molecules play a key role in the cross-talk between BMSC and lymphocytes, it is conceivable that BMSClymphocyte interactions are strongly affected by incubation of BMSC with statins. Indeed, the inhibition exerted by BMSC on T lymphocyte proliferation in response to polyclonal mitogens or anti-CD3 monoclonal antibodies was abrogated when BMSC were pre-incubated with statins. The statin-mediated effects were time and dose-dependent, as they occurred in the presence of 10microM by 48h or after 72h with 1microM concentration of statin. Of note, mevalonic acid, the metabolic product of (HMG-CoA)-reductase could revert morphologic, phenotypic and functional effects of statins. Altogether, these findings indicate that alterations of cholesterol metabolism can interfere with BMSC functional behaviour and thus statins may be useful in regulating BMSC-mediated immunosuppression. Granulocytes recovery post autoHSCT is associated with the stromal cell-derived factor -1 (SDF-1/CXCL12) gene polymorphism A. Gieryng* (1), S. Madej (2), J. Werkun (2) , E. Nowak (2) It has been previously shown that the presence of A at position 801 in the 3'-untranslated region (3'-UTR) within the stromal cell-derived factor -1 (SDF-1/CXCL12) gene is associated with a higher yield of CD34+ mobilized for haematopoietic stem cell transplantation (HSCT) in autologous setting and allogeneic donors. These findings prompted us to investigate whether accommodation of haematopoietic stem cells in the marrow, reflected by the granulocytes recovery (expressed as the day post transplant when the number of granulocytes exceeded 0.5x10 9 /l) may be also influenced by the SDF-1 gene polymorphism. Forty pts (F/M=19/21; age 19-64, median 46 yrs; NHL=20, HD=9, MM=6, AML=4, Ca testis=1) undergoing autologous HSCT were investigated. SDF-1-3' UTR genotyping was performed using a PCR-RFLP technique and digestion with MspI restriction enzyme. It appeared that: (1) As it was found in previous studies also in this group of patients a higher mobilization potential characterised patients having allele A (median fraction of CD34+ cells x10 6 : 9.50, 6.05, and 4.07 for patients carrying AA, AG and GG genotype, respectively, although this relationship did not reach statistical significance).(2) SDF-1 gene polymorphism was significantly associated with the pace of granulocytes recovery post HSCT (p=0.024 assessed by median test taking into account the days of reaching >500/ul of granulocytes in blood after transplantation in patients carrying AA, AG and GG genotypes).(3) Number of transplanted CD34+ cells was associated with the pace of granulocytes recovery (R Spearman: -0.38; p<0.016) (4) When the patients were divided into two subgroups with respect to the presence or absence of the SDF-1-A allele, patients carrying the SDF-1-A allele (in homozygous or heterozygous form) had quicker recovery of granulocytes than those having GG genotype (median: 15 vs 17 day post HSCT, p<0.032 Mann-Whitney test). Objectives: Granulocyte colony-stimulating factor (G-CSF) is a pleiotropic cytokine playing a major role as regulator of hematopoiesis and innate immune responses. Recent data indicate that G-CSF also exerts regulatory function in the adaptive immune system and may promote tolerogeneic cells at both poles of APC/ T cell interaction. Interestingly, T lymphocytes may express a functionally active G-CSF receptor. Here, we characterize a regulatory T cell subpopulation directly generated after in vitro stimulation with G-CSF. Methods: Human peripheral CD4+CD25-T lymphocytes were isolated by MACS separation from healthy donors and kinetic assays were subsequently performed by stimulation with G-CSF (± anti-CD3 monoclonal antibody). Marker molecules of regulatory T cells were analyzed by realtime RT-PCR in the stimulated T cell populations. Proliferation and inhibition assays were performed in order to determine the suppressive capacity of G-CSF stimulated T cells. Results: In vitro stimulation of CD4+CD25-T lymphocytes with G-CSF revealed a time dependent up-regulation of FOXP3 gene expression which was significant after 4h of stimulation (p<0.05 vs. non stimulated control cells; n=8). Gene expression analysis of further regulatory T cell marker resulted in increased mRNA levels for CD73 and CD83. In contrast, mRNA expression of GATA3, GITR, CD127, GRP83 and CLTA4 was not affected by G-CSF. Furthermore, functional assays demonstrated that G-CSF may slightly increase the proliferation of stimulated CD4+CD25-T lymphocytes. However, CD4+CD25-T cells preincubated with G-CSF suppress anti-CD3 induced proliferation of autologous T cells in independent inhibition assays. Conclusions: Our results demonstrate that G-CSF modulates CD4+CD25-T lymphocytes in terms of gene expression and functional capacities exhibiting an immunoregulatory phenotype. Most importantly, G-CSF stimulated T lymphocytes seem to have a suppressive effect on the proliferation of autologous T lymphocytes and up-regulate important marker molecules of regulatory T cells. Thus, G-CSF represents an interesting candidate for specific immune modulation in transplantation medicine and autoimmune diseases. Association of cytokine levels in unrelated haematopoietic stem cell transplantation outcome A. Prieto-Hinojosa (1) HLA matching is critical for the overall outcome in both the HLA-matched related and unrelated donor (MUD) haematopoietic stem cell transplant (HSCT) settings. Other non-HLA-encoded genes have been investigated for their role in overall survival after HSCT transplant. Amongst these are the cytokines, whose levels in patients can vary both as the result of genetic polymorphism -for example, polymorphisms in the promotor regions of the cytokine genes -and of epigenetic factors influencing the nature and extent of the immune response. In HSCT, cytokine polymorphisms have been associated mainly with graft versus host disease (GvHD) and transplant related mortality (TRM). High levels of TNF-alpha have showed an inconsistent association with GvHD. However, the lack of INF-gamma and low production of IL-1 can accelerate aGvHD. We measure the levels of different cytokines (IL-1b, IL-2, IL-4, IL-10, IL-12p70, IL-15, IL-17, INF-gamma, TNF-alpha and TGF-beta) in plasma from 62 patients undergoing an unrelated donor-HSCT and we correlated with transplant outcome. The median follow up was of 1049 days. The overall survival was 40.3%. We observed that high levels of regulatory cytokines was correlated with higher incidence of relapse, less disease free survival and less overall survival where higher levels of inflammatory cytokines correlated with better survival and less relapse. None of the cytokines studied here was associated with acute or chronic GvHD or TRM. We found that higher levels of IL-10 and TGF-beta correlated with worse survival (p=0.001 and p=0.026 respectively). This higher proportion also correlated with higher incidence of relapse (p=0.020 and p<0.001 respectively) and with decrease in disease free survival (p=0.003 and 0.029 respectively). No effect was seen in the incidence of acute or chronic GvHD. Conversely, inflammatory cytokines had the opposite effect, patients with high levels of TNF alpha had less relapse and better DFS(p=0.020 and p=0.025 respectively). In a multivariate analysis, high levels of regulatory cytokines resulted in worse OS (IL-10 Relative Risk (RR), 4.475; p=0.002 and TGF beta RR, 3.206: p=0.019) in reduced DFS (IL-10 RR, 3.531; p=0.009 and TGF beta RR, 4.040: p=0.005) and an increase in disease relapse (IL-10 RR, 5.121; p=0.017 and TFG beta RR, 14.491, p<0.001). Pre-transplant cytokine measurements may be an important indicator that can allow the stratification of patients into highrisk and low-risk categories for relapse. CD4+ lymphocytes under the influence of a local environment differentiate into Th1 and Th2 cells as well as for cells exerting suppression (FoxP3 Treg cells) and those involved in autoimmunity characterized by a production of interleukin 17 cytokines (Th17). IL-6 (i) plays a role in promoting CD4+ cells differentiation into Th17 helper cells and (ii) is frequently elevated in HSCT pts. Therefore, we studied the cytoplasmic expression of IL-17 and that of FoxP3 in stimulated PBMC of alloHSCT patients (14 pts, median age: 45 yrs, 13 leukemias, 1 SCID). Seven of them developed aGvHD. Peripheral blood lymphocytes were examined at the beginning of either aGvHD manifestation or hematological reconstitution and then in one week intervals. Nine healthy individuals served as controls. PBMC were stimulated with BD Leukocyte Activation Cocktail (PMA, Ionomycin and BFA) in the presence of GolgiStop, then stained with CD4, IL-17A and FoxP3 MoAb and analyzed in CD4+ and CD4-lymphocytes. FoxP3 mRNA was measured in unstimulated PBMC with the use of qPCR. Peg-filgrastim is a G-CSF form characterized by a increased plasma half-life, approved for clinical use. The availability of this drug allowed us to test the ability to improve myeloid S327 recovery after autologous peripheral blood stem cell transplantation (aPBSCT) for hematological malignancies in comparison with standard growth factors. We also investigated the kinetics of appearance of CD34+ cells in peripheral blood after transplantation and its relation with growth factors administration. From February 2004 to November 2007 we enrolled 86 patients submitted to aPBSCT, divided in three groups: A group, B group, C group who received respectively a single dose of peg-filgrastim on day +1, daily filgrastim starting from day +1 and daily filgrastim starting from day +6 until engraftment respectively. Characteristics of patients of three group are shown in table 1. CD34+ cell dose infused was similar in three groups while age was significantly higher in patients of C group, affected by multiple myeloma. All patients achieved engraftment as showed in table 2. Statistical analysis showed a statistically significant improvement of neutrophil recovery in A group compared with both B and C group: day 9 (range 7-11) to 500 Polymorphonuclear cells/microL in A group, while day 10 (range 8-13) and day 11 (range 9-15) in B group and in C group respectively (p=0.0002); furthermore we found a statistically significant difference in number of days with absolute neutrophil count (ANC) <100 cells/microL: 4 days (range 2-8) in A group, while 5.5 (range 3-10) and 5 (range 3-10) in B and C groups respectively (p=0.0028). We did not observe significant differences concerning other endpoints analyzed but, interestingly, we found a statistically significant higher CD34+ percentage on day +10 in patients receiving peg-filgrastim: 0.095 % vs 0.025 % (p=0.01).In conclusion, peg-filgrastim administration was well-tolerated, efficacious and significantly improved myeloid recovery compared with filgrastim administration both at day +1 and +6 after aPBSCT. The reduction of time with ANC<100/microl could be very crucial even if in our small series did not result in significant clinical improvement. These data together with higher post-transplant CD34 mobilization needs to be confirmed in a larger series in order to clarify effective benefits and justify economical impact of peg-filgrastim. Objectives: High Mobility Group Box 1-Protein (HMGB1) is a nuclear chromosomal protein occurring ubiquitary in all mammalian tissues. When HMGB1 is actively released by haematopoietic cells or passively by necrotic cells it demonstrates cytokine function and induces inflammation. The role of HMGB1 in induction of allogeneic graft vs. tumour effects and/or in vaccination is little understood. In this study we investigated the intensity of HMGB1 expression in different canine haematopoietic cells and the influence of HMGB1 on T-cell proliferation. Methods: Monocytes (CD14+), T-lymphocytes (CD3+) and Granulocytes were isolated from EDTA blood (n=6) and CD34+ stem cells were isolated from bone marrow (n=5) of normal Beagle dogs by Ficoll density gradient and magnetic cell sorting. CD34+ were thereafter differentiated into Dendritic Cells (DC). From CD14+, CD3+, Granulocytes, CD34+ and DCs mRNA was isolated and TaqMan qPCR was performed. Relative quantification was done using the 2E-ddCt method. For proliferation assays PBMC were cultured in Quantum 007 for 72 hours in the presence of 2µg/ml Concavalin A (positive control) or rhHMGB1 (0, 1, 5, 10, 25 and 50ng/ml), respectively. [3H]Thymidine was added and incorporation was measured on a scintillation counter. The stimulation index (SI) was calculated as mean cpm of stimulated wells divided by mean cpm of negative control. Responses were considered positive if SI was ≥2. Results: The mean relative HMGB1 mRNA expression was lower in CD14+ and Granulocytes compared to expression in CD3+ T-lymphocytes (CD14+: mean 15±6%; Granulocytes: mean 29±18%). HMGB1 expression was high in CD34+ stem cells and decreased during differentiation from CD34+ stem cells into DCs (mean 44±17%). Exogenous rhHMGB1 at concentrations of 1, 5 and 10ng/ml increased T-cell proliferation compared to negative controls. The SI reached 2.6±1.97, 2.1±0.93 and 2.0±0.83, respectively. At higher concentrations (25ng/ml and 50ng/ml) rhHMGB1 had only limited influence on T-cell proliferation (SI 1.5±0.57; 1.1±0.22). Conclusion: HMGB1 is highly expressed in canine CD3+ Tlymphocytes and CD34+ stem cells. Of interest, HMGB1 expression decreases during CD34+ stem cell derived DC S328 differentiation. In addition, exogenous rhHMGB1 shows a stimulatory effect on T-cell proliferation at low concentrations. Therefore, our data suggest that HMGB1 might be a useful immune adjuvant in lymphocyte stimulation and graft vs. tumour reactions in vivo. Role of cytokine gene polymorphism in both donor and recipient in acute graft-versus-host disease after reducedintensity conditioning haematopoietic stem cell transplantation I. Sakellari*, A. Hatzidimitriou, D. Mallouri, P. Kaloyannidis, I. Batsis, D. Mpartzoudis, C. Vadikoliou, C. Smias, A. Anagnostopoulos, A. Fassas G.Papanicolaou (Thessaloniki, GR) There has been clear evidence that certain genotypes of cytokines influence the occurrence of acute graft versus host disease (aGVHD) after conventional hematopoietic stem cell transplantation (HCT). The aim of the present study was to investigate both donor and recipient cytokine gene polymorphisms and their contribution to aGVHD incidence after reduced intensity conditioning hematopoietic stem cell transplantation (RIC-HCT). DNA samples of 24 recipients and their donors were analyzed for genotype of proinflammatory, inflammatory cytokines or their receptors. The 24 patients suffered from malignant hematological disease and all but one received grafts from siblings. Results were correlated with the incidence of GVHD grades II to IV, which occurred at 12 patients. In terms of patient genotypes, the polymorphism of IL1beta 511promoter/position+3962, IL2 promoter/position 330 (p≤0,05). and IL2 166position (p≤0.06) were significant factors for developing aGVHD in univariate analyses. Patients with IL1beta C/C genotype developed lower incidence aGVHD (13,5%) v Sigma C/T (54%) p(0,05) and T/T (75%) (p≤0,02). In patients with G/G genotype Iota L2330 higher rate aGVHD appeared vs in those with genotype G/T, 70% vs. 30% respectively (p≤0,01).The single nucleotide polymorphism in theIL2 gene promoter region results in the T to G transition which is related to overproduction of IL2 as in vitro studies have shown. On multivariate analysis, aGVHD was related to: full donor chimerism, IL1beta and IL2330 gene polymorphisms and the non administration of ATG (p≤0,05). In terms of donor genotypes, on univariate analyses significant factors for aGVHD, were the following polymorphisms were determined: IL2166 G/T genotype (66%) vs G/G (26%), IL1beta 511-T/T 72% vs C/C 0% and IL4RA G/A 60% vs A/A 33% (P≤0.02). Disparities between donor-recipient polymorphisms and minor histocompatibility antigens HLA-2 did not influence the aGVHD rate. Reduced intensity conditioning regimens spare many residual immune recipient cells and immune reactions are activated by both donor and survived recipient T lymphocytes and the cytokine expression seems to be dependent on gene polymorphisms. The widening of the research field and the reproducibility of the above results may lead to intensification of aGVHD prophylaxis Objective: The aim of study was to investigate the impact of patient's and donor's Interleukin-6 (IL-6) gene polymorphism on outcomes of allogeneic hematopoetic stem cell transplantation (allo-HSCT).Materials and methods: Analysis of the IL-6 174G>C promoter polymorphism genotype in 51 transplant recipients and their HLA-identical donors was performed. The median age of patients(pts) was 15 years (from 1 to 47 years). 24 pts were females, 27 -males. 32 pts had ALL, AML-13, CML-4, NHL-2. Unrelated allo-HSCT were performed in 37 pts, related allo-HSCT -in 14 pts. The reduce intensity conditioning regimes were used in 26 pts (51%), myeloablative conditioining -in 25 pts (49%). In the majority of allo-HSCT cyclosporin A and methotrexate were used for aGVHD prophylaxis. The IL-6 genotypes were determined by an allele-specific PCR. Results: The distribution of polymorphism of IL-6 gene was significantly different in healthy donors compared with pts suffering from ALL, AML, CML, and NHL. The frequency of C/C genotype was higher in healthy donors (15,7%) than in pts (3,8%) (p=0,043). Incidence of relapse (Rel) was increased for IL-6 G/G patient's genotype (p=0,042). Relapse developed in 12 of 20 (60%) pts with G/G genotype of IL-6 gene compared with 29% (9 of 31pts) with C/G and C/C patient's genotype. Donor's genotype C/C was favorable for complete remission (CR) after allo-HSCT(p=0,015). Eight patients whose donors had C/C genotype of IL-6 remained in CR (Rel rate 0%) compared with pts whose donors had C/G and G/G genotype (Rel rate 49%, 21 of 43 pts). Logistic regression analysis confirmed the association of Rel frequency with recipient's IL-6 genotype (p=0,025), in addition to age of pts and disease's stage. The 5-year overall survival (OS) was 42% and 86% in pts with G/G and C/G genotype, transplanted in remission (n=24). Summary: The IL-6 174G>C polymorphism is a marker of an outcome after allo-HSCT. The presence of IL-6 C allele in pts and also in donors of hematopoietic stem cells is associated with the lower incidence of relapse after allo-HSCT. Background: Viral infections are the leading cause of nonrelapse mortality after unrelated cord blood transplants (UCBT). Post-transplant lymphopenia, lack of established antiviral memory, and poor cytotoxic function of CB lymphocytes contribute to ineffective immunity. Ex vivo T cell expansion could serve post UCBT as non-specific immunoprophylaxis in the form of DLI utilizing IL-2 + CD3/CD28 co-stimulatory beads. However, there is high rate (>10%) of activation induced cell death if IL-2 is the sole growth factor. We tested our hypothesis that IL-7 may aid T cell expansion and survival, while preserving the capacity of expanded T cells to home to primary lymphoid organs despite maturational events towards a more cytotoxic phenotype. Materials and Methods: Following an IRB-approved protocol T cells were isolated from frozen/thawed specimens and cultured with CD3/CD28 artificial beads (ClinExVivoR, Invitrogen Corporation) in 5% serum replete media + IL-2, ± 10ng/ml of IL7 (R&D Syst). Media, cytokines were replenished to maintain a concentration of ~0.75 x10 6 cells/ml. Automated cell count, trypan blue viability assessed overall cell growth and viability at each feeding. Lyse/no wash FACS staining with Trucount beads quantified viable CD3+ T cells. Multiparameter FACS was employed to characterize the S329 evolution of surface and intracellular (ic) immunophenotype. Cytotoxicity profile of the day 0 and D12-14 progeny was tested. Results: After 12-14 day expansion, T cells expanded on average 98-fold (n=15). Expansion was significantly enhanced by IL7 (mean 156 vs 47 fold, p=0.027) by increasing the fraction of T cells entering cell cycle (75% of T cells ic Ki67+ with IL7 versus 64% without, p<0.01) and by reducing T cell apoptosis, quantified by activated ic Caspase-3 detection (7% with IL2 alone vs 4% with IL7 added, p=0.027, 2-tailed paired t-test). Regardless of IL-7 in the media, Th1/Tc1 cytokines, Granzyme A, B, perforin expression was enhanced comparably, while cytotoxicity (Europium release assay) was absent against a Burkitt lymphoma cell line (IM9) or primary fibroblasts from UCB recipients. Over 90% of expanded T cells are CD62L, CD27, CD28 and CD45RA positive. Conclusions: IL7 boosts expansion of UCB T cells while preserving a "naïve" surface phenotype. However,cytokine production and granzyme/perforin expression suggests partial maturation. These results pave the way to donor-derived UCB T cell expansion suitable for DLI to boost immunity. Infusion of cryopreserved stem cell grafts containing DMSO results in adverse effects such as vomiting, dyspnoea, and cardiovascular effects. Furthermore exhalation and bodily fluid excretion of sulfite-containing metabolites affects hospital environment and necessitates patient isolation regimes and intensive ventilation due to unpleasant odeurs. In order to reduce these effects, we removed DMSO by washing 108 thawed autologous stem cell grafts prior to reinfusion in 101 patients receiving chemotherapy for hematological malignancies during 2005-06. As a control group we retrospectively included 90 patients receiving un-washed autologous stem cell grafts during 2003-04. Grafts were selected for washing if the CD34+ cell count was above 4.5 x 10 6 /kg BW in the freshly harvested graft. The frozen graft was thawed at 37°, adding pulmozyme and MgCl2, and kept cold after thawing. Washing was performed in 2 cycles in a Cobe 2991 device, using cold isotonic NaCl with 3% Macrodex and 2% human albumin achieving a mean 1.6 log reduction in DMSO content, and was followed by filtration due to residual DNA. The median viability of the cells in the washed grafts were 86 % (interquartile range 79-90.5) and the cell recoveries were: TNC 72.1% (interquartile range 58.8-81.1); CD34+ cells 93.4% (interquartile range 78.4-116.9). The washed cells contained a median of 5,3x10 6 CD34+ cells/kg BW (interquartile range 4.4-6.8). Technical problems due to clotting arose in 2% of the washed grafts, requiring supplementary filtration. All patients engrafted satisfactorily in the neutrophil line, whereas 6 patients experienced prolonged thrombocytopenia, including a single patient receiving less than 2.0x10 6 CD34+ cells/kg BW. The median time to reach a neutrophil count >0.5x10 9 was 11 days (interquartile range 10-11), and for platelet count >20x10 9 the median was 14 days (interquartile range 12-15, excluding the 6 patients with prolonged thrombocytopenia). This was not significantly different from engraftment times in the control group (log rank test). Adverse effects were significantly milder upon infusion of washed compared with un-washed stem cell grafts (fishers exact test, p<0.05). Hospital environment benefited from the disappearance of the sulfite smell. We conclude that the washing procedure was feasible and safe in 95% of the patients. The possible effect of washing on megakaryocytic engraftment will be further investigated. Background: Recent clinical studies have indicated that grafts consisting of two unrelated umbilical cord blood (UCB) result in more consistent and accelerated engraftment compared to single unit UCB transplants, but the mechanism for enhanced engraftment is unknown. Two hypotheses can explain higher frequencies and more rapid engraftment observed with transplantation of double UCB units compared to single UCB transplants: 1) immunological interactions between competing UCB units that release hematopoietic cytokines; and 2) a stochastic mathematical model that predicts increased probability of engraftment when multiple units are present. A previous study demonstrated that the observed 4 day acceleration in the time to reach an ANC>500/mcL with double versus single UCB grafts was explained by the stochastic model (Waller 2007 . Blood 110:2034 . To test these hypotheses in mice, we transplanted limited numbers of donor bone marrow (BM) plus donor splenocytes (SP) from C57Bl/6 (H2b) and Balb/c (H2d) mice into lethally irradiated B10.BR (H2k) recipients and measured survival and engraftment. Methods: Three treatment groups were compared, recipients of 1 x 10 6 BM and 1 x 106 SP from H2b donors; 1 x 10 6 BM and 1 x 10 6 SP from H2d donors; a mixture of 0.5 x 10 6 BM and 0.5 x 10 6 SP from both H2b+ H2d donors. We tested whether overall engraftment and survival were greater in the recipients of mixed grafts compared with mice receiving equal numbers of donor BM and SP from a single donor. Results: There was a trend favoring better survival for recipients for double allografts, as the 60-day survival was 30%, 60%, and 60% for recipients of H2b, H2d, and H2b+ H2d grafts, respectively (p=ns). T-cell chimerism in surviving transplant recipients showed mean values of >95% donor Tcells in recipients of either H2b or H2d grafts, with 5/6 recipients of H2b+ H2d grafts with >95% H2b T-cells and 1/6 recipients with >95% H2d T-cells. Conclusions: In a murine model of double allogeneic transplantation, engraftment and survival of a mixed H2b+ H2d or single H2b or H2d hematopoietic grafts containing limited numbers of donor BM cells and SP were comparable. Similar to what is observed clinically, cells from only one donor establish durable engraftment among recipients of a double hematopoietic cell graft following myeloablative conditioning. These data are consistent with the stochastic model of engraftment in which each unit of a double hematopoietic graft act independently. Selective allodepletion (SD) is a strategy to eliminate hostreactive donor T-cells from allografts to prevent graft versus host disease (GvHD) while conserving useful donor immunity. We developed a semi-closed, GMP-quality, clinical scale SD process where donor-derived lymphocytes are stimulated with patient-derived T-antigen presenting cells in an ex vivo mixed lymphocyte reaction (MLR) [Blood 2007 ]. Host-alloactivated T S330 cells are targeted by activation-based changes in pglycoprotein that result in an altered efflux of the photosensitizer TH9402 and are then eliminated by exposure to visible light in the photodepletion device (Kiadis Pharma Inc, The Netherlands). After "Food and Drug Administration" and "Institutional Review Board" approval we initiated a clinical trial where HLA-identical sibling recipients with hematological (non T-cell) malignancies received a CD34-cell selected transplant (Miltenyi,Germany) containing less then 1 x 10(4) T cells/kg together with 5 x 10(6) /kg viable SD donor T cells on day 0, using an age-adapted, radiation-based preparative regimen (FluCyTBI). Low-dose cyclosporine was used as sole immunosuppression in the absence of GvHD. The first five patients transplanted (median age 45 (28-53) years with ALL, ALL, MCL, MDS/AML, or MDS) have a median follow-up of 111 (55-167) days. Patients received a stem cell product containing a median of 6.0 (5.4-9.5) x10(6)/kg CD34+ stem cells and a median of 8.5 (0-25.7) x10(2)/kg residual unselected T cells in addition to 5x10(6)/kg SD T cells. Absolute lymphocyte recovery was rapid (median 1144 (677-2486) cells/µL day 30 post transplant). Early T cell chimerism was donor-dominated (median of 77% (40-79) on day 14, and 98% (94-100) on day 30, and 100% (96-100) on day 60 and thereafter. Two patients developed grade I skinonly aGvHD. No grade II-IV acute or chronic GvHD occurred. Three patients reactivated CMV and three developed hemorrhagic cystitis with BK-or Adeno-Virus. All patients remain relapse-free. One patient received an unmanipulated DLI to treat a delayed fall in T cell chimerism. Although results are preliminary, this is the first demonstration of the clinical feasibility of photodepletion-based SD stem cell allotransplants in matched siblings. Robust lymphocyte recovery and early donor chimerism provide early evidence for the functionality of SD T cells in the absence of clinically significant GvHD, allowing a reduction of immunosuppression for sufficient disease control. Introduction: Haploidentical transplantation is an important therapeutical option in patients with severe diseases, if there is a lack of donor or less time for donor search. Different graft manipulations like CD34 or CD133 positive selection or CD3/CD19 depletion were introduced in the last years. Method: We report about a protocol which was adapted for children and was first introduced for adults by one of us (HJK). This protocol consists of transplantation of non-depleted bone marrow of one of the haploidentical parent, followed by CD6depleted peripheral blood stem cells (PBSC) on day +6. Result: By this kind of depletion in our 8 children a purity of 97% can be achieved in regard to CD6 positive cells with a recovery rate of 99% for CD34 positive stem cells resulting in a megadose graft (>20x10 6 /kg body weight). Nearly almost of the CD4-positive cells could be removed (99,7%) , where as about 6,8x10 6 CD8-positive, CD6-negative cells/kg body weight remain. This cells might have immunosuppressive function because acute and chronic Graft-versus-Host Disease (aGvHD;cGvHD) occur not more frequent (7 patients included: aGvHD I-II: 3x, III-IV: 1x, extended cGvHD: 2x) and responses in all cases to immunosuppressive treatment. In addition 33% of B-cells were removed using this kind of depletion and treatable reactivation of Epstein-Barr virus has occurred in 2 of the 7 patients. NK-cells remain completely in the grafts and are presumably responsible for the antileukemic effect before in vivo T-cell reconstitution occurs. In all patients a full chimerism could be achieved (median: d+24), although only one of seven patients was transplanted in haematological remission (4x refractory AML and 3x ALL in aplasia). Two of the ALL-patients are still in remission (d+165; d+550) and median time of remission in the AML patients was 212 days. Conclusion: CD6-depletion seems to be a feasible graft manipulation with induction of stable engraftment, chimerism and tolerance. In contrast to other graft manipulation a higher antileukemic effect is postulated, because clearance of the leukemia could even be observed in patients who were not in remission at the time of transplantation. D.N. Eissens, M.P. Hendriks, F.W.M.B. Preijers, H. Dolstra, N.P.M. Schaap, A. van der Meer*, I. Joosten RUNMC (Nijmegen, NL) NK cells are important for the anti-tumor response after allogeneic stem cell transplantation (SCT) that primarily occurs within the first three months following SCT. Impaired reconstitution of NK cells correlates with poor clinical outcome. As part of a single-centre randomized phase III study, CD34+ graft selection was compared to a CD3/19 negative selection procedure. As CD34+ selected grafts are devoid of NK cells, in contrast to the CD3/19 depleted grafts, we set out to study the effect of NK cells in the graft on NK cell repopulation after peripheral blood SCT. Blood samples were taken from each patient (10 patients per group) prior to SCT and 0.5, 1, 2, 3, 6, and 12 months after SCT and analyzed for absolute numbers of NK cells and the presence of inhibitory and stimulatory receptors. In the CD3/19 group, already at 14 days post-SCT, absolute numbers of NK cells in the peripheral blood reached similar levels as compared to numbers prior to SCT (80±41.10 6 /L vs. 86±54.10 6 /L). Moreover, reconstitution of the classically defined CD56bright and CD56dim NK cell subsets had already taken place. Over 40% of these NK cells expressed the activation receptor NKp30 and 80% expressed NKp46. About half of the NK cells expressed the inhibitory receptor NKG2A. One month after SCT, the absolute NK cell numbers increased even further (266±103.10 6 /L) and the repertoire of inhibitory and stimulatory receptors was fully reconstituted. After this, numbers dropped slowly but remained above pre-SCT levels. In the CD34 group, absolute numbers of NK cells reached pre-SCT levels at 1 month after SCT (121±71.10 6 /L vs. 98±72.10 6 /L) and these levels were maintained throughout. Meanwhile, reconstitution of NK cell subsets was beginning to take place and inhibitory and stimulatory NK cell receptors were expressed at a level similar as observed pre-SCT. In conclusion, compared to conventional CD34+ graft selection, CD3/19 depleted grafts in allogeneic SCT result in higher NK cell numbers and faster NK cell reconstitution following SCT. In addition, inhibitory (NKG2A, KIRs) and stimulatory (NKp30, NKp46) receptors are already present within the first fourteen days after SCT suggesting that these NK cells can mediate anti-tumor responses starting within the first month post-SCT. Additionally these NK cells may reduce GVHD allowing a higher T-cell dose in the graft and as such a higher GVL activity. Functional studies will have to confirm the NK cell-mediated anti-tumor reactivity. Overcoming various co-morbidities by G-CSF-primed unmanipulated bone marrow stem cell transplantation in patients with adult AML W.S. Min, H.J. Kim, B.S. Cho, S.Y. Kim, Y.J. Kim, C.K. Min, S. Lee, S.G. Cho, J.W. Lee, C.C. Kim Catholic University of Korea (Seoul, KR) Objectives: It was reported that the use of G-CSF primed unmanipulated bone marrow(uBM) would allow rapid engraftment without increased risk of GvHD. We hypothesized that uBM transplantation may result in successful engraftment without increasing the incidence of transplant-related complications(TRC) and transplant-related mortality(TRM), specifically in patients with adverse co-morbid conditions pretransplant. Methods: Between January 1998 and August 2006, thirty seven patients with a variable risk of AML undergoing allogeneic stem cell transplantation(SCT) from an HLAidentical sibling have participated in this prospective study. The presence of a various preceding treatment-related sequelae before SCT included fungal infection with cavitary pneumonia or pleural effusion, perianal or liver abscess during chemotherapy, toxic or viral hepatitis reactivation, weight discrepancy between the donor and the recipient greater than 10kg, and/or presence of major organ dysfunction. The median follow-up duration for all patients was 18 months(range, 4~125). The majority of patients received a standard conditioning regimen consisted in cyclophosphamide plus total body irradiation with GvHD prophylaxis. Results: The median CD34+ cells infused were 3.3 x 106/kg(range, 0.7 ~ 15.6). The median day for recovery of absolute neutrophil count to >500/ul and platelet count to >20,000/ul without transfusion were 13(range, 6~22) and 18(range, 11~29), respectively. Incidences of acute and chronic GvHD were 43%(16/37), and 47%(16/34). However, we found only 1 patient of steroid-resistant high grade(III) acute GvHD. Together, 4 patients showed extensive type(3 of them were progressive from acute GvHD) chronic GvHD, but none of them have been fatal. As expected, 16%(6/37) died due to 3 of critical pneumonia with ARDS, 1 of intracranial hemorrhage, 2 of severe acute GvHD. Among the 3 cases of pneumonia, only 1 patient developed the same course of infectious complication as before SCT. There was no particular finding in the context of CMV infection or other posttransplant complications. Overall, 11(30%) patients were relapsed so far. The estimated probability of 2-year overall survival rate was 55%. Conclusion: Thus, this may suggest a possibility that we can overcome various pre-transplant co-morbid conditions of patients using uBM without increasing both TRC and TRM. (2) Haploidentical hematopoietic cell transplantation (HHCT) after high dose conditioning with a megadose of CD34-selected stem cells has been complicated by regimen related toxicities, slow engraftment and delayed immune reconstitution leading to high treatment related mortality (TRM). A new regimen using reduced intensity conditioning (RIC) and immunomagnetic graft CD3/CD19 depletion may allow HHCT with lower toxicity and faster engraftment. CD3/CD19 depleted grafts not only contain CD34+ stem cells but also CD34 negative progenitors, dendritic-, natural killer-and graftfacilitating cells which may allow stable engraftment even without a megadose of CD34+ cells. A multicenter phase I/II study of HHCT using RIC with fludarabine (150-200 mg/m 2 ), thiotepa (10 mg/kg), melphalan (120 mg/m 2 ), OKT-3 (5 mg/day, day -5 to +14) and graft CD3/CD19 depletion has been initiated. No post grafting immunosuppression was applied if the graft contained <5x10 4 CD3+ cells/kg. To date, 41 patients (median age=46 [range, 21-65] years) have been enrolled in this study. Diagnosis were AML (n=24), ALL (n=7), NHL (n=4), MM (n=2), CML (n=2) and MCL (n=2). Patients were high risk with refractory disease (n=21) or relapse after preceding HCT (auto=6, allo=12). The CD3/CD19 depleted haploidentical grafts contained a median of 7.0 x 10 6 (range, 3.4-18x10 6 ) CD34+cells/kg, 3.5x10 4 (range, 0.4-44x10 4 ) CD3+T-cells/kg and 3.5x10 7 (range, 0.02-37.3 x10 7 ) CD56+cells/kg. The regimen was well tolerated with maximum acute toxicity being grade 2-3 mucositis. Five cases of reversible peripheral neuropathy and 3 cases of progressive multifocal leukencephalopathy (PML) occurred posttransplant in heavily pretreated patients. Engraftment was rapid with median time to >500 granulocytes/µL of 12 (range, 9-50) days, >20000 platelets/µL of 11 (range, 7-30) days and full donor chimerism after 2-4 weeks in all but two patients. Incidence of grade II-IV GVHD was 41% with grade II=12, III=3 and IV=2. TRM in the first 100 days was 10/41 (24%). Overall survival is 15/41 patients (37%) with deaths due to relapse (n=13), infection (n=9), PML (n=2), GVHD (n=1) and cardiac failure (n=1) with a median follow-up of 230 days (range, 56-908). So far, we did not observe a statistical significant survival advantage for patient transplanted from a KIR-mismatched donor (22/41 patients). This regimen is promising in high risk patients lacking a suitable donor, and a prospective phase I/II study is ongoing. Purpose: In ABO incompatible bone marrow transplantation an erythrocyte depletion of the bone marrow is necessary. For this purposes the MNC collection procedures of various cell separator systems are used. The AMICUS™ system was introduced in the field of peripheral blood mononuclear cell collection years ago, showing a good performance regarding efficiency and safety. In order to evaluate the performance of the MNC collection program of the AMICUS device for the bone marrow procedure we retrospectively analysed our data obtained from the AMICUS and from the Fenwal CS3000+ device. Methods: From 2005 to 2007 we performed 10 procedures with the AMICUS and 12 with the Fenwal CS3000+ (F) device in ABO mismatched allogeneic bone marrow transplantations in our institution. For the AMICUS (A) device the normal MNC program was used. The anticoagulant was prepared 500 ml saline solution plus 2000IE Heparin instead of ACD-A solution. Parameter setting were blood flow rate 25 ml/min, anticoagulant blood ratio 1:12-15, cycle volume 300 to 800 ml, RBC offset 7.0, number of cycles 3 -4, to process 4 times the volume of the original bone marrow. The procedure on the Fenwal CS3000+ was performed according to the S332 manufacturer's instructions. Cell counts, erythrocyte volume, total volume and calculation of the efficiencies were obtained before and after the procedure. Results: There were no statistical differences in donor age, reciepient age, type of ABO mismatch and CD34+ cell yield [6,82≥4,78 (A) versus 8,55≥6,2 (F) 10E06cells/kg bw] before and after the procedure the procedure for both devices. The efficiency for the CD34+ cell collection was lower but not statistically significant in AMICUS device (70%±19 versus 84%±18; U-Test p=0.139). The erythrocyte volume in the end product was higher but not statistically different in the AMICUS device (8.97±3.5 versus 6.98±4.9 ml; U-Test p=0.27). During the evaluation period no technical problems were observed. Almost all patients showed an sustained engraftment. One patient died at d 11 from multiple organ failure and was not available for further engraftment analysis. Conclusions: We conclude that the AMICUS device could be used for MNC collection from bone marrow in order to deplete erythrocytes. For depletion of plasma the AMICUS is not applicable. Therefore the AMICUS is not recommended for routine use, and the manufacturer is called to develop a program applicable for the needs of bone marrow processing. Allogeneic stem cell transplantation (SCT) with T-cell depleted grafts reduces the incidence of Graft-vs-Host disease (GVHD) and consequently decreases transplant related mortality (TRM). However, post-transplant immune reconstitution is slower resulting in more infections, increased graft failure and higher relapse rates. By using T-cell depletion techniques with an add-back of a fixed dose of CD3+ T-cells (partial T cell depletion), these drawbacks may be overcome. Furthermore, the lower incidence of GVHD after SCT also enables the introduction of post-transplant immune therapy in order to reduce relapse rates. In a randomized study, we compared CD34+ cell selection with the CD3+/CD19+ cell depletion method. By using CD3+/CD19+ depletion not only more CD34+ cells are transplanted, but also CD34-negative stem cells as well as NK cells, dendritic cells and monocytes. These subsets may generate beneficial effects on longterm engraftment, infections and may improve the Graft-versus-Leukemia effect. Methods: Patients with acute or chronic leukemia or myelodysplastic syndrome, eligible for allogeneic SCT, were included in this study and randomized for either CD34+ selection or CD3+/CD19+ depletion. Patients were stratified for diagnosis. From May 2006 until November 2007 patients were included in the study with a median follow-up of 4.5 months. All stem cell grafts contained a fixed dose of 1 x 105 CD3+ T cells/kg bw. At 0.5, 1, 2, 3, 6, and 12 months after SCT, respectively, we evaluated immune reconstitution and clinical outcome. Results: The number of CD34+ cells in the grafts were significantly higher in the CD3+/CD19+ depleted grafts compared to the CD34+ selected grafts (median 4.3 vs 6.5 x 106/kg, p<0.05). The number of T and B lymphocytes in the grafts were not different. All patients engrafted and the time to engraftment was not different between the two selection procedures. In the first 3 months after SCT, the immune reconstitution of lymphocytes, especially CD4+ T cells and NK cells was faster in patients transplanted with CD3+/CD19+ depleted grafts. Already at day 14 after SCT these marked differences were clearly apparent ( Figure 1 ). GVHD (12% > grade 2) and TRM (10%) were comparable in both groups. Conclusion: The CD3+/CD19+ depletion procedure resulted in stem cell grafts containing more CD34+ stem cells and showed a faster immune reconstitution of CD4+ T cells and NK cells. This may improve longterm outcome of partially Tcell depleted SCT. Bochennek (1) Allogeneic T cells are known to exert both beneficial and adverse effects in haematopoietic transplantation. Thus, in certain settings clinicians require maximum or exact doses of T cells administered. Manipulations of cell preparations such as immunomagnetic selection or depletion or freeze storage may cause considerable damage to T cells. We therefore propose a 4-colour-flow-cytometric strategy for correct absolute vital T cell enumeration in manipulated cell preparations for clinical use. The gating strategy is based on the ISHAGE single-platform stem cell enumeration method in combination with experiences from lymphocyte subtyping, using low scatter, high expression of CD3 and CD45 antigens and 7-AAD staining in a no-wash-preparation with counting beads. In spiking experiments, the detection limit was set at 0.7±0.5 CD3+ cells/ul with at least 50 T cell events acquired. The cell preparations analysed contained a median absolute CD3+ T cell number of 221x10³ (0.09%, CD34 selected grafts, n=187), 900x10³ (0.004%, CD3/CD19 depleted grafts, n=15) and 283x10³ (0.012%, CD3 depleted/CD56 enriched NK-cells, n=14), respectively. In addition, we could improve our new strategy for enumeration of residual T cells on the base of 5colour flowcytometric analyses (CD45FITC/CD3PE/ CD4ECD/7AAD/CD8PC7) and the change from the EPICS-XL-MCL to the FC-500 (both Beckman Coulter) using a limited number of manipulated cell preparations (n=40). This resulted in a detection limit of 0.1±0.03 CD3+ cells/ul. The results differed of those from conventional T cell measurement in cell products after extensive manipulation e.g. in freeze-thawed cell preparations used for donor lymphocyte infusions, the difference was up to 50 % due to dead T cells contained. In S333 case of stem cell grafts, the method provides reliable residual T cell enumeration even at extremely low concentrations. Fully automated, clinical grade processing of leukapheresis and bone marrow harvests using the Sepax system S. Urbani*, S. Dal Pozzo, P. Anedotti, L. Lombardini, M. Biscardi, P. Bufano, B. Mazzanti, A. Bosi, R. Saccardi Careggi Hospital (Florence, IT) Background: Implementation of new regulatory bodies in the manipulation of Haematopoietic Stem Cells (HSC) for transplantation results in an increasing need for standardization and efficiency of laboratory procedures. The Sepax cell separation system (Biosafe SA, Eysins, Switzerland) is a cell centrifugation device aimed at the automated processing of blood or blood components in a closed and sterile environment. Volume reduction before cryopreservation, manipulation for AB0 mismatching and washing of thawed units before transplantation are the major applications either for allogeneic or for autologous HSCT. Methods: Three protocols were used after appropriate validation: 1) Plasma removal was carried out either to reduce the cryopreserving volume or to eliminate natural antibodies in case of AB0 mismatching (PBSC programme) 2) Collection of bone marrow (BM) Buffy Coat (BC) either for AB0 major incompatibility or BM cells concentration in Regenerative Medicine and before cryopreservation (GVR program). In both 1) and 2) protocols the operator can implement the output volume.3) Washing of a thawed cellular product to prevent infusionassociated toxicity (PBSC washing programme) From December 2001 to December 2007 we processed 556 leukapheresis and 38 BM for HSCT. In addition we processed 44 BM harvests from patients undergoing autologous BM stem cells implant for the treatment of osteopeneic disorders. Results are summarized in the table. Recovery/reduction are expressed in percentage of initial value. Data are expressed as a median (range). Conclusions: Time processing and especially intervention of the technician are remarkably reduced as compared to the manual procedure. As the machine works in a closed system connected to the collection and cryopreservation bags, the risk of bacterial contamination during processing is low. Sepax provided a satisfactory degree of feasibility and reproducibility in all the tested conditions. E. Fronkova, K. Muzikova, E. Mejstrikova, M. Kovac, R. Formankova, O. Hrusak, J. Stary, P. Sedlacek, J. Trka* on behalf of the CPH Relapse is the major cause of failure of allogeneic transplantation in childhood ALL. Minimal residual disease (MRD) detection using quantitation of clone-specific immunoglobulin (Ig) or T-cell receptor (TCR) rearrangements before and after transplantation in children with high-risk ALL proved to be an important predictor of outcome. An early initiation of an adoptive immunotherapy (donor lymphocyte infusions -DLI, or immunosuppression withdrawal) in patients with molecular relapse may avert or postpone haematological relapse but may cause GVHD. The method and the guidelines set for MRD interpretation by European Study Group on MRD in ALL are very precise to avoid both false negative and positive results. So far, this method proved to be reliable leaving only a theoretical possibility of potential non-specific amplification in regenerating non-malignant lymphocytes. We observed in the group of 22 patients after transplantation (median follow-up 42 months) detectable MRD positivities in Ig/TCR-based qPCR not leading to further molecular progression of the disease (16 of 107 (15%) total samples). Simultaneous RT-PCR detection of BCR/ABL, TEL/AML1, or MLL/AF9 in 12 samples from patients with fusion genes gave negative MRD result. We hypothesized positivities in Ig/TCRbased assays were false, most likely resulting from nonspecific amplification despite the application of internationally agreed strict measures. Thus, we applied two non-self specific clonal IgH systems to our cohort of samples and received similar number of false positivities (21.5% and 15%). Nonspecific products amplified in these qPCR systems differed from the specific ones both in length and sequence. Statistical analysis proved excellent correlation of this phenomenon with B-cell regeneration in bone marrow as measured by flow cytometry and Ig light chain kappa excision circles quantitation (p<0.0001). We conclude that despite Ig/TCR quantitation is a reliable method for MRD detection after transplantation. isolated positivities in Ig-based qPCR systems at the time of intense B-cell regeneration must be viewed with caution to avoid wrong indication of treatment. Supported by MZ00064203 and MSMT 0021620813. The role of autologous hematopoietc stem cell transplantation (autoHSCT) in the treatment of adult acute lymphoblastic leukemia (ALL) is a subject of controversies as several prospective studies failed to prove its advantage over maintenance chemotherapy. Those studies, however, did not take into account the status on minimal residual disease (MRD), which is now recognized a potent predictor for relapse. The goal of this analysis was to determine the impact of MRD on outcome of autoHSCT. Data on 126 autoHSCT recipients collected from 5 national study groups cooperating in the European Leukemia Net were analyzed. Median age of 78 B-lineage and 47 T-lineage highrisk ALL patients was 26.5 (16-58) years. Ph+ ALL was recognized in 10 cases. All patients were in first complete remission (CR) lasting 6 (1.5-69) months. Peripheral blood was used was used as a source of stem cells in 64 patients whereas bone marrow, in 61 cases. Conditioning was based on chemotherapy alone (n=83) or TBI (n=42). MRD was evaluated in bone marrow with the use of either multiparametric flow cytometry (n=94) or molecular techniques (n=31). MRD level at autoHSCT was <0.1% in 101 patients and ≥ 0.1% in 24 cases. With the median follow up of 5.1 years, the probability of leukemia-free survival (LFS) at 5 years for the whole group equaled 44% (±5). Two patients died of transplantation-related complications. The LFS rate was significantly higher for patients with the MRD level at transplantation <0.1% compared to those with MRD ≥ 0.1% (52% vs. 15%, p=0.006). The difference was particularly pronounced for peripheral blood HSCT (56% vs. 0%, p=0.01) and for T-lineage ALL (53% vs. 11%, p=0.03). In a multivariate analysis adjusted for other potential prognostic factors (age, CR duration, Ph+ ALL, immunophenotype, source of stem cells, type of conditioning), the MRD status <0.1% remained the only independent factor associated with increased LFS (HR=0.43, p=0.004) Conclusions: MRD status is the most important predictor for LFS after autoHSCT in adults with ALL. More than half of patients with high risk disease and low MRD level may be cured. This observation should be taken into account when planning prospective trials. It may also contribute to reevaluation of the role of autoHSCT in the therapy of adult ALL. Longitudinal quantitative evaluation of WT1 gene expression as monitoring of minimal residual disease in acute myeloid leukaemia following allogeneic bone marrow transplant A. Candoni*, E. Toffoletti, A. Chiarvesio, C. Pipan, A. Michelutti, M. Tiribelli, E. Simeone, D. Damiani, F. Barbone, R. Fanin University of Udine (Udine, IT) Introduction: WT1 is identified as a tumor suppressor gene encoding a transcriptional regulator and playing a role in the development of Wilms Tumor. WT1 overexpression is described in several oncological diseases including leukemias. The majority of acute myeloid leukemia (AML) patients don't have a suitable specific molecular marker for monitoring minimal residual disease (MRD). Quantification of WT1 in bone marrow samples before and after allogeneic BMT can be useful as a marker of MRD. Methods and results: Here we report the results of our study aimed to evaluate the dynamic expression of WT-1 in AML patients (pts) following allogeneic BMT. The expression of WT-1 was measured at distinct intervals of treatment, using Real Time Quantitative RT-PCR with the specific TaqMan probe; the WT-1 expression was related to the control gene ABL. The cDNA level of WT-1 was detected in bone marrow samples from 30 AML pts (13 males and 17 females) at diagnosis, at the time and after the allogeneic BMT. All cases showed high WT1 expression levels at diagnosis with a mean of 4648 (SD 3847) and a median of 3679 (range 658-13923) copies of WT1/10000 copies of Abl. At transplant 21 pts (70%) were in complete cytologic remission (CcR) and 9 (30%) had refractory or relapsed AML. Bone marrow samples from pts in CcR at BMT showed significantly lower WT1 expression levels (mean 114±150), compared to the samples from pts with relapsed or refractory disease (mean 4544±3347) (P=0.004). After BMT a rapid decline of WT1 expression levels was observed in all pts that attained or maintained a condition of CcR (FIGURE A). After a median follow up of 7 months from transplant, 4 out 30 pts relapsed (13%) and all of them had an increase in WT1 expression. Two of these pts died with leukemia and two were successfully reinduced with DLI ± chemotherapy with a rapid reduction of WT1 levels. Besides we found a complete concordance between WT1 expression levels and other disease markers (when available). Conclusions: In our experience there was a concordance between WT1 expression levels (measured by quantitative RT-PCR) and status of AML before and after BMT. Longitudinal quantitative evaluation of WT1 may be useful as a non-specific leukemia marker (NSLM) for monitoring MRD and as a predictor of AML cytologic relapse. Based on this results cases with a rapid increase of WT1 levels after BMT and without GVHD should be candidate to discontinuation of immunosuppressive therapy and/or DLI. Molecular-genetic monitoring of haematopoietic chimerism and minimal residual disease in AML patients after stem cell transplantation O. Blau, C. Gentilini, A. Nogai, M. Reinwald, A. Sindram, E. Thiel, L. Uharek, I.W. Blau Charite University Berlin (Berlin, DE) In this study we analyzed 83 patients (48 male, 35 female) with AML who underwent related (28) and unrelated (55) allogeneic stem cell transplantation (ASCT). The mean age was 49 (17-68). Twenty seven patients (33%) received standard myeloablative and 56 (67%) reduced intensity conditioning (RIC). All patients were investigated with standard cytogenetic and molecular genetic methods at the time of initial diagnostics and all through different stages of the disease. To detect genetic aberrations we used G-banding, FISH, PCR, and sequencing. For quantitative chimerism investigation we used AmpFlSTR® Identifier® PCR Amplification KIT (Applied Biosystems) contains fluorescentlabeled primer pairs for simultaneous amplification of 16 different loci each. We evaluated chimerism in bone marrow aspirate (BM) and in CD34+ cells on days +28, +56, +100, +180, +360, and at end of investigation. After sex-mismatch ASCT FISH with specific probes for sex chromosomes were exercised. The data was compared with minimal residual disease (MRD). Both standard and RIC regimens were well tolerated. All patients engrafted. Relapse was developed in 31 (45%) patients; 26 from them (84%) received RIC conditioning. Interestingly, all patients after RIC SCT with CC at day +56 in BM did not develop relapse. By contrast, 70% of patients which had a relapse showed mixed chimerism (MC) on day +56 after RIC SCT. We suggest that the chimerism data at day +56 is important factor in sense of relapse in Allogeneic hematopoietic stem cell transplantation (Allo-HSCT) is a powerful immunological strategy to maintain and improve remission rates in core-binding factor acute myeloid leukemia (AML) after relapse. In high-risk AML, minimal residual disease (MRD) usually persists after Allo-HSCT. A graft-versus-leukemia (GVL) effect may be crucial in eliminating residual leukemic cells. Monitorization of minimal residual disease (MRD) by quantitative real-time polymerase chain reaction (RT-PCR) allows a very sensible method of monitoring MDR in patients with CBF-related AML, including inv (16). Purpose: The direct anti-leukemic effect of the conditioning regimen and a possible GVL effect may be measurable by MRD techniques, but data regarding AML with inv(16) are scarce. We describe 7 AML patients with inv(16) and CBFB/MYH11 transcripts, in an advanced disease status who received an Allo-HSCT. MRD was quantified at various time points before, during and after transplantation using quantitative RT-PCR. Results: Four patients were conditioned with reduced intensity and 3 with conventional regimens. Before Allo-HSCT, all patients showed high levels of CBFB/MYH11 transcript proteins. The conditioning effect was observed with a median decrease of 3 log in all patients, and could be separated from the GVL effect in 3 patients. Acute and chronic GVHD were observed in four and seven patients, respectively. In all evaluable patients molecular data showed a molecular complete remission when GVHD occurred, indicating that the GVL effect was strong enough to obtain and maintain complete molecular remission in this setting. Patients with no GVHD or with GVHD in remission (n=5) showed a progressive increased in the MRD. Tapering the IS and/or DLI led to further molecular complete remission (CR) in 4 patients. Conclusion: These data indicate that RT-PCR may be a useful technique in monitoring CBFB/MYH11 transcripts after Allo-HSCT, separating the antileukemic effect of the conditioning from a later GVL effect which can be elicited by rapid tapering of the IS and/or DLIS. J.L.Piñana is supported by grants from the Instituto de Salud Carlos III (expedient CM06/00139, Ministerio de Sanidad. Spain) The price of survival: are all patients and transplants equal? I.H. Gabriel*, A. Chaidos, M. Klammer, E. Olavarria, E. Kanfer, A. Rahemtulla, D. Miljokovic, J.F. Apperley Imperial Health Care NHS trust, Hammersmith Hospital (London, UK) In the UK hospitals are currently reimbursed for SCT according to prices negotiated at a regional level and are based on historical costs and activity. In 2008 this system will change to a fixed national tariff (FNT), based on a national average cost for SCT. The proposed level of reimbursement seemed inadequate and it was difficult to ascertain how this figure had been calculated. In order to inform the process we collected comprehensive financial data taking into account the variability in transplant costs. We studied 10 autologous (ASCT), 10 sibling, and 10 unrelated donor transplants (MUD). In each group we selected those with shortest and longest inpatient stays, and the other 8 were selected to include different diseases, disease status, age, and preparative regimens. The costs were calculated from the transplant workup until 6 months (ASCT) and 12 months (allografts) post transplant. We reviewed all clinical activity using the case notes, medication charts, and our computerised pathology and imaging results systems. Using these data plus overheads and utility costs, we produced accurate figures for each patient. In the case of unrelated allogeneic transplants, donor search and stem cell procurement were also included. In all 3 groups the costs varied considerably. The average price for an ASCT was 50,575 Euros (E) (range E28,932 -E127,083), for an HLA matched sibling SCT, E105,577 (72,818 -265,038 E) and for a MUD SCT, E154,953 (E56,401 -E265,038). These costs are remarkably similar to those currently reimbursed (as opposed to the proposed new national tariff) with the exception of ASCT (E36,000). A wide range in costs was seen for all aspects of the SCT procedure including donor search and cell procurement (E13,807 to E35,328), pharmacy costs (E2,881 to E108,580) and post-SCT follow up. SCT is inherently heterogeneous and complications are often unpredictable so high and wide ranging costs are expected. However, the earliest suggestions for the FNT underestimate cost. Our experience of this consultation exercise is that centres calculate costs differently and therefore achieve varying results. Failure to include all costs falsely underestimates a FNT. As a result, hospitals may have to select patients to adhere to fixed prices and novel transplants will become increasing difficult from a financial perspective. Lack of appropriate and accurate reimbursement is likely to have a detrimental effect on the UK transplant experience. State-of-the-art unrelated cord blood banking as of 2007: regulatory issues and requirements of transplant centres L. Körschgen, A. Meyer, M. Aktas, J. Rox, P. Wernet, G. Kögler University of Dusseldorf Medical School (Dusseldorf, DE) The Düsseldorf cord blood (CB) bank has started unrelated and related directed CB banking in 1992/1993. Hitherto (December 2007) a total of 12951 CB samples have been cryopreserved and 462 CB units were provided for transplantation (54% for children and 46% adults). The Düsseldorf CB bank has the production permission by local authorities, was the first to receive the license by the Paul-Ehrlich Institute in 2001 in Germany and is one among 3 CB S338 banks worldwide who received the international FACT-NETCORD accreditation already in March 2004. Due to the regulation of the EU and the licensing criteria in Germany, but also mainly due to the requirements of the transplant centers, the policy of our cord bank has changed during the last years. Transplant centers only request cord blood units with a high nucleated cell count and a substantial number of CD34+ cells: the last 100 DUCB transplants provided last year had a mean NC-count of 1.62x10 9 and a mean CD34+ count of 5.67x10 6 after thawing. All total transplants (n=462) provided had a cell count of 1.49x10 9 . This is the result of establishment of CB transplantation in adults, but also the knowledge that >3x10 7 NC/kg body weight are required for a fast speed of engraftment and survival. .As a consequence for banking the Düsseldorf CB bank switched to the banking of units with high NC and CD34+ counts: During the last year 75% of the cryopreserved units had a NC count of >9x10 8 after cryopreservation and 23% over 1.5x10 9 and 875 of the units a CD34+ value of ≥2x10 6 . Due to this policy of the CB bank, 65% of the CB units must be discarded before processing mainly due to low volume/NC number<1x10 9 and only a minority due to other exclusion criteria on the basis of the EU guidelines and the national legal authorities. At the beginning of the CB banking, 1940 CB units were stored unseparated, all the remaining 10826 units volume reduced using HES. In order to perform the volume reduction under strict standardized GMP-criteria, since 2005 the automated cell processing system Sepax (BIOSAFE) with the CS490 separation kit was introduced into our routine banking. DUCB units were shipped to 132 different TX-centers in 28 countries. The Kaplan-Meier estimate of survival at two years is 42%. The Baldrige National Quality Program criteria as a framework for a comprehensive quality plan facilitates FACT/JACIE accreditation J. Schriber* (1), J. Alvarnas (1) With increasing complexity, regulation,financial pressures and oversight of the transplant field, continuous quality improvement is becoming essential to the success of the transplant team. The latest version of FACT/JACIE standards require a much more comprehensive quality plan then prior standards. Since 2005,the COH-Banner BMT Program has used the Baldrige National Quality Program criteria to improve clinical and service processes and quality. This award which recognizes role model behaviors,is the highest achievement in the USA for business and service excellence. Baldrige criteria are based on core values of visionary leadership, customer driven excellence, organizational and personal learning, valuing workforce and partners, agility, focus on the future, managing for innovation, management by fact, social responsibility, focus on results and creating value and a systems perspective. The criteria are organized into 6 areas (Leadership, Strategic Planning, Customer and Market focus, Measurement Analysis and Knowledge Management, Workforce Focus, and Process Management)and a results component that correlates with each process. Subcomponents address issues such as vision, ethical practices, sustainability and organizational effectiveness and offer a more comprehensive template to enhance several areas addressed in the FACT/JACIE standards (i.e. work design/process development incorporating input from key customers, suppliers and partners, minimizing costs of inspections, tests, prevention of errors and rework as well as organizational learning). Importantly the criteria do not suggest "how to" but merely ask "how" so that organizations can utilize approaches that work best for them. By incorporating these into our daily work we have empowered the workforce to identify opportunities for improvement(OFI's)and redesign processes with appropriate measures in their specific area. During our successful 2006 FACT reaccreditation less than 1% of significant changes were made 6 months prior to inspection despite 23% of 378 SOP's being revised between inspections. The Baldrige criteria can be used as a self assessment tool to recognize significant OFI's and allow for continual change and improvement rather than prior to planned inspections. They can be used as a framework to augment FACT/JACIE requirements,improve value to patients,the workforce and other stakeholders while improving health care quality and organizational sustainability. Use of a comprehensive national database as a quality control instrument J. Passweg (1) The primary goal of JACIE is quality improvement in all aspects of hematopoietic stem cell transplantation (HSCT). Reporting of all transplants and regular analysis of outcome is integral part of a quality management system. Few analyses have been done to assess the impact of a comprehensive HSCT data capture system on a national level and to use data comparison as a quality control tool. We made use of the legal requirement that all 9 HSCT teams in Switzerland, 1 allogeneic, 3 combined allogeneic-autologous and 5 autologous HSCT teams, to adhere to JACIE guidelines and to report all transplants to the STABMT (Swisstransplant Group for Blood and Marrow Transplantation) registry. From 1997 -2006 .4 % allogeneic, 70.6% autologous were performed for 2987 patients, 60% male, 40% female, median age 46 (0-76.years). Main indications were leukemia (767 allo, 220 auto), lymphoproliferative disorders (138 allo, 1508 auto), solid tumors (8 allo, 333 auto) and non malignant disorders (92 allo, 10 auto). As of December 2007, 1899 patients (64 %) were alive, 1079 (36%) had died, 249 (8.4%) from transplant related mortality (TRM) or 830 (27.9%) from disease progression. This corresponds to a survival probability of 50 ± 3% in recipients of autologous HSCT and 57 ± 4% in recipients of allogeneic HSCT at 5 years whilst TRM probabilities were 7 ± 2% and 20 ± 3% respectively. Each team was assessed for survival and TRM data for each disease category and transplant type and data were compared with outcomes of all other teams combined. Data were adjusted in a multivariate regression model for the impact of transplant center adjusting for important factors associated with outcome, e.g. disease, disease stage, age, and histocompatibility. Separate models were constructed for allogeneic and autologous HSCT. There were no significant differences among teams in overall survival of allogeneic and autologous HSCT. There was a significant difference in models of TRM due to 3 events in a small center for autologous HSCT but no center difference in TRM for allogeneic HSCT. Reasons for this outlier are being evaluated with the respective teams. These data show that a consistently high quality may be achieved in data monitoring within a country by a comprehensive database. Deviations are recognized rapidly and discussed with the respective teams. Such regular comparisons within the professional HSCT organizations may serve as a model for quality assurance. Cost effectiveness of posaconazole versus standard azole therapy for the prevention of invasive fungal infections in high-risk patients in Switzerland R. Greiner (1) Two randomized phase 3 trials demonstrated primary antifungal prophylaxis with posaconazole (POS) decreased the incidence of invasive fungal infections (IFIs) in patients at high-risk for IFI (Cornely et al. and Ullman et al., NEJM 2007) . Moreover, primary prophylaxis with POS reduced the overall mortality in neutropenic patients. Objective: A cost-effectiveness analysis was conducted based on the phase 3 trials results from a Swiss perspective. Methods: Two decision-analytic models were developed to estimate the cost-effectiveness of antifungal prophylaxis for hematological patients with graft-versus-host disease (GVHD) or neutropenia. The trial results were extrapolated to lifetime horizon by extending the models with 1-month Markov cycles including specific mortality risk for underlying disease. In both models patients obtained prophylaxis (POS or standard azoles, ie, fluconazole in GVHD and fluconazole/itraconazole in neutropenia). The probabilities of IFI, IFI-related death and death from other causes were estimated based on the phase 3 trial results. Both models were run with long-term mortality data for the Swiss population. Costs for IFI treatment were estimated with official prices, tariffs, and primary statistics of Switzerland. Total costs, IFIs avoided, life-years gained, and the incremental cost-effectiveness ratio (ICER) of POS versus standard azoles over a lifetime were calculated. Results: In the GVHD and neutropenia models, POS is associated with fewer IFIs (0.05 vs. 0.09 and 0.05 vs. 0.11) and increased life-years (8.00 vs. 7.86 and 0.744 vs. 0.728) per patient compared with standard azoles. POS is associated with higher total costs in the GVHD model (CHF 17,720 vs. CHF 10,679) but saves costs in the neutropenia model (CHF 9,089 vs. CHF 10,207). ICER in the GVHD model amount to CHF 48,324 per life year saved. A second-order probabilistic Monte Carlo sensitivity analysis was conducted to assess the effects of parameter uncertainty particularly relating to treatment efficacy and the costs of an IFI. Both models indicate there is an 18% (GVHD) and 80% (neutropenia) probability that POS is cost-saving. Moreover, the probability that the ICER ratio for POS is below an estimated CHF 50,000 per life-year saved threshold is 85% and 90%. Conclusions: This study suggests that in Switzerland prevention of IFIs with POS is the dominant strategy among high-risk neutropenic patients and a cost-effective strategy among HSCT with GVHD patients. Collection of bone marrow (BM) or peripheral blood (PB) grafts for unrelated donor transplantation depends on the geographic location of the donor and subject to variations in transport time to transplant centers (TC). We evaluated the impact of transport times on outcome after transplantation, adjusting for patient, disease and transplant characteristics. Transport times examined: time from end of collection to receipt at TC and from receipt at TC to infusion. All patients received T-replete grafts, myeloablative conditioning regimens and calcinuerin-inhibitor graft-versus-host disease (GVHD) prophylaxis. Collections were facilitated by the NMDP in 2000 to 2004 for patients with acute or chronic leukemia in 1st or 2nd remission and myelodysplastic syndrome (refractory anemia); approximately 60% of patients were in 1st remission. 115 centers collected 938 BM grafts and 80 centers, 507 PB grafts. 93% of BM grafts were transported at room temperature; 98% of PB grafts were transported in a refrigerated gel-pack. Median time from collection of BM grafts to receipt at TC was 9.3 hours and from receipt at TC to infusion, 3.2 hours. Corresponding times for PB grafts were 10.4 and 2.8 hours. Most patients achieved neutrophil recovery by day-28, 93% after BM and 96% after PB transplants. Transport times were not associated with neutrophil recovery for either graft. Platelet recovery was lower after transplantation of BM grafts but not PB when interval from end of collection to receipt at TC was over 20 hours (odds ratio 0.42, p=0.005) and interval from receipt at TC to infusion was over 6 hours (odds ratio 0.55, p<0.001). GVHD rates were not associated with transport times. Overall mortality rates were higher, adjusted for other significant factors, after transplantation of HLA-matched BM grafts (matched at HLA A, B, C and DRB1; allele-level typing) received at TC was over 20 hours from end of collection (relative risk 2.67, p<0.001). Interval from receipt at TC to infusion did not impact survival. Transport times were not associated with mortality risks after PB transplants. Longer transport times occurred primarily when grafts were collected outside of the US for transplantation at a US TC or grafts collected in the US transported to a TC outside the US. Review of current procedures for anticipated longer transport times for BM grafts is required to optimize outcomes after unrelated donor transplantation. Donor NKT cells and outcome following HLA-identical haematopoietic stem cell transplantation D. Nachbaur*, J. Auberger, B. Kircher, P. Schumacher, J. Clausen Hematology and Oncology (Innsbruck, AT) Background: Natural killer T (NKT) cells represent a small but significant population of T lymphocytes which have been demonstrated to play a protective role in autoimmune disease as well as to reduce or prevent graft-versus-host disease by harnessing graft-versus-leukaemia activity following allogeneic stem cell transplantation. Methods: The number of CD3+CD56+ NKT cells in the graft was retrospectively correlated with clinical outcome of thirtysix patients receiving a first allogeneic peripheral blood stem cell transplant (myeloablative conditioning, n=23; reducedintensity conditioning, n=13) from their HLA-identical sibling donors between 2004 and 2007. Results: A median number of 0.2 (range, 0.04-0.57) x 108 CD3+CD56+ NKT cells was transplanted with the graft. NKT cells within the graft significantly correlated with CD3+ T cells (r=0.46, p=0.005). Overall survival for the entire cohort at two years was 45% (25%-65%, 95% CI), the relapse incidence was 41% (25%-66%, 95% CI), and the transplant related mortality was 19% (9%-39%, 95% CI). OS and TRM was identical in patients receiving high or low NKT numbers. By univariate analysis patients receiving high NKT numbers had a trend for a lower relapse incidence (34% vs 50%). Overall, the incidence of acute GVHD grades II-IV was 48% (34%-68%, 95% CI). Patients receiving a higher NKT cell number had a trend for a lower cumulative incidence of acute GVHD II-IV (39% vs 61% for patients receiving higher NKT cell numbers). By multivariate analysis including CD3, NK, NKT, TREG, and CD34 cell numbers, and recipient age, and time interval between diagnosis and SCT as numerical variables, Calabria cord blood bank organisation: a unique example in Italy of collaboration between birth centres, transfusion medicine units and cord blood bank A. Pontari* (1), G. Pucci (1) The Calabria Cord Blood Bank (CCBB), created in January 2006, allows the cord blood (CB) donation in the Birthing Centres (BC) scattered in the regional territory. CB collection and cryopreservation is subjected to strict rules in order to have products with high quality and safety. This result is achievable thanks to the collaboration among the Departments of Obstetrics and Gynaecology, the Transfusion Medicine Units (TMU), the Departments of Paediatrics, the Laboratories of Immunogenetics, Microbiology and Virology in several regional facilities. The CCBB network is hereafter described: in each involved facility a person is identified as responsible for the Project; CCBB staff organizes the training for gynaecologists, obstetricians, professional nurses to collect CB in the BC and distributes standard operative procedures and registration forms to the 23 BC and 12 TMU follower to the Project. CCBB furnished BC with homologated transport case, with control temperature thermometer. Despite the long distance between CCBB and several of BC and TMU (until 300 km), with the aid of volunteer associations, the CB units (CBU) arrive at CCBB within 24 hours after collection. The BC send immediately the collected CBU to the territorial TMU, that perform a function of control and verification of the documents and CBU integrity, clot presence, volume, transport requirements, temperature check, deliver and arrival time. Next, the CBU are shipped to TMU in Reggio Calabria, where a further control is carried out and, at last, they arrive to CCBB. Here, the CCBB staff is responsible to verify the cellularity (TNC>1,1 x 10 9 ), to perform microbiological test to guarantee the product sterility at the collection time, clonogenic tests and viability of cryopreserved samples.After 6-12 months from the collection, in order to highlight possible infectious diseases set in the window-period, mothers refer to TMU closer to their residence to take a sample of blood, carry out the mandatory tests and deliver the letter from the Paediatrician confirming the good health of the baby: all these documents are sent to CCBB, that proceeds with the final cryopreservation after quarantine or eliminates the CBU. As well, CCBB staff performs counselling service both for the mothers interested to donate the CBU and BC and TMU personnel. Thanks to this organization, in September 2007 CCBB obtained the quality certification ISO 9001 related to collection, manipulation and cryopreservation of CBU. Cord blood donation in Calabria: a quality management system G. Pucci* (1), A. Pontari (1) Calabria Cord Blood Bank (CCBB) has its office at the Regional Bone Marrow Transplant and Cell Therapy Center "Alberto Neri", Azienda Ospedaliera "Bianchi-Melacrino-Morelli" in Reggio Calabria (RC). In January 2006 CCBB started its activity for cord blood (CB) collection; in September 2007 obtained the certificate of quality ISO 9001:2000 related to collection, manipulation and cryopreservation of CB units. In order to obtain the quality certification, CCBB implemented standard procedures for providing assurance about the ability to satisfy quality requirements, involving the facilities follower of the project "Cord Blood Bank" in Calabria: Birthing Centres (BC) and Transfusion Medicine Units (TMU). The Quality Management Plan (QMP) includes the control of the organisational structure; personnel qualifications and training; outcome analysis; audits; detection and reporting of errors, accidents and adverse events; record review and document control; product tracking. To date, 23 BC and 12 TMU are involved in the project. The BC staff (gynaecologists, obstetricians, professional nurses) were trained to collect cord blood during the afterbirth phases after spontaneous birth with placenta in situ and post placenta removal during caesarean birth. The training is managed by CCBB and documented. CCBB issued standard operative procedures describing the activities of CB collection, transportation, document control, validation, manipulation and S342 cryopreservation. Specific registration forms were created to control all the process steps. In order to be transplanted in patients suffering from oncohaematological diseases without sibling donor, the collected CB units must have elevated quality and safety standards (volume, cellularity, sterility). To guarantee the mentioned characteristics, a protocol has been drafted defining the following limits for CB units: minimum cell content N° TNC >1,1 x 10 9 , volume ≥100 ml, absence of clots and arrival time at the CCBB laboratory in Reggio Calabria within 24 hours. In 22 months, CCBB received n°1333 CB units collected at the 23 BC, validated by the 11 TMU and afterwards by the TMU in Reggio Calabria. Among 1333 CB units received, n° 701 (53%) were eliminated because of volume <100ml, n° 467 (35%) for poor cellularity N° TNC < 1,1 x 10 9 and n° 165 (12%) were banked. After 12 months of activity, the CCBB staff started audits at the BC and TMU to verify compliance with the procedures. 17% of adolescents above the age of 15 years treated for acute lymphoblastic leukaemia in the ALL-BFM 95 trial protocol suffered from avascular necrosis of the bone (AVN). If conservative measures fail, core decompression surgery is required. The success rate of core decompression surgery varies depending on the stage between 29% and 84% (Castro et al., 2000) . It is evident from these data that improved intervention strategies are warranted. Therefore, we evaluated biological and clinical aspects of implanting autologous MSC during surgical intervention in order to improve bone regeneration. AVN represent an area of absent blood perfusion. Therefore, we cultivated MSC under oxygen tensions of 3% or lower. These cells showed slowed proliferation and differentiation into bone, but secretion of significant amounts of VEGF as well as IGFs and their binding proteins. 10 ml of heparinized bone marrow were drawn from the iliac crest of the patient and MSC were expanded as described earlier (Müller et al., 2006) . MSC could be expanded from 5 out of 5 juvenile bone marrow aspirates. Within three weeks of culture, sufficient numbers of cells, i. e. 112•10 6 ± 67•10 6 , were generated in a certified GMP lab. MSC grown under these conditions were shown to be able to differentiate into osteoblasts. No chromosomal aberrations were detected by matrix-based comparative genomic hybridization using DNA microarrays containing >6,400 genomic DNA fragments. At the day of core decompression surgery, cells were harvested and resuspended to a final volume of 3 ml in saline. As core decompression implies drilling a hole into the necrotic area, the MSC were implanted through the same channel, which was sealed thereafter. Hence, instillation of the MSC did not require additional surgical procedures. Application of MSC was feasible and successful during core decompression surgery in all of the 5 patients. Follow up of the patients is between 18 and 12 months. None of the patients experienced adverse side effects, most importantly there were no infectious complications after surgery and instillation of MSC. Clinically, no patient deteriorated and all patients reported less or no pain two months after surgery. Although the number of patients in this pilot trial is small, initial data for safety and feasibility of employing MSC in juvenile steroid-induced osteonecrosis is very promising. E. Chernykh, N. Starostina*, A. Paltsev, A. Ostanin, O. Leplina, E. Shevela, I. Lisukov, A. Kulagin, V. Kozlov Institute of Clinical Immunology (Novosibirsk, RU) Objectives: Currently, orthotopic liver transplantation is the main therapeutic option for patients with liver cirrhosis. However, a shortage of suitable donor organs, requirement for immunosuppression and high cost restrict its usage. The potential of bone marrow stem cells to differentiate into hepatocytes open new possibilities for their use for regenerative liver therapy. The present study was devoted to the evaluation of tolerance and efficacy of bone marrow cells transplantation in the complex treatment of liver cirrhosis (LC). Methods and Materials: Forty seven patients between 15 and 65 years with a diagnosis of LC were enrolled in the study. According to Child-Pugh score the present subjects included 12 patients with class A, 28 -with class B and 7 -with class C. Bone marrow was harvested from the ilium by standard procedure under general anesthesia. Mononuclear cells (MNCs) were isolated by Ficoll density separation. The mean number of 1.81 ± 0.17 x 10 7 MNC, containing 78 ± 10 x 10 6 CD34-positive cells, was infused intravenously and into the liver. Results: No major complication or specific side effects related to the procedure were observed. Autologous bone marrow cells therapy (ABMT) in class A patients resulted in reduction of asthenia syndrome, decrease of cytolysis (AST and ALT level) and enhancement of serum albumin level during 12 months of observation. Besides, abdominal ultrasonographic study revealed portal hypertension decrease. Significant Child-Pugh score decrease was seen after 1, 6 and 12 months in 17 (48.6%) of 35 patients with noncompensated LC. ABMT in this group was also accompanied with decrease of total serum bilirubin, GGTP and ALT and temporary enhancement of platelet count in a 1 and 6 months after treatment. Conclusions: ABMT in patients with advanced chronic liver disease is safe and feasible. Overall, observed results are encouraging for the future development of stem cell therapy for patients with liver cirrhosis. Effect of acute inflammation induced by prolonged exercise on circulating stem/progenitor cells that mediate tissue repair I. Papassotiriou (1) The "Spartathlon" ultradistance foot race (246 Km continuous, prolonged, brisk exercise for up to 36 hours) provides a unique model of prolonged duration exercise that reveals dramatic systemic inflammatory changes. Endothelial progenitor cells (EPCs) have been shown to participate in vascular repair and angiogenesis, while circulating bone marrow originated fibrocytes represent multipotent cells mediating tissue repair and remodeling after injury. In this study we investigated the effect of this type of exercise on the number of circulating EPCs and fibrocytes along with S343 molecules of endothelium dysfunction and chemotactic proteins in 10 "Spartathlon" athletes before, at the end and at 48 h post race. The EPCs were obtained by culturing peripheral blood mononuclear cells (PBMC) under endothelial cell conditions (EndoCult) and were measured as colonyforming units (CFUs). Circulating fibrocytes were cultured from PBMCs in IMDM medium supplemented with IL-3 and M-CSF and identified as CD45+CD14+CD34lowCollagen-I+ fibroblastic cells. We also determined the plasma levels of E-, L-and P-selectins, sICAM-1, sVCAM-1, thrombomodulin, lipocalin-2, IL-8 and MCP-1 with appropriate methodology. Circulating EPCs increased by nearly ten-fold in peripheral blood at the end of the "Spartathlon" race (from 48±15 cells/ml to 464±36 cells/ml) and they remained increased (420±28 cells/ml) even at 48h post race. The percentage of the CD45+CD14+CD34lowCollagen-I+ fibrocytes cultured from PBMCs before, at the end, and 48 h post race did not reveal any significant difference (64.5±6.2% vs 70.8±8.5% vs 68±4.8% respectively). Plasma levels of lipocalin-2, IL-8, MCP-1, E-selectin, sICAM, sVCAM and thrombomodulin were increased significantly at the end of the race and returned at the pre race levels 48 h post race, while L-and P-selectins remained unaffected before and at the end of the race, presenting a similar decline at 48 h post race. Our study demonstrates that acute inflammatory tissue damage induced by exhausting exercise increases EPCs but not fibrocytes. Given the ability of EPCs to promote angiogenesis and vascular regeneration and the association of fibrocytes with tissue fibrosis after persistent inflammation, we conclude that this kind of repair cell mobilization may serve as a physiologic repair mechanism in acute inflammatory tissue injury.