key: cord-0034140-b31ovd5y
authors: nan
title: Physicians Poster Sessions
date: 2004-03-21
journal: Bone Marrow Transplant
DOI: 10.1038/sj.bmt.1704499
sha: 8cffd97c4b0f3c1d9a3f8e0950892899711864a6
doc_id: 34140
cord_uid: b31ovd5y

nan

The role of cytokines including Interleukin-1 (IL-1) has been associated with the incidence and severity of aGVHD. A modified human in vitro skin explant model was utilised to determine the role of the three IL-1 proteins; IL-1 alpha and beta as agonists and IL-1Ra present in mixed lymphocyte reaction (MLR) supernatants with graft versus host reaction damage in skin explant by the supernatant alone. Mixed lymphocyte reactions were generated by using combinations of HLA Class I and Class II mismatched normals (n=21). The levels of each of the three IL-1 proteins was measured by ELISA to determine the optimum neutralisation of these proteins in the MLR supernatants by specific antibodies when co-cultured with third party skin explants for 3 days (n=21) . Controls consisted of untreated MLR supernatants and isotype antibody treated explants. All experiments were performed in duplicate. Histopathological damage was graded as graft versus host reaction (GVHR I-IV) in-house and also by an independent histopathologist.

Effective neutralisation by the specific antibodies was confirmed by the measurement of the three IL-1 proteins present in the skin explant culture supernatants. Statistical analysis was performed using non-parametric Wilcoxon Matched Pair tests (2-tailed) . There was no significant difference in GVHR grade was observed between untreated explants and those treated with; anti-IL-1 alpha, anti-IL-1beta and the isotype control antibodies (P=0.28, P=0.25 and P=0.5 respectively).

There was a significant increase in GVHR grade of skin explants when treated with anti-IL-1Ra when compared to the untreated skin explants (P=0.0001). In summary, the neutralisation of IL-1Ra further illustrates the role of the IL-1 family in the damage mediated in this modified human in vitro model of graft versus host disease irrespective of the presence of TNF-alpha and IFN-gamma.

Pegfilgrastim is able to mobilise autologous CD34+ peripheral blood progenitor cells in patients with lymphoid malignancies and solid tumours F. Kroschinsky, K. Hölig, U. Platzbecker, A. Haack, M. Schaich, M. Bornhäuser, G. Ehninger, University Hospital (Dresden, D) Myelosuppressive chemotherapy followed by daily injections of granulocyte-colony stimulating factor (G-CSF) is the common procedure to mobilize autologous CD34+ peripheral blood stem cells (PBPC) in patients in whom a later myeloablative high-dose therapy is intended. Pegfilgrastim (Neulasta, Amgen Inc., Thousand Oaks, USA) is a covalent conjugate of filgrastim and polyethylene glycol with an increased elimination half-life due to decreased serum clearance. After chemotherapy a single injection of pegfilgrastim (PEGFIL) is equivalent to daily filgrastim in enhancing neutrophil recovery. We examined the ability of PEGFIL to mobilize PBPC in 22 patients (17 male, 5 female, median age 53 years) who received myelosuppressive chemotherapy for non-Hodgkin´s lymphoma (NHL, n=13), Hodgkin´s lymphoma (HL, n=3), multiple myeloma (MM, n=4) or soft tissue sarcoma (STS, n=2). PEGFIL 6 mg was given subcutaneously 48 hours after the end of cytotoxic treatment (day 0). CD34+ cells in the peripheral blood (PB-CD34) were measured if white blood cells (WBC) exceeded 1.0 Gpt/L after nadir. PBPC collections started at a PB-CD34 cell count >10/µl and were performed as large-volume apheresis (4x blood volume) using a Cobe Spectra (Gambro BCT Inc.). Additional conventional filgrastim (FIL) was given at a dose of 2x5µg/kg if PB-CD34 count was found <10/µl. The apheresis could be started between day +7 and day +13 (median day +9) after the administration of PEGFIL. Median PB-CD34 peak was 79/µl (range 2-539/µl). Additional FIL administrations were necessary in 6 patients. All of them were heavily pretreated including a previous autologous transplant in two of these patients. A median CD34-yield of 7.0x10 6 /kg (range 2.2 -59.2x10 6 /kg) could be harvested in 1 or 2 apheresis. We conclude that, except for heavily pretreated patients, the administration of 6 mg pegfilgrastim after chemotherapy is able to induce the mobilization of a sufficient number of CD34+ PBPC for stem cell collection. The further investigation should include a randomized comparison with conventional filgrastim as well as economic aspects.

DLI is an efficacious treatment for patients in relapse after allogeneic stem cell transplantation (SCT). However, some patients are refractory or respond only partially to this treatment. In this setting interleukin-2 (IL-2) may be of therapeutic benefit, since it can augment natural and adaptive immunity. A total of 13 patients (median age 40) with a variety of hematological diseases (8 with CML, 2 with AML, 2 with MM and one with NHL) relapsed after allogeneic SCT (4 from VUD). The median time between SCT and relapse was 13 months and the median interval between relapse and DLI administration was 7 months. Four patients received DLI according to a bulk dose regimen (BDR) whereas in 9 patients an escalating-dose regimen (EDR) was used. All patients had not responded or had responded only partially to DLI. Recombinant IL-2 (rIL-2) was administered by subcutaneous injection. Ten patients followed a pulse regimen of IL-2 administration (18 MU, three times a week, every other week) whereas 3 patients received a continuous regimen consisting in 3 MU three times a week, every week. Five patients achieved CR and 4 PR after DLI/IL-2 therapy. Three CML patients have remained disease free after a median of 59 months. Those achieving CR appeared to have a survival advantage as compared to partial or non responders. The most frequent side effects were fever (100%), lethargy (69%) anorexia and vomiting (31%). Five patients experienced GVHD (grade II-IV) after the treatment. In order to investigate the in vivo effects of rIL-2 a number of immunological parameters were investigated. An increase in the number of CD56+ NK cells in the peripheral blood was documented in 2 patients during the treatment, although in vitro NK cell activity did not show any increment. Interestingly, the number of virus-specific CD8+ T lymphocytes did not change during DLI/IL-2 treatment and their function, as measured by intracellular IFN-gamma staining, appeared to diminish. However, the proportion of AnnexinV+ apoptotic T cells was not increased in these patients. We conclude that rIL-2 appear to enhance the clinical efficacy of DLI in a proportion of patients. However, at the doses used in our study rIL-2 does not induce a significant effect on NK and T cell functions.

Early-acting cytokines induce the generation of dendritic cell precursors from CD34+ cells A. Encabo, P. Solves, P. Sepúlveda, F. Carbonell-Uberos, M.-D. Miñana, Agencia Valenciana de Ciencia y Tecnología, Centro de Transfusión de la Comunidad Valenciana (Valencia, E) There is a growing interest in generating dendritic cells (DC) for using as vaccines. Several cytokines, specially stem cell factor (SCF) and FLT3-ligand (FL) have been identified to be essential to produce large numbers of myeloid precursors, and even to increase DC yield obtained by the action of granulocytemacrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha). However there are few studies on the effect of the early-acting cytokines, commonly used to expand CD34+ progenitor cells, on DC generation. Objectives: To assess whether early-acting cytokines employed to expand CD34+ cells were able to induce the generation of DC precursors in the absence of serum, and if so, to evaluate the possible differences induced by IL-6 and IL-3. Methods: CD34+ cells from umbilical cord blood were isolated by immunomagnetic methods and cultured in serum-free medium in the presence of SCF, TPO, FL and IL-6 or IL-3, named as STF6 and STF3, respectively. After seven days, the expanded cells were harvested, replated and cultured the following 7 days with GM-CSF and IL-4. TNF-alpha was added to the culture for the last 48 hours to induce terminal maturation of DC. Flow cytometric analyses were performed after CD34+ cell expansion, and before and after addition of TNF-alpha. FITC-dextran assay and mixed leukocyte reaction were performed to evaluate the functionality of DC generated. Results: In the absence of serum, STF6 and STF3 cytokine combinations were able to generate CD14+CD1a-and CD14-CD1a+ myeloid DC precursors from CD34+ cells, but IL-6 had an inhibitory effect on the generation of CD14-CD1a+ cells, and IL-3 on CD14+CD1a-cells. Both DC precursors differentiated into mature DC by GM-CSF, IL-4 and TNF-a. DC obtained from both types of culture exhibited equal allostimulatory capacity that was closely related to CD83+ cell content. Conclusion: This two-step procedure consisting of expansion of hematopoietic progenitors and further induction of differentiation and maturation of DC, avoiding the effects of serum, provides a system to study the developmental pathway of DC generation, and results in the generation of mature DC joined to a continuous generation of DC precursors, as assessed by antigenic expression, useful for clinical purposes. Supported by FIS 01/0066-03

I.L. Barbosa, P.A. Benevides, F. Gomes, C. Sousa, S. Roncon, A. Ávila, A. Campos, C.P. Vaz, F. Campilho, P. Pimentel, A. Carvalhais, Instituto Portugues Oncologia Porto (Porto, P) The aim of this study was to determine the effect of G-CSF used in the PBPC mobilisation had on the cell populations of the haematopoietic grafts. The parameters evaluated were the balance between T lymphocytes and their naive (CD45RA) and memory (CD45RO) subpopulations and ype1/type2 cytokine production. Peripheral blood (PB) prior to mobilisation and PBPC grafts were collected from healthy individuals (HI) (n=22) and patients candidates to autologous transplantation (n=17) (HD, NHL and MM) . For PBPC mobilisation HI received 10µg/kg/day of G-CSF, while patients had two daily injections (NHL and HD 12µg/kg/day and MM 16µg/kg/day). Cell phenotype was evaluated by flow cytometry and the number of IL4 and IFN-g secreting cells was evaluated using the ELISPOT technique. HI demonstrated a decrease in the percentage of lymphocytes and T cells expressing CD45RA and CD45RO in PBPC grafts in comparison with PB (Table 1 ). In the patient group there were no changes in the expression of CD45RO and a decrease in the expression of CD45RA in PBPC which was more pronounced on T lymphocytes (Table 1) . However, if patients were subdivided according to diagnosis, no changes were observed in these cell subpopulations on MM patients (n=6), while the reported changes occurred on NHL/HD patients (n=11). To date, the cytokine study was performed on cells from 5 HI and 6 patients. For both groups the IFN-g production was identical for mononuclear cells from PB and PBPC graft. For the majority of HI and patients there was an increase in IL-4 production by mononuclear cells from PBPC graft in comparison with PB. This rise was more significant for MM patients than for the HI and the other patients. Further studies are required to establish if the G-CSF effect on IL-4 secreting cells is dose dependent, as MM patients were mobilised with the highest dose . In summary, G-CSF mobilisation in HI and patients alters the relationship between naive/memory lymphocytes (CD45RA/CD45RO) on PBPC grafts. The IFN-g and IL-4 production by PB and PBPC graft cells may suggest an immunomodulatory role for G-CSF in the balance between type 1/type 2 immune response.

Maintenance therapy with rituximab (CD20 antibody) following autologous stem cell transplantation for multiple myeloma B. Georgievski, L. Cevreska, M. Ivanovski, N. Siljanovski, A. Stojanovik, O. Karanfilski, Z. Stojanoski, I. Panovska, A. Pivkova, Medical Faculty (Skopje, MK) Maintenance therapy for multiple myeloma (MM) after autologous stem cell transplantation (SCT) has been used in the past 15 years. Various regimens have utilized that in most instances it may prolong disease-free survival but compromise the quality of life. MM is usually not considered for anti CD20+ treatment, but it has been also suggested that circulating CD 20+ are able to play a significant role in determining relapse after effective treatment. The malignant plasma cells of MM are frequently CD20+ and therefore CD20+ antibodies have been evaluated as maintenance therapy after autologous HSCT. We report 4 patients with MM undergoing autologous stem cell rescue in our institution in a 3 years period (from September 2000 till October 2003 , that were provided maintenance therapy with Rituximab. After treatment with VAD regimen (4 cycles), peripheral blood progenitors were previously mobilized with Cyclophosphamide 4gr/m2 + G-CSF 5 mcg/kg/day and collected in range from 3,0x10 8 /kg to 7,5x10 8 /kg (median 4,65) . Patients received single high-dose intravenous Melphalan (200mg/m 2 ) followed by autologous HSCT. Rituximab 375mg/m 2 was started on day +30 after autologous transplantation and repeated every 3 months for a total of 2 years. The median age at transplantation was 55 years (range 46-64), M: F=3:1, disease status prior transplant was CR (3), PR (1) . There were no treatment related deads. The study is still ongoing, so far only 1 patient received complete therapy with 9 Rituximab infusions, 2 patients received 3 infusions and one patient received 1 infusion. Rituximab infusions were well tolerated, no side effects were reported. With median follow up of 12, 5 months (range 2-28 months), all 4 patients are alive in CR with good quality of life. Although the number of patients is too low, our results suggest that Rituximab can be administered safely, early after autologous HSCT and may eradicate any minimal residual disease. The therapeutic role of Rituximab in MM after autologous SCT remains exclusive and requires further evaluation.

Peripheral blood progenitor cells (PBPC) grafts are widely used for haematopoietic transplantation. The aim of this study was to evaluate NKbright and NKdim cell subsets and their NK receptor (NKR) expression in G-CSF mobilised PBPC grafts. Thus, NK population from peripheral blood (PB) prior to mobilisation was compared with PBPC grafts collected on the 5th day of mobilisation. This study was performed on healthy individuals (HI) adult PBPC donors (n=22) and patients candidates to autologous transplantation (HD, NHL and MM) (n=19). NK cell phenotype was evaluated by flow cytometry using 4 colour staining. The following NKR were studied: NK lineage (CD16, CD57), killer cell immunoglobulin like receptor (KIR) (CD1582, NKb1) and C-type lectin receptors (KLR) (CD69, CD94 and CD161). Based on the CD56 and CD16 expression NK cells were divided into NKbright cells (CD56++CD16+) and NKdim cells (CD56+CD16++). Both HI and patient NKdim cells were the major cell population present on PB and PBPC grafts (Table1). A higher percentage of NKbright cells were present on PB and PBPC grafts from patients in comparison with HI (Table1). In HI, PB NKbright cells were characterised by low expression of CD16 and CD57 (<13%+ve cells), no expression of either KIR or CD69 and a high expression (>50%) of KLR (CD94 and CD161). PB Nkdim cells expressed CD57 (median-45%+ve cells), high expression of CD16, CD94 and CD161, low expression of KIR and no expression of CD69. Following G-CSF mobilization, PBPC grafts showed an increased %CD56++CD16+ and decreased %CD56++CD161+ and %CD56+CD57+ cells. No other changes occurred on NKbright and NKdim cells present in the grafts. Patient PB NKbright cells were characterised by increased expression of CD16 and decreased expression of CD161 in comparison with HI. On the other hand, no other major differences in NKR expression were found on PB NKdim cells. Patients PBPC grafts showed decreased %CD56++CD161+ and %CD56+CD57+ cells as observed in HI and a further decrease in %CD56+CD161+ cells. In summary, G-CSF mobilisation does not affect the ratio between NKbright/NKdim cells present in the grafts from HI and patients. The major effect of G-CSF on NKR expression was the decrease in CD161 expression on NK cells from both study groups. Further investigations are required to determine if the functional activity, namely cytotoxic capacity of these cells is affected by this procedure.

S. Kang, Y. Jung, J. Park, J. Choi, H. Lim, H.C. Kim, Armed force capital hospital, Ajou university school of medicine (Seongnam, Suwon, KOR) Myeloablative conditioning regimens used in hematopoietic stem cell (HSC) transplantation can damage the stromal cells as well as HSCs resulting in poor engraftment. Animal studies suggested that transplantation of healthy stromal cells enhanced engraftment of HSCs and down-regulated alloimmune responses. We examined whether co-transplantation of cultureexpanded mesenchymal stem cells (MSCs) in HSC transplantation is safe, and also whether it can improve engraftment of HSCs. Four weeks prior to HSC transplantation, we obtained five ml of bone marrow aspirates from the original donors, isolated MSCs and culture-expanded for four weeks with conventional method. The culture-expanded MSCs were infused on day 0 into twelve patients (CD34+=3.71 ± 7.19 x 10 6 /kg, MSCs=6.19 ± 2.19 x 10 5 /kg). Of twelve patients, four patients were AML, two CML, two ALL, two Myelofibrosis, one MDS, and one renal cell cancer. Six patients were male and another six were female with mean age of 29. 3 (ranges 4 -50) . Eleven patients received HLA matched sibling donor cells and one patient received HLA haplo-mismatched sibling donor cells. Nine cases used BM and three used PB as their source of stem cells. After HSC tranplantation we evaluated the time to granulocyte engraftment and the degree of acute GVHD. No serious complications related to infusion of both MSCs and HSCs were observed. Engraftment day of granulocytes was mean 12.0 ± 2.9 days after transplantation, whereas engraftment day was mean 13.1 ± 3.1 days with conventional HSC transplantation (p=0.047 by non-parametric independent T test). Acute GVHD was noted in five patients; 3 patients with grade I, 1 with grade II, and 1 with grade III, which were well controlled with steroid therapy. The patients with grade II and III subsequently progressed to chronic GVHD, limited and extensive stage, respectively. There was no graft failure. Co-transplantation of MSCs and HSCs seems to be safe without serious toxicity, while enhancing engraftment of HSCs, and may decrease the risk of acute GVHD and graft failure. Additionally, chimerism studies of both HSCs and MSCs were available in five cases after transplantation. Although complete donor chimerism of HSCs was documented in all five patients, no one showed donor chimerism of MSCs. The reason for difficulty to achieve donor chimerism of MSCs even with culture-expanded MSCs infusion remains to be determined.

Human bone marrow contains pluripotent mesenchymal (MSCs) and Haematopoietic stem cells (HSC) . MSCs have been shown to be capable of differentiating into a variety of cell phenotypes including bone, cartilage, fat, muscle and neural cells. We have identified, in human umbilical cord blood, cells that strongly resemble bone marrow derived MSCs, as defined by expression CD73, CD90 and CD105 cell surface markers. These cells have been successfully expanded and maintained in culture for over one year in their undifferentiated state MSC-like cells also express the intermediate filament protein nestin, which is expressed by early neuronal and pancreatic progenitor cells. These cells also showed a strong expression of the von Willebrand factor (vWF) which is a characteristic marker of endothelial cells. However, other endothelial cell related markers CD31(PECAM-1), CD62E (E-selectin) and CD54 (ICAM-1) were not detected. When placed in an adipogenic medium they developed adipocyte characteristics as depicted by staining of the lipid vacuoles with oil-red-O and increased expression of PPAR as detected by Real-Time PCR. Furthermore, following incubation in neural differentiation medium containing retinoic acid and neurotrophic factors, umbilical cord derived cells expressed the neuronal cell marker b-tubulin III or the glial cell marker, glial fibrillar acidic protein (GFAP). These results indicate that cord blood may provide an additional source of MSCs phenotypically similar to those found in bone marrow. The presence of nestin suggests that these cells may have the capacity to differentiate into neural and/or pancreatic islet-like cells.

F.M. Sanchez-Guijo, L.I. Sanchez-Abarca, M.D. Caballero, P. Menendez, E.M. Villaron, M. Alberca, N. Lopez-Holgado, A. Orfao, J.F. San Miguel, M.C. del Cañizo, Hospital Universitario de Salamanca, Centro de Investigacion del Cancer (Salamanca, E) To date there are no studies which have addressed whether after allogeneic stem cell transplantation (Allo-SCT) the proportion of CD34+ cells and their subsets are similar o different in bone marrow between donor and host when the engraftment is stable (more than 1 year after the transplant). Moreover, is yet to be assessed if the presence of chronic graft versus host disease (cGVHD) modifies the proportion of this precursors. In the present study, we have analyzed total bone marrow (BM) CD34 positive cells and their subpopulations by flow cytometry from 40 patients undergoing HLA-matched sibling Allo-SCT and we have compared it to that of their respective 40 donors. In 11 cases, patients were under treatment for active cGVHD. Bone marrow (BM) samples were simultaneously obtained from both patient and donor. In all cases time from engrafment had exceeded one year (mean ± SD: 928 ± 490 days after the transplant). Complete chimerism in BM was confirmed in all cases by PCR. BM samples were stained for the following quadruple monoclonal antibodies (McAb) combinations: CD71-FICT/CD13-PE/CD45-PC5/CD34-APC and CD90-FITC/CD133-PE/CD38-PC5/CD34-APC. With these combinations, four CD34+ subpopulations were defined: lymphoid, erythroid, granulomonocytic (GM) and immature. Overall CD34+ cells proportion was lower in the patient's group when compared with the donors(0.45±0.24% versus 0.60±0.26%; p=0.013). Upon comparing CD34+ subsets in patients and donors we found significant differences with regard to the lymphoid subset (20.8±15 vs 12.5±10, respectively; p=0.003) and the immature subpopulation (5.9±2 vs 9.3±3, respectively; p<0.001), whereas there were no differences in both the erythroid and GM subsets. When the presence of cGVHD was evaluated, we found a significant lower proportion of CD34+ cells in the active cGVHD group (0.23 ± 0.19 %versus 0.52 ± 0.23 % ; p=0.001). In this regard, we only found significant differences in the lymphoid subpopulation's percentage, which was lower in the cGVHD group (10 ± 10 % versus 23 ± 16 %; p= 0.015). In summary, our results show that in the long term period after Allo-SCT the proportion of BM hematopoietic progenitors is lower compared to their respective donors, and even more, these progenitors are mainly committed with a lower immature progenitor proportion. Interestingly, patients with active cGVHD under immunosuppressor treatment exhibit an even lower BM CD34+ cell proportion. our laboratory, a direct selection method to obtain MSC from fresh murine BM. This was achieved by an immunomagnetic sorting with an anti-CD49a antibody : the cell population expressing CD49a (CD49a+) generates 100% of the CFU-f potential of the BM. The aim of this study is to further characterize the murine CD49a+ cell phenotype and their BM homing potential after systemic infusion in mice. CD49a+ cells were directly selected from male BALB/c and C57BL/6 murine fresh BM. Stem cell phenotype of CD49a+ fraction was determined by the : i) flow cytometrics studies, and ii) evaluation at day 10 of cloning efficiency (CE=nb of CFU-f obtained for 10E5 mononuclear cells seeded) of cultivated CD49a+ cells. Residuals CD49a+ cells were banked in nitrogen before in vivo experiments. BALB/c male CD49a cells were infused into no irradiated or sub lethally irradiated BALB/c. Three to six months after transplant, recipient mice were sacrificed. BM and blood cells were recovered in order to detect male origin cells using real time polymerase chain reaction (RT-PCR) and fluorescence in situ hybridation (FISH) . In addition, a fraction of BM cells was cultivated in order to generate CFU-f which were subjected to RT-PCR and FISH analysis. CD49a+ fraction represented approximately 4 to 11% of mononuclear cells dependant on the strain of mice. MSC sorted directly had a stem cell phenotype since : i) CD49a+ cells expressed c-kit, CD34 and Sca1 molecules and ii) Sca1+ cells were exclusively founded in the CD49a+ fraction. This result was confirmed by the culture of CD49a+/Sca1+ cells which generated the totality of the BM CFU-f potential. Finally, CD49a+ infused cells : i) were able to home to the BM in only irradiated recipients : FISH analysis showed that 40% of the BM cells were male origin and ii) they conserved their CFU-f potential in female recipients three months after infusion : RT-PCR showed that 25% of the CFU-f cells were donor origin. Systemic infusion of CD49a+ cells in to irradiated mice provides a model for bone reconstruction.

M. Ramírez, C. Regidor, I. Marugán, J. García-Conde, J. Bueren, M.N. Fernández, CIEMAT, Clinica Puerta de Hierro, Universidad de Valencia (Madrid, Valencia, E) We hypothesized that the strategy of co-transplantation of human cord blood (CB) and low numbers of haploidentical mobilized peripheral blood (MPB) CD34+ cells could shorten postransplant neutropenia in human patients. Highly purified MPB stem cells would cause an early first wave of engraftment without interfering with the slower engraftment of CB cells. Recently we reported results supporting this hypothesis (Fernández et al. Exp Hematol 2003; 31:535) . To investigate the effect that the proportion of MPB to CB cells may have on engraftment kinetics, we have assayed co-transplantation of different ratios of human hematopoietic stem cells from these 2 sources into NOD/SCID (Non Obese Diabetic/Severe Combined ImmunoDeficient) mice. This model, although not suitable for evaluating the role of immune mechanisms in co-transplantation, allows us to functionally study the contribution of each source of cells, in terms of both timing and percentage, to the human hematopoietic engraftment of the recipient mice. Fixed numbers of human CB CD34+ cells were mixed with MPB stem cells at different ratios (1:5, 1:10, 1:20, 1:50; CB:MPB) and transplanted into sub-lethally irradiated NOD/SCID mice. At periodic intervals after transplantation (from days +15 to +35), marrow samples were aspirated from one femur by puncture through the knee joint. At the end of the experiments (day +50) the mice were killed and single-cell suspensions prepared from the marrows. Cell samples were analyzed by flow cytometry for quantifying human hematopoietic cells. Contribution of MPB and CB hematopoietic stem cells to the human engraftment was determined by PCR amplification of 10 different single tandem repeat (STR) loci. Overall, CB stem cell contribution predominated, even at the earliest analysis, except with the highest MPB:CB cell ratio, 50:1, which resulted in marginal CB stem cell engraftment at all assay times. MPB stem cell contribution progressively decreased from day +15 to the end of the experiments. Therefore, our results show that in this xenogeneic model human MPB stem cells contributed mainly in the first wave of engraftment and had a marginal role in long-term engraftment, only interfering with CB engraftment in the experiments with a very high number of MPB cells. In all other cases CB cells predominated both in the short and long term engraftment.

The potential of autologous bone marrow stem cells for stroke patient management I. Lisukov, A. Kulagin, A. Krivoshapkin, P. Pilipenko, E. Netesov, E. Chernykh, A. Ostanin, O. Leplina, E. Melidy, A. Petrovskaya, P. Semin, V. Kanygin, V. Kozlov, Institute of Clinical Immunology (Novosibirsk, RUS) Recent observations have been demonstrated haematopoietic stem cells plasticity point towards their use in repairing nonhaematopoietic tissues and organ cell replacement therapy. The purpose of the study was to estimate the efficacy and safety of autologous bone marrow stem cell injection directly to brain lesions in stroke patients. Seven patients (6 male, 1 female) after getting the informed consent were included in this study. Mean age was 44+/-10 years (24-67). Three patients suffered from the ischemic lesions and four patients had the consequences of hemorrhagic stroke. Median period of patients follow-up before this procedure was 15 month. All patients had severe preoperative neurological deficits and were severe disables. On the day of procedure, patients had 200 ml bone marrow extracted, which then underwent Ficoll gradient centrifugation to obtain the mononuclear cells (MNC). The number of injected MNC was 0,8-1,4x10 9 cells for each patient. The total volume of cell suspension was 1-2ml. In four cases frameless stereotactic (Voyager SX, Marconi) stem cell injections into the area of posthemorrhagic cyst and perifocally were performed. In three cases injections into postischemic brain lesions were used during bypass surgery. Neurological examination, Bartel's scale and functional independency scale assessment, transcranial magnetic stimulation, somatosensory evoked potentials, computed tomography (CT) and magnetic resonance imaging (MRI) data were evaluated before and 3, 6, 12 months after the procedure. The median follow-up period was up to 6 months. No complications were observed postoperatively. There was significant reduction of spasticity in early postoperative period in all patients. Two patients demonstrated strength improvement in paralytic extremities. One patient recovered completely from moderate preoperative hemyparesis. Speech recovery was noted in two cases. CT showed posthemorrhagic cyst volume reduction by 30% in two cases. Bartale's scale score increased by 20 points. Functional independent scale score improved by 5 point in average. We conclude that autologous bone marrow stem cell injection directly to brain lesions is safe and may reduce the neurological deficits in stroke patients.

It has been previously reported, that not only embryonic but also hematopoetic (HSC) and mesenchymal stem cells (MSC) have the capability to differentiate into various cell types. In order to investigate whether HSC may differentiate also into MSC, we assessed chimerism in peripheral blood, mononuclear cell fractions (MNC) of bone marrow, and MSC derived from bone marrow applying fluorescence in situ hybridization (FISH) using X/ Y gene probes in sex-mismatch and STR-PCR in sexmatched patients after allogeneic hematopoietic cell transplantation. Of 55 sample units scheduled to evaluate, a confluent monolayer of bone marrow MSC could only be generated in 26 samples. MSC were characterized by flow cytometry. Bone marrow aspirates and peripheral blood samples were obtained from 4 female recipients with male donors, from 9 male recipients with female donors and from 7 sex matched transplanted patients. Thirteen patients have received peripheral blood stem cell transplants, 2 patients CD 34-selected peripheral stem cells, and 5 patients bone marrow, respectively. Five patients were analysed repeatedly during the post transplant period. Of 26 sample units, 18 analyses were carried out between the first 12 months after transplantation (median time after transplantation 99 days; range 27-365), and 8 analyses were performed at later time points (median time after transplantation 2920 days; range 476-4225). All patients had chimerism in peripheral blood and MNC of the bone marrow. In all but one patient bone marrow derived MSC were of recipient origin. This one patient showed reproducibly MSC of donor origin in a frequency of 1% after having received CD34-selected peripheral stem cell transplantation 5 years ago. Our results indicate, that although HSC have been found to differentiate into a variety of nonhematological cell types, a differentiation into mesenchymal stem cells usually does not occur. B. Rüster, B. Grace, R. Bistrian, O. Seitz, R. Henschler, Blutspendedienst Frankfurt am Main (Frankfurt, D) MPC have previously been shown to engraft in mice after i.v. administration. We have asked whether transplanted MPC might use similar adhesion and chemotactic migration mechanisms as are known for hematopoietic cells. MPC were grown from human bone marrow aspirates by serial passaging of low density cell fractions and culture in FCS-supplemented medium over a period of three to four weeks. The resulting homogenous fibroblast-like cell population by flow cytometric analysis showed expression of the mesenchymal cell antigens, SH-2 (CD 105) and SH-3 (CD 73), but was negative for the expression of CD 45, CD 34 and AC133. Of the known adhesion receptors, MPC were positive for CD 44, VCAM-1 and, to a lesser degree, CD 62P. In chemotaxis assays using 8 µm micropores in Boyden chambers, MPC did not respond to the chemokine SDF-1 alpha over a wide concentration range, but migrated in a chemotactic fashion towards human plasma (1-40%), indicating that MPC are able to follow guided movements. We therefore injected 1x10 6 MPC or as a control 1x10E AC133+ into 2.5 Gy preirradiated NOD/SCID mice and analyzed bone marrow, speen, liver, brain and lungs after a period of 8 weeks. Donnor cell engraftment was quantitated using human specific TaqMan RT-PCR for the GADPH gene. We fownd human engraftment of MPC in the livers and in the bone marrow, but negative result in spleen, lung and brain. Quantitatively, engraftment of MPC and AC133+ cells was at comparable levels in liver and bone marrow ; however, AC133+ cells also strongly engrafted in speen, brain and lung.

Our data indicate that MPC show regulated chemotactic response in vitro and regulated organ engraftment, which however follows a different organ distribution in MPC and HPC. Barcelona (Barcelona, E) Background Present knowledge of the mechanisms underlying the adhesive processes that lead to homing and/or mobilization of hematopoietic progenitor cells, and the influence of blood rheology is still limited. Objectives and Methods We studied the adhesion of CD34+ peripheral blood progenitor cell (PBPC) to the adhesive proteins fibronectin, laminin, and collagen, and to stromal cells, under flow conditions. The effect of granulocyte colony-stimulating factor (G-CSF) on the adhesive properties of the stromal cells was also analyzed, evaluating changes in the expression of VCAM-1, the adhesion of CD34+ PBPC, and the activation of p38 MAPK. Results Under static conditions, all the adhesive substrata assayed promoted adhesion of CD34+ PBPC, being higher on the stromal cells. Under flow conditions, we observed significant adhesion only onto the stromal cell monolayers. Exposure of the stromal cells to G-CSF notably increased the expression of VCAM-1 on the cell surface and promoted the activation of p38 MAPK, effects paralleled by an augmented adhesion of CD34+ PBPC under flow conditions. Conclusions CD34+ PBPC attachment to the bone marrow implies not only cell-cell but also cell-extracellular matrix interactions, where the reologic conditions present in the bone marrow microenvironment may play a determinant role in these processes. In our in vitro conditions G-CSF increases the Widespread use of CB for transplantation is limited by low content of hematopoietic stem cells (HSC) and prolonged thrombocytopenia, due both to the low amounts of Mk progenitors transplanted and their delayed engraftment. Therefore, there is great interest in ex-vivo expansion of CB HSC especially as concerns Mk lineage. Coordinate gene expression during HSC differentiation toward the mature Mk lineage is controlled by an array of transcription factors (TF) regulating cell proliferation, endoduplication, and terminal maturation. Studies employing targeted gene disruption have shown that TF GATA-1, GATA-2, FOG and NF-E2 are actually required at different cellular levels and developmental steps for the proliferation and maturation of Mk progenitors up to platelet shedding. We have observed that CD34+ cells re-isolated (eCD34) from ex vivo 4 wexpanded CD34+ in the presence of FL, SCF, TPO, produce less mature Mk when compared to freshly isolated CD34+ (bCD34+). To verify whether changes in TF expression profile might be involved in the apparent Mk maturation defect of eCD34+, expression levels of the above TFs were quantified by TaqMan analysis. We found that in vitro expansion of bCD34+ to obtain an eCD34+ cell population led to a 1-1.5 log reduction in absolute mRNA levels for those TFs; however, when eCD34+ were primed to Mk differentiation in vitro by incubation with IL6, IL3, SCF and TPO (63ST), TF expression in immunoselected CD41+ cells after 14 d of culture was restored up to levels comparable to those attained in CD41+ cells obtained from the bCD34+. Notably, TFs mRNA levels in CD41+ cells, derived either from bCD34 or eCD34, were comparable to those measured in bCD34+, while, as expected for maturing Mk, there was a 1-2 log increase in mRNA for GPIIb. Overall, these data indicate that ex vivo expansion of CD34+ is accompanied by down-regulation of GATA-1, -2, NF-E2 and FOG-1, to suggest that expression profile of eCD34 shifted towards a less characterized phenotype. However, these genes could be promptly reactivated by incubation with 63ST to indicate the persistence of a fully competent differentiation program in eCD34+, as also suggested by the efficient platelet production when eCD41+ cells were injected into NOD/SCID mice. These findings, together with a more comprehensive ongoing microarray analysis, might have relevance for the design of protocols for ex vivo expansion of CB HSC and their proper differentiation towards the Mk lineage.

S. Urbani, R. Caporale, L. Giabbani, L. Lombardini, P. Romagnani, R. Saccardi, A. Bosi, Ospedale di Careggi (Florence, I) Mesenchymal stem cells are multipotent progenitors retaining the capability to undergo multilineage differentiation towards all the mesodermal cellular lineages, displaying a great degree of orthodox and unorthodox plasticity and playing an important role in a wide range of clinical applications. In our set of experiments, MSCs were obtained from bone marrow buffy-coats of healthy donors. The number of CFU-F we found was in mean 48/1*106 bone marrow TNCs. The purity of our cultures was ascertanined evaluating the expression of classical hematopoietic and mesenchymal markers both by flow cytometry and by RT-PCR (TaqMan): they rapidly lost the expression of the typical hematopoietic markers CD34, CD45 and CD14 and expressed variable amount of CD105 and CD73; they did not express CD133 antigen. The expansion potential of MSCs was up to a 109 factor (over 5 months culture). In our particular culture conditions, we analyzed by RT-PCR (TaqMan) the expression of the transcription factor Runx-2, specific for osteogenic differentiation, with a progressive increase in the transcript amount as cultures underwent successive trypsinizations (P1, P3, P6). Osteogenic and chondrogenic differentiations were obtained after 21 days of culture in the presence of specific stimulation factors, and were evaluated by multiple techniques, including standard stainings, immunofluorescence for collagen II expression, scanning and transmission electron microscopy. Flow cytometric analysis was performed excluding dead cells by 7-AAD staining; the general polulation appeared on a dimensional scatter as divided in 2 subpopulations, one consisting of small and agranular cells and one of large and complex cells. To analyze the proliferation kinetics of primary culture cells, combining FBS and UltroserG, in Iscove's MDM, we used CFSE to evaluate, at different time intervals, the number of cell generations and the achieved proliferation index. The subpopulation of "small cells" showed a higher proliferation index and the combined use of UltroserG and FBS resulted in a lower loss of this subpopulation after 28 days of culture. In clinically feasible conditions of extended cultures, MSCs progressively lose their multipotentiality and proliferation potentials: the evaluation of the effects of manipulation of cells in culture and the definition of conditions to retain MSCs in a functional progenitor state at adequate numbers are the prospect for an effective self-cell regeneration therapy.

V. Martinez-Muñoz, M. Garcia-Escarp, I. Sales, J. Barquinero, J.C. Domingo, P. Marin, J. Petriz, IDIBAPS, Vall d'Hebron, Universitat de Barcelona (Barcelona, E) Recent data point to the existence of different classes of human stem cells with variable self-renewal potential and short-or longterm repopulating capacity. Telomeres are nucleoprotein structures at the chromosomal ends, essential to maintain chromosomal integrity and genomic stability, and are critical for the proliferative activity of cells. These observations led us to investigate the telomere length in highly purified human hematopoietic CD34+ cells, obtained from the mobilized peripheral blood of normal donors, and patients with multiple sclerosis (MS) undergoing autologous stem cell transplantation. We have used flow cytometry-based fluorescent in situ hybridization (Flow-FISH) and cell sorting (MoFlo, DakoCytomation) for the measurement of relative telomere length. Analysis of the CD34+ cell samples showed a surprising heterogeneity in the relative telomere length. Strikingly, a very small fraction of cells with very large telomeres (40 kb) was systematically identified in all samples of purified CD34+ cells, but not in those containing non-selected cells. Mean values for five different CD34+ cell subpopulations on the basis of the telomere length increase were: 1/ 33 ± 24.99 kb, 2/ 2.4 ± 1 kb, 3/ 0.5 ± 0.1 kb, 4/ 0.1 ± 0.02 kb, and 5/ baseline. Average telomere length is 7.6 kb in peripheral blood CD34+ cells. The highest estimated telomere length of CD34+ cells was approximately 85 kb. Our results lend support to previous observations on the heterogeneity of the stem cell compartment. Here we show by flow cytometry and confocal microscopy that CD34+ cells comprise different subpopulations that differ in their telomere length. The finding of CD34+ cells with very long telomeres could provide new clues to the biology and the pathology of these intriguing cells. Grant #SAF2002-0261 (MCYT).

I. Rasmusson, O. Ringdén, B. Sundberg, L. Lönnies, K. Le Blanc, Karolinska Institutet (Stockholm, S) Mesenchymal stem cells (MSC) have immuno-modulatory properties. They inhibit T cell proliferation to mitogens and allogens, prolong skin graft survival and lower the risk of graftversus-host disease. T cells can be inhibited even when separated from the MSC by a semi-permeable membrane, indicating that the inhibition is mediated by a soluble factor. We have analyzed cytokine profiles of MSC, peripheral blood lymphocytes (PBL) and co-cultures of PBL and MSC. Using ELISA, western blot and ELISpot we saw that MSC increased the level of interleukin-2 (IL-2) and soluble IL-2 receptor (sIL-2Ralpha, CD25) in supernatants of mixed lymphocyte cultures (MLC). The increased level of IL-2 was not the source of inhibition, since addition of rhIL-2 stimulated PBL. rhIL-2-induced lymphocyte proliferation was abrogated in the presence of MSC. Real-time quantitative PCR showed an increase in IL-2 and sIL-2R mRNA in MLC. In contrast, IL-2 and sIL-2R transcription decreased when MSC where added to cultures stimulated by the mitogen Phytohemagglutinin (PHA), indicating that MSC control IL-2 signaling in T cells by different mechanisms depending on the T cell stimuli. In MLC, MSC also affected IL-10 levels, causing increased production on day 3 and decreased production on day 5 compared to the control without MSC. MSC were also added to unstimulated responder cells in MLC to confirm that the increased levels we see do not come from the MSC. To conclude, MSC have significant effect on IL-2, IL-2R and IL-10 after lymphocyte stimulation by alloantigens and/or mitogen.

Kinetics of human platelet reconstitution in NOD/SCID-mice following transplantation with ex vivo expanded CD34positive cord blood cells Y. van Hensbergen, L.F. Schipper, A. Brand, M.C. Slot, W.A. Noort, W.E. Fibbe, Sanquin Bloodbank, LUMC (Leiden, Amsterdam, NL) Transplantation with cord blood (CB) stem cells is characterized by a delayed hematopoietic reconstitution in the peripheral blood. The low number of hematopoietic stem cells in CB may be one of the factors causing this delay. In this study we evaluated whether in vitro expansion of CD34+ CB cells with TPO could accelerate platelet recovery. Subsequently, we studied the effect of in vitro expansion on the residual normal multilineage engraftment capacity of the expanded cells and the self-renewal capacity of the engrafted stem cells in the bone marrow. In addition to engraftment, also the homing of the expanded CD34+ CB cells was evaluated. CD34+ cells were isolated from CB and cultured in 50 ng/ml TPO for 10 days. Sub-lethally irradiated NOD/SCID mice were transplanted with non-expanded CD34+ cells or with the total fraction of expanded CD34+ CB cells. During the first 6 weeks after transplantation blood was obtained via tail incision and human platelets were measured by flow-cytometry using anti-humanCD41-PE antibody. The detection limit of the method we developed to quantify the number of human platelets in the blood of NOD/SCID mice was 0.001x10 presence of donor-derived haematopoeitic cells we isolated and cultured BM-CD015+ cells to study the origin of MSCs after allogeneic SCT. BM-samples with a volume varied between 3-6 mL were obtained from 9 transplant recipients and mononuclear cells were separated by Ficoll gradient centrifiguration. CD105+ cells were selected using magnetic beads (MACS) conjugated to monoclonal anti-human CD105 antibody. Purified cells were then subjected to long-term culture in myelocult media (Stem Cell Technologies) supplemented with bFGF. The phenotype of the cultured MSCs was characterized by immunohistochemistry and the origin of the cells was determined using a polymerase chain reaction (PCR-based assay for polymorphic short tandem repeat (STRs). More than 90% of selected cells expressed CD105 antigen. All samples gave rise to MSCs in 10 days. The cultured cells expressed myofibroblastic markers such as Vimentin and SMA but lacked expression of certain haematopoeitic and endothelial markers such as CD34, CD45 and vWF. For each patient and at all time points (ranging from 1 to 12 months after transplantation), STR-PCR analysis showed that cultured cells came exclusively from the recipient's bone marrow. BM-MSCs were obtained easily and quickly from immunomagnetically isolated CD105+ cells. By this method we could avoid monocyte-macrophage contamination without performing repeated trypsinization. Finally, MSCs of donor origin do not engraft at all in recipient's marrow after allogeneic SCT.

Expression of microsomal carnitine palmitoyl transferase and potential for mobilisation of peripheral blood stem cells M. Fillitz, H. Karlic, H. Tüchler, A. Lohninger, E. Pittermann, M. Pfeilstöcker, Hanusch-Hospital Vienna, L 

Besides their role in fatty acid metabolism, some carnitine associated enzymes are also involved in signal transduction processes which may play a role in differentiation of blood cells. The microsomal carnitine palmitoyltransferase (mCPT) has multiple activities including a phospholipase C activity which may have a striking effect on growth factor dependent signal transduction involved in hematopoiesis. Despite increasing progress in peripheral blood stem cell (PBSC) mobilization procedures there are still patients who have an unpredictable low potential for sufficient stem cell yield. Based on pilot studies mRNA-synthesis of mCPT was evaluated as a possible predictive factor for stem cell yield representing hematopoietic regeneration in patients undergoing stem cell mobilization. Using quantitative reverse transcriptase Real Time PCR we determined relative mRNA levels of mCPT and of further genes involved in lipid metabolism (MCAs). Analyses were performed before mobilization therapy, before first PBSC apheresis and from a sample of the apheresis product. We analysed a cohort of 23 consecutive patients who were undergoing PBSC mobilization for different hematological malignancies (12 NHL, 8 Hodgkin´s Disease, 3 Multiple Myeloma). All but one patients achieved sufficient mobilization of PBSC (>2.45x10 6 CD34 positive cells/kg body weight). In 1st apheresis PBSC yield was 1.08-48.1 (median: 6.1) x10 6 /kg body weight. In peripheral blood taken before mobilization chemotherapy, median relative mRNA levels of mCPT were 34.5 % (=copies/100 copies of ß-actin) in cases with an total apheresis outcome of lower than 10x10 6 CD34 positive cells / kg body weight (CD34+/kgBW). In patients with a CD34 count higher than 10x10 6 CD34+/kgBW it was 128%. In first apheresis samples, median relative mRNA levels of mCPT were 77% in cases with a CD34 count lower than 10x10 6 CD34+/kgBW and 368% in patients with a CD34 count higher than 10x10 6 CD34+/kgBW. Only one patient did not mobilize PBSC (PB CD34 not reaching treshold for apheresis). In this case the relative mRNA level of mCPT was 7% (vs. 78% in healthy adults), indicating a relation of mCPT to a systemic potential for a sufficient hematopoietic reconstitution. This was further confirmed by statistical analysis of mCPT expression which presented a significant correlation between number of CD34 positive cells (rel% of WBC) in peripheral blood at time of first apheresis, yield of CD34 positive cells in 1st apheresis and total harvest outcome. We conclude that determination of mCPTlevels within the scope of possible autologous stem cell collection has the potential to predict the fraction of CD34positive cells in the apheresis product. For patients with low mRNA levels for mCPT, an alternative stem cell harvest procedure should be chosen. Acknowledgement:

Supported by Jubiläumsfonds der Österreichischen Nationalbank

Temporal changes in gene expression profile of healthy donors after G-CSF mobilisation C. Castilla, N. C. Gutierrez, I. Isidro, E. Ocio, M. Delgado, P. Maiso, M. C. Cañizo, J. M. Hernandez, J. F. San Miguel, Hospital Universitario de Salamanca (Salamanca, E) Introduction: G-CSF is the most commonly used agent for haematopoietic stem cells mobilization into the periphery in healthy donors for allogenic transplantation. Thought, it is well known that G-CSF stimulates proliferation and developing of myeloid cells, many of its effects in healthy donors are still unclear. The possibility of studing thousand of genes in the same experiment by microarrays tecnology could provide powerful information previously unkown. Aims: To evaluate G-CSF effects on transcriptome of peripheral blood cells of healthy donors. Material: Seven donors mobilized with G-CSF (5 microg/kg/q12h for 4 days) were included. Peripheral blood samples were collected before starting G-CSF (day 0), the day of collection (day +5) and 6 months after mobilization. Methods: Total RNA (5-10 microg) was labeled and hybridized to Human Genome U133A microarray (Affymetrix). Hierarchical clustering algorithm and multidimensional scaling method were used in the unsupervised approach. For supervised learning, Significance Analysis of Microarrays (SAM) was performed. Results: Two-way hierarchical cluster analysis showed a dendrogram with 2 major branches whith the samples before G-CSF and on the day +5, after G-CSF. Supervised analysis identified 815 significant genes that distinguished the two groups: 352 genes were up-regulated and 463 down-regulated after G-CSF. The most relevant functional categories were adhession, apoptosis, celular cycle and extracellular matrix. Hierarchical cluster analysis performed 6 months after G-CSF, showed a dendrogram with 2 branches: samples on day of collection on one branch, and samples of 6 months after G-CSF and before mobilization together on the other branch. No changes in the gene expression profile of peripheral blood cells before G-CSF and 6 months after were observed. Conclusions: The use of G-CSF as hematopoietic stem cells mobilization agent in healthy donors induces changes in expression of multiple genes involved on important mononuclear blood cell functions. These gene expression changes are temporal and returned to the normal profile after six months. /l were 17,9 and 31,5 days, respectively (p<0,001) . Univariate analysis revealed influence of stem cells source, number of CD34+ cells on kinetics of short engraftment in both groups. Number of MNC correlated with recovery in acute leukemia pts, but no in lymphoma pts. Number of CFU-GM influenced engraftment in lymphoma group only. There was no correlation between sex, previous therapy duration and parameters of short term recovery in both groups.

Step-wise analysis confirmed the significant influence of stem cell source on kinetics of recovery after HDT in both groups-shorter recovery occurred after PBSCT vs. BMT. ROC curves analysis determined cut-off values of infused CD34+ cells connected with delayed WBC recovery and PLT recovery in both groups of pts. In acute leukemia group most discriminating CD34+ cut-off values for rapid WBC and PLT recovery were 2,87x10 6 /kg and 1,66x10 6 /kg CD34+, respectively. For lymphoma pts these figures were 2,9x10 6 /kg and 2,11x10 6 /kg, respectively. The dose of MNC in acute leukemia group and number of infused CFU-GM in lymphoma pts were tested. Most discriminating MNC cut-off value for rapid WBC and PLT recovery were 2,03x10 8 /kg and 1,99x10 8 /kg respectively. In lymphoma pts most discriminating CFU-GM cut-off values for rapid WBC and PLT recovery were 71,9x10 4 /kg and 98,3x10 4 /kg, respectively. Conclusions: These results confirm that the time of recovery after HDT depends of diagnosis -the engraftment is significantly slower in leukemia pts then in lymphoma pts. The influence of quantity and quality of the graft (CD34+, MNC and CFU-GM) on engraftment kinetics are different in analyzed groups of pts.

V. Martinez-Muñoz, I. Sales, M. Garcia-Escarp, J. Barquinero, J.C. Domingo, P. Marin, J. Petriz, IDIBAPS, Vall d'Hebron, Universitat de Barcelona (Barcelona, E) Stem cells from different tissues can be identified by the Side Population (SP) phenotype. CD34-negative hematopoietic stem cells can be identified based on the efflux of Hoechst 33342 (Ho342), and comprise a 0.05% of the murine bone marrow cells. The use of two markers, Ho342 (blue and red) and propidium iodide (three-color flow cytometry) is limited to instruments equipped with UV lasers (351 nm excitation). Here we did experiments based on multicolor flow cytometry technology (MoFlo, DakoCytomation) to simultaneously measure 7 fluorescences from individual human SP cells in a single sample tube. Surface expression markers used in this study were: FITC-CD45, PE-CD90, PE-CD117, PE-CD133, PE-CD54 and PE-pancytokeratin, PerCP-CD34 (488 nm excitation) and APC-CD38 (633 nm excitation). Among the cells identified as SP cells (median=0.09%; range: 0.004-0.96%), all were CD34 negative. Although there is some controversy as to whether CD34-are more primitive than CD34+ cells, here we show a highly defined Side Population for the CD45(+)CD34(-)CD54(-)CD90(-)CD117(-)CD113 (-) pancytokeratin(-)CD38(-/low) phenotype, suggesting a more primitive origin for the SP fraction. Grant #SAF2002-02618 (MCYT).

Refrozen cord blood haematopoietic progenitors induce multilineage engraftment in NOD/SCID mouse F. Timeus, N. Crescenzio, F. Sanavio, A. Doria, L. Foglia, P. Saracco, W. Piacibello, M. Berger, F. Fagioli, University of Turin (Turin, I) Objectives: Since to date most units in cord blood (CB) banks are stored in individual bags, the refreezing of CB progenitors might offer new options in transplant settings. The objective of the present work was to evaluate human engraftment in NOD/SCID mice transplanted with twice frozen-thawed human CB progenitors. Methods: Six previously cryopreserved by a standard method CB units, were thawed at 37°C and quickly refrozen within 15 min. Two successive freeze-thaw cycles were performed with an interval of one month or more. Samples were tested for the expression of CD34 and their subsets, for in vitro growth and for the capacity of repopulating irradiated NOD/SCID mice. Animals were killed 5 weeks after transplantation and bone marrow (BM) was analysed for human multilineage engraftment. Results: There was no significant difference in the recovery of mononuclear cells (MNCs), absolute count and apoptosis rate of CD34+ cells, CD34+/38-/13-cells, CFU and LTC-IC between the first and second thawing. Mouse BM after transplantation showed an equivalent percentage of human CD45+ cells, associated with multilineage engraftment in basal condition as well as after the first and second thawing (table 1). The engrafting potential (total number of human cells/mouse, divided by the number of cells transplanted) of CB MNCs in basal conditions and after the first and second cryopreservation procedure was respectively 2.6±2.1, 3.2±2.8 and 3.1±2.5. Human CFU and LTC-IC from BM cells did not show a significant difference after the second thawing. Conclusion: The present work confirms our previous clonogenic studies (1) and shows that refrozen CB progenitors substain human multilineage engraftment in SCID mice. Therefore, under controlled conditions, it is possible to manipulate an aliquot of cells before transplantation even when the CB unit is cryopreserved in a single bag. This might, for example, optimize the infusion timing of expanded and unexpanded progenitors in transplant settings. References: 1. F Timeus et al, Haematologica, 88:74-79,2003 .

M. Mohyeddin, M. Bashtar, K. Alimogaddam, B Ansari, A. Ghavamzadeh, Shariati Hospital (Teheran, IR) Objectives: Mesenchymal stem cell (MSC) that are capable of self-renewal and differentiation into various connective tissue lineages attract a lot of attention from investigators in the context of stem cell therapies. In our ongoing study, we have evaluated the frequency, phenotype and differentiation potential of MSC in bone marrow (BM) cord blood (CB) and mobilized peripheral blood stem cell (PBSC). Material and methods: Nineteen CB were collected from the umbilical cord following full-term normal delivery, 10 BM and 19 PBSC were obtained from normal BM and PBSC donors at the time of collection. Mononuclear cells were obtained by ficoll and washed in RPMI, suspended in culture medium [Dulbecco,s Eagle,s medium with low glucose containing 20% fetal bovine serum (FBS) for CB and PBSC, 10% FBS for BM cell, and penicillin-streptomycin] and seeded (T-25 or 75 flasks) at a concentration of 2x10 6 cells/ml (CB and PBSC) and 1x10 6 cells/ml (BM). Cultures were incubated at 37ºC a humidified atmosphere containing 5% CO2, with a change of culture medium every 4 days until the fibroblast-like cells reached confluence. On reaching confluence. the adherent cells(MSCs) were re-suspended using 0.25% trypsin and re-seeded at 1x10 4 cell/ml (passage).Every other passage the trypsinized MSC were examined for CD13, CD44,CD34 by flow cytometry and induced to differentiate into adipocytes and osteocytes. Results: All BM samples produced MSC that survived multiple passages in mesenchymal culture medium, primary culture took between 11 to 25day (mean15d) and every passage took 4 to 25 d (mean12d). PBSC and CB samples produced a non-confluent adherent layer of heterogeneous cells, which were relatively resistant to trypsinization and did not proliferate beyond the first passage. Immunophenotype of BM-derived MSC in every other passage were CD34-, CD13+ and CD44+ the adipogenic and osteogenic differentiation were confirmed by oil-red O and Von Kossas staining respectively. The mentioned evaluation for PBSC and CB were not attempted because these were not confluent even in the first passage . Conclusion: In our on going study, human BM cells produce adherent homogeneous confluent monolayer in the MSC culture. These cells are positive to MSC surface proteins and differentiate to MSC lineage, whereas PBSC and CB culture produce a heterogeneous mixture of non-confluent cells, which cannot be passaged under the same conditions.

The effect of cryopreservation on mesenchymal stem cells M. Mohyeddin, M. Bashtarr, K. Alimogaddam, B. Ansari, M. Khalili, M. Solamani, A. Ghavamzadeh, Shariati Hospital (Tehran, IR) Objectives: The ability of mesenchymal stem cells (MSCS) to self-replicate and differentiate into mesenchymal tissues, and the ease with which MSC can be cultured and expanded in vitro, make these cells an attractive tool in stem cell therapy for the repair of damaged organs and tissue engineering. Because, In vitro expanding of MSC is time consuming, having a pool of already expanded cells will be helpful. In this study we have evaluated the effect of cryopereservetion on ex-vivo expansion of MSC. Materials and methods: Bone marrow (BM) cells were taken from 5 normal adult BM transplant donors at the time of harvest. Mononuclear cells were isolated by ficoll and cultured in T-75 flasks with 21x106cells/flask in DMEM-LG medium, consisting of penicillin-streptomycin and 10% FBS. Flasks were incubated at 37oC in a humidified atmosphere containing 5% CO2 and the culture medium was changed every 4 days. When the confluences of MSC reached over 90% they were detached with 0.25% trypsin, counted and re-seeded at 21x10

The aim of our studies was to evaluate whether in our expansion protocol, involving the use of combination of haemopoietic growth factors, pre-treatment with HESW might enhance the efficiency of expansion and possibly of trasduction. Material and Methods: The HESWs were generated by a piezoelectric devise, kindly provided by Med & Sport (Torino, Italy). CD34+ cells were obtained from the cord blood of full term pregnancies. CD34+ cells underwent HESW treatment with a high range of energy and a number of shots (from 0.22 mJ/mm2 to 0.43 mJ/mm 2 and from 200 to 1,500 shots). Cells were monitored for their ability to expand with FLT3 ligand, c-kit ligand, thrombopoietin and IL6 in terms of CD34+, CD34+CD38-, CFCs. We then investigated the role of HESWs to enhance the transfection mediated by lentiviral vectors. Finally, we performed serial transplantation in immunodeficient mice (NOD/SCID) of HESW treated cells to evaluate whether HESW treatment enhanced the engraftment capacity Results: A significant advantage of HESWs cells was obtained in terms of CD34+CD38-expansion in a short term suspension culture, while the significant differences for the total cell expansion and colony generation were noted only in very long term stroma-free cultures. Also the transfection efficiency was successfully affected by the HESW treatment, in particular the 0.32 mJ/mm 2 energy per 500 shots guaranteed an expansion of up to one log to the untreated cells following eleven weeks of culture (a 55 fold increase compared to a 5 fold increase for the controls). Moreover, following transplantation in NOD/SCID, the HESW treated cells engrafted the primary marrow with up to 62% of human cells in the murine marrow, while the control had a 27% of human engraftment. Finally, we demonstrated here that HESW treatment could enhance the human GFP+ transduced cells in the xenogenic murine marrow. Conclusion: HESW enhance the expansibility of the very early progenitors, and after lentiviral vector transfection, we were able to find many more human GFP+ cells in the murine marrow.

Predictive factors for full donor chimerism after reducedintensity conditioning for HLA-identical allogeneic stem cell transplantation M. Mohty, J. Eliaou, C. Faucher, K. Bilger, O. Avinens, V. Bardou, N. Vey, J. Gastaut, D. Maraninchi, D. Blaise, Institut Paoli Calmettes, CHU de Montpellier (Marseille, Montpellier, F) The kinetic of lineage-specific chimerism of donor origin (DC) is an important issue after RIC allogeneic transplantation, because of its major impact on transplant-related events. Predictive factors for full DC are not yet well defined in this setting. Here, we investigated the impact of different factors on the establishment of full myeloid and lymphoid DC (>95% cells of donor origin) after RIC transplantation. 38 pts from a single center receiving HLA-identical allogeneic transplantation were analyzed. Pts and graft characteristics are: age 47 y (18-63), diagnosis: 11 myeloid malignancies (29%), 20 lymphoid malignancies (53%) and 7 metastatic solid tumors (18%). 34 pts (89%) were considered as high risk. 31 pts (82%) received a fludarabine, busulfan and antithymocyte globulin-based RIC, while 7 pts received fludarabine and low-dose TBI (2 Gy). All pts received a PBSC graft, with 26 (68%) receiving CSA alone for GVHD prophylaxis and 12 (32%) receiving CSA and MMF. Median time for ANC and platelet recovery was 16 (0-23) and 12 d (0-99). 26 pts had acute GVHD [7 grade I (18%), 11 grade II (29%) and 8 grade III-IV (21%)] at a median of 35 d (20-85). At day 30 after transplantation, 91% of pts had a full myeloid DC, and by day 90, all pts but one (97%) achieved a full myeloid DC. The latter did not allow depicting predictive factors for full myeloid DC. In contrast, 24 pts (63%) did not achieve full CD3 lymphoid DC by day 30, and 8 pts (21%) were still in mixed lymphoid DC (median 90% cells of donor origin) by day 90 after transplantation. Univariate analysis including: patients and disease characteristics, patient immune status at time of transplantation as reflected by WBC and lymphocyte counts, RIC type and GVHD prophylaxis regimen, PBSC graft composition (CD34+, CD4+, CD8+, CD19+, NK cells), and early transplantrelated events such as acute GVHD and time to engraftment, showed that increasing CD34+ stem cell and CD4+ T cell doses infused with the graft, were significantly associated with the establishment of full lymphoid DC by day 90. In multivariate analysis, CD4+ T cell dose (analyzed as a continuous variable) was the strongest parameter significantly predictive of full lymphoid DC in the first 3 months after transplantation (P=0.04; OR=1.013). Overall, these results suggest that the graft composition might represent an attractive tool towards harnessing donor chimerism and thereby other transplant-related events after RIC allogeneic transplantation.

M. Bornhäuser, C. Thiede, U. Oelschlaegel, T. Illmer, J. Schetelig, A. Kiani, B. Mohr, G. Ehninger, Universitätsklinikum Dresden (Dresden, D) In a phase I study we investigated whether a sufficient number of CD133+ cells can be isolated by the CliniMACS system from G-CSF stimulated blood of healthy donors to allow for allogeneic engraftment. Primary clinical end-points were safety, speed of hematological engraftment and donor chimerism in various cell subsets. So far, four patients with acute myeloid leukemia in first or /l) engraftment was achieved by all patients after 14 (10-21) and 20 (14-45) days, respectively. GvHD grade I occurred in only one patient. Using a quantitative STR-PCR, rapid increase of donor chimerism could be observed for CD15+ myeloid cells, CD34+ progenitors and CD3-/CD56+ NK cells. In contrast, CD4+ and CD8+ cell numbers remained low with persistence of recipient signals until donor lymphocytes were infused in 4 out of 7 patients. Interestingly, significant numbers of myeloid (CD11c+/HLA-DR++) and plasmocytoid (CD123+/HLA-DR++) blood dendritic cells of donor origin could be detected as early as 28 days after transplantation. One patient died from early cardiac failure on day 22. Six patients are alive between 30 and 360 days after transplantation. In conclusion, this study provides preliminary evidence that allogeneic CD133+ cells enriched by large-scale immunomagnetic selection can be used as an alternative source of hematopoietic cells to achieve multi-lineage engraftment in recipients with hematological malignancies. The quality of T cell depletion is comparable to the results achieved with CD34+ selection resulting in a very low risk of acute graft-versus-host disease.

J.H. Won, H.J. Cheong, S.J. Kim, G.H. Jang, C.K. Kim, J.T. Cheong, N.S. Lee, K.T. Lee, S.K. Park, D.S. Hong, H.S. Park, Soon Chun Hyang University Hospital (Seoul, KOR) Objectives: Ex vivo expansion of hematopoietic stem and progenitor cells is a very ambitious idea that would have major implications in the areas of stem cell transplantation and somatic gene therapy. However, successful ex vivo expansion has evaded and frustrated scientists for a number of years. Fas antigen (Fas Ag; CD95) is a cell surface molecule that can mediate apoptosis. Bcl-2 is a cytoplasmic molecule that prolongs cellular survival by inhibiting apoptosis and Bax is a pro-apoptotic member of the Bcl-2 family that targets mitochondrial membranes and induces cytochrome-C release. In this study, we evaluate the effect of the expression of Fas Ag, Bcl-2, and Bax on CD34+ or AC133+ hematopoietic progenitor cells during in vitro expansion. Methods: CD34+ cells and AC133+ cells isolated from human cord blood were cultured in serum free medium supplemented with 100 ng/mL SCF, 100 ng/mL IL-3, 100 ng/mL IL-6, 100 ng/mL G-CSF, and 4ng/mL IL-1-beta for 7 days. After expansion culture, we re-isolated CD34+ cells and AC133+ cells and compared the number of CFU-GM and CFU-GEMM, and expression of Fas Ag, Bcl-2, and Bax with unexpanded cells. Results: The number of CFU-GM was expanded 23.92 fold in CD34+ cells and 15.21 fold in AC133+ cells at day 7 of culture but the number of CFU-GEMM was not expanded (1.47 fold in CD34+ cells and 1.18 fold in AC133+ cells). The expression of Fas Ag and Bax was 7.44% and 2.75% in fresh isolated CD34+ cells and significantly increased to 19.7% and 33.67%, respectively, in expanded CD34+ cells at day 7 of culture but Bcl-2 was not changed (from 76.71% to 76.56%). In case of AC133+ cells, the expression of Fas Ag and Bax was also significantly increased from 5.87% and 4.27% to 24.85% and 28.67%, respectively, and Bcl-2 was not changed (from 74.77% to 72.80%). Conclusions: These results indicate that the nature of expansion was similar between CD34+ cells and AC133+ cells and expression of Fas Ag and Bax is induced on CD34+ cells and AC133+ cells expanded in vitro in the presence of hematopoietic growth factors. Induction of Fas Ag and upregulation of Bax may be expressed as part of the differentiation program of hematopoietic cells.

Partial in vitro T-cell depletion with CAMPATH-1H in combination with DLI for CML molecular relapses decreases GVHD with 70% 5-years DFS in HLA-identical sibling PBSC for haematological malignancies Y. Chalandon, E. Roosnek, L. Waelchli, C. Helg, B. Chapuis, Hopital Cantonal Universitaire (Geneva, CH) Transplantation with stem cells from an HLA-identical sibling donor is a well-established therapeutic modality for hematological malignancies. Unfortunately, the donor T cells in the graft may cause severe forms of GVHD. T cell depletion diminishes this risk but is associated with an increased frequency of relapse. We report here the results of a prospective nonrandomized study of 37 successive patients transplanted between January 1998 and August 2002, which focused on the question of whether partial T-cell depletion in vitro with CAMPATH-1H in combination with DLI used for treatment of molecular relapses in CML would prevent GVHD while preserving the graft-versus-leukemia effect. Eighty-nine percent of the patients (median age 40 y [range 18-60]; diagnoses AML=12, ALL=2, CML=11, MDS=3, NHL=5, MM=2, AA=2) received a TBI-based conditioning. GVHD prophylaxis was CSP-MTX±MP in 30 patients (81%) and CSP-MP±ATG in 7 patients (19%) . All patients received PBSC that had been mobilized with G-CSF for 6 days and harvested on day 4, 5 and 6. Partial T cell depletion was obtained by treating the pooled phereses of days 4 and 5 with CAMPATH-1H in vitro , then infused into the patients followed the next day (6) by the unmanipulated pheresis. The median nb of CD3+ cells was 124 x 10e6/kg (range 28-304). Results: All patients engrafted successfully. Neutrophil recovery occurred at a median of 14 days (range 8-21) and platelet recovery at 13 days (range 8-27). Acute GVHD was limited to grade II in 3 (8%) and to grade I in 10 (27%) patients. cGVHD occurred in 9 patients (24%), 1 of them had extensive disease. Eight CML patients and 1 NHL patient were treated with DLI at the first signs of recurring disease, resulting in CR in all but 2 CML patients who kept their minimal molecular residual disease with a BCR-ABL/ABL ratio <0.05% as determined by a nested PCR. Day 100 OS and DFS were 95% (95% CI:88-100%). With a median follow up of 39 months (range 15-70), the actuarial 5 year OS is 74% (95% CI:53-94%) and DFS is 70% (95% CI:51-88%). The 100-day and 5-year TRM are 3% and 6% respectively. Conclusion: The results of this pilot study show that partial T-cell depletion in vitro with CAMPATH-1H decreases the incidence of GVHD and TRM, without having a negative impact on the frequency of relapse or progression except for CML. However, all these patients could be rescued with low doses DLI resulting in a 5 year OS and DFS + DLI of 100%. /l. When cells were frozen and then thawed at time 0, the average GM-CFC recovery exceeded 37% regardless of the cell concentration or cryoprotectant used. After storage in the liquid state for 16 hours and then subsequent freeze-thawing however, there were major losses in the samples that had been stored at 500x/l (p=0.00000002), but not in these stored at 100x10 9 /l (p=NS). Furthermore, there were major differences in the recoveries of the more concentrated cells according to which cryoprotectant was used. With H.S.A. the recovery of total viable cells and GM-CFC was 34% +/-15% and 4% +/-3%, with FFP 30% +/-17% and 9% +/-18%, but with PS was 74% +/-25% and 43% +/-20%, a highly significant improvement (p= 0.0005 and 0.0002 compared to both H.S.A. and FFP). This study indicates that storage damage in the liquid state arises if cells are maintained at high cell concentrations and only becomes fully apparent after freeze thawing, and that under these exacting conditions, 5%PS 4%H.S.A. and 5%DMSO is a superior cryoprotectant. We recommend its use wherever cryopreservation is not immediate or the PBSC have been extensively manipulated.

T-cell depletion of stem cell grafts with campath-1H 'in the bag'' followed by cyclosporin immunosuppression remains a cost effective strategy in transplantation N. Novitzky, V. Thomas, G. Hale, H. Waldmann, UCT Medical School, The Dunn School of Pathology, Oxford University (Cape Town, ZA; Oxford, UK) Introduction: We tested the strategy of prophylaxis by T-cell depletion (TcD) of stem cell grafts with anti CD52 antibodies followed by post-transplant with cyclosporin (CSA) immunosuppression. Patients and Methods: The myeloablative conditioning was radiation based. Following apheresis, grafts were incubated ex vivo with CAMPATH-1H and infused intravenously. Post transplantation patients received therapeutic doses of CSA for 90 days. In addition, patients with CML received a pre-emptive protocol of escalading DLI starting at 6 months. Results: 54 patients received grafts from HLA identical siblings for malignancies (n= 44) or bone marrow failure (n=10). Following mobilisation of PBPC, a median of 2.98 (range 1.03-18.4) CD34+ x10^6/kg were harvested. Grafts were treated with a median of 10 mg (7.5-50) CAMPATH-1H. Recovery of leukocytes and platelets was at a median of 11 (8-35) days.

There were 13 deaths. GvHD was seen in 6 patients and in 3, associated infection caused their death. 3 patients relapsed and died. Another with CML developed hepatic lymphoma and died. Of the remainder 9, 7 patients with CML have remained in molecular remission following pre-emptive DLI. All patients with marrow aplasia survive disease free. Median follow up of all patients is 612 days (8-1917) and 76% are alive, with normal blood counts in remission. Survival was significantly higher in male patients (p< 0.03), absence of GvHD (p= 0.04) and in lower dose of CAMPATH (10 vs. 20 mg; p= 0.04). Conclusions: TcD with anti-CD52 followed by CSA resulted in low rates of GvHD and mortality. In CML patients, pre-emptive DLI gave long-term protection from relapse.

Bone marrow transplantation after processing by gradients and negative immunomagnetic T-and B-cell selection to prevent GvHD without loss of engraftment potential F. Preijers, A. Schattenberg, W. Ruijs, C. Trilsbeek, University Medical Center St. Radboud (Nijmegen, NL) T-cell depletion of bone marrow (BM) transplants strongly reduces GVHD, but almost complete elimination compromises engraftment and increases relapse rate after BMT. NK cells are often simultaneously eliminated. In contrast, B cells that induce PTLD in absence of T cells, must be eliminated. This implicates that a balance of cell populations in the graft and in the patient after conditioning should be composed so that only cell populations with harmful effects are depleted. BM cells possessing beneficial activity in engraftment and against tumor cells should remain in the graft. The most widely applied depletion methods, CD34 selection, results in elimination of all cells, except stem cells and progenitors, and thus also populations with beneficial effects. Therefore we assessed negative immunomagnetic selection (NIS) of T and B cells from BM transplants. The NIS elimination was evaluated and compared with the historically used counterflow centrifugation elutriation (CCE). Method: BM Transplants were volume-reduced and preseparated by gradient centrifugation on clinical grade PureCell (Medicult, Denmark) to eliminate erythrocytes and granulocytes. T and B cells were depleted by NIS on Isolex 300i (Baxter; CD2 and CD19) and on CliniMACS (Miltenyi; CD3 and CD19). MNC's were added to the negative fraction in order to achieve a T cell dose in the graft of 0.5 x 10 6 T cells/kg body weight. Results: Since November 2001 transplants of 57 patients are processed as mentioned. Before CCE was applied (>500 transplants). The CD3/CD19 resulted in the strongest depletion of T cells (3.0 log). The number of CD34 (FCM), HPC's (cultures), T and B cells were comparable in the ultimate grafts due to add back of MNC. However, the NK cell number was fivetimes higher by using CD3/CD19 NIS. The incidence and severity of GVHD and the relapse rate were comparably low for all T-cell depleted transplants. In order to eliminate specifically those cell populations that hamper BMT we conclude that NIS using anti-T and anti-B MoAbs is a recommendable processing method for BM transplants. NK cells are better recovered by CD3 than CD2. Follow up is too short to draw definitive conclusions regarding relapse rates. /L, 53(72) days; and time to hemoglobin >110g/L, 70(67) days. Significant correlation was observed between short and long term engraftment and increased CD34 cells (p<0.03), viability-adjusted CD34 cells (p<0.02), and CFU-GM (p<0.05) counts. Only CD34 count >3x10 6 /Kg had the highest predictive value for short and long term hematopoietic recovery (p<0.017). Conclusions: In this series of patients undergoing autologous PBPC transplantation, increasing doses of CD34 cells, viabilityadjusted CD34 cells and CFU-GM were correlated with faster hematopoietic recovery. The infusion of CD34 cells >3x10 6 /Kg was the best parameter to predict short and long term hematopoietic recovery. Supported in part by grant P-EF/2003 from FIJC and C03/03 from FIS.

Long-term culture impairs engraftment of normal and 5fluorouracil treated bone marrow in sub-myeloablated hosts L. D 'Hondt, C. Grimaldi, J.F. Lambert, P.J. Quesenberry, Centre Hospitalier Notre-Dame et Reine Fabiola, Roger Williams Medical Center (Charleroi, B; Providence, USA) We demonstrated that day 1 to day 6 post 5-Fluorouracil (5-FU) bone marrow (BM) was defective in short and long term engraftment in both non and submyeloablated host mice. We evaluated here the effect of long-term culture in Dexter's condition on normal and 5-FU treated BM. BALB/c male mice were given 5-FU (150 mg/kg) 1, 2, 6, 8 or 12 days prior to sacrifice. Sixteen million BM cells of each group of mice were seeded in T25 flasks in 5 cc Dexter's medium. Each week half of the supernatant was removed and replaced by fresh medium. After 6 weeks in culture, cells were scraped, resuspended and 5 million cells were injected into BALB/c female recipient irradiated with 100 cGy. Each group consisted of 5 female recipients. Engraftment of cultured BM was evaluated and compared to normal uncultured BM at 10 weeks post injection. BM engraftment was quantified using FISH analysis and spleen and thymus engraftments were assessed by Southern blot using a Y chromosome-specific probe. Table 1 shows the percentage ± SEM of BM, spleen (SP) and thymus (THY) engraftments.

These data showed the rapid induction of an engraftment defect 1 day post 5-FU with a significant nadir by day 6 and a partial recovery by day 12. The 6 week culture into Dexter's condition did not appear to correct this engraftment deficit observed not only in BM but also in SP and THY. Results were comparable when engraftment levels were corrected for the level of cellular amplification observed during culture. In addition cultured normal BM cells also displayed engraftment deficit (data not shown). This suggested that long-term culture does not correct engraftment deficit induced by 5-FU.

Use of CD3+/CD19+ depleted grafts from mismatched related donors in children P. Lang, J. Greil, M. Schumm, M. Pfeiffer, W. Bethge, R. Handgretinger, D. Niethammer, P. Bader, University Children's Hospital (Tuebingen, D) Positive selection of stem cells from mismatched donors is an established method for effective prevention of GvHD. However, also CD34 negative progenitors and effector cells other than T cells may influence engraftment and relapse probability. Therefore, we investigated the feasibility of T cell and B cell depletion by using anti CD3 and anti CD19 coated microbeads and the CLINIMACS device. 10 clinical scale separations were performed with leukapheresis products (median cell number 4x10 10 ) from mismatched related haploidentical donors (parents). The selected cells were used for initial transplantation or as posttransplant donor leukocyte infusions (DLI). Median depletion of T and B cells was 4.6 log and 3.5 log, respectively. Recovery of CD34+ stem cells was 73% . 3 patients with refractory relapsed AML were transplanted with CD3+/19+ depleted grafts, consisting of 6.2x10 6 /kg CD34+ cells, 78x10 6 /kg NK cells, 180x10 6 /kg monocytes and 8x10 3 /kg residual T cells. Moreover,10x10 6 /kg positive selected stem cells were added. A conditioning regimen with reduced intensity (melphalan 140mg/m², fludarabine 200mg/m², thiotepa 10mg/kg) was administered due to intensive pretreatment. Two patients received CSA prophylaxis. All patients had sustained engraftment, median time to ANC >500 was 12 days. No GvHD >grade I and no transplant related toxicity occurred. T cell recovery was rapid. 4 other patients with ALL who relapsed after transplantation of haploidentical positive selected stem cells, received CD3+/19+ depleted cells as donor leukocyte infusions (48.8x10 6 /kg NK cells, 260x10 6 /kg monocytes, 3,7x10 3 /kg T cells and 10,4x10 3 /kg B cells), combined with mild chemotherapy. The infusions were well tolerated without any side effects . No GvHD occurred, although immune suppression was avoided. Activity of NK cells against K562 was higher in grafts from non mobilized donors than in grafts from mobilized donors. Conclusions: We present preliminary results, which demonstrate the feasibility of C3+/19+ depleted grafts in the haploidentical setting in a small number of patients. With the same method, high doses of negative selected NK cells and other effectors for clinical use could be obtained. Thus, the method represents a new approach for graft manipulation in mismatched transplantations.

Feasibility of a novel clinical-scale CD3/CD19 depletion procedure, and how it compares to conventional CD34+ selection G. Fritsch, S. Matthes, C. Peters, V. Witt, J. Pichler, D. Scharner, C. Eichstill, E. Zipperer, H. Gadner, CCRI (Vienna, A) Within the last years, CD34 positive selection has emerged as method of choice to deplete T-and B-cells prior to transplantation of hematopoietic stem cells from HLAmismatched donors. However, a growing body of evidence suggests that a considerable number of stem cells do not express CD34. In addition, the infusion of other donor cells, like mesenchymal precursors or NK cells may be advantageous in terms of engraftment, GvL and GvHD. Therefore we aimed to establish a clinical-scale procedure that sufficiently depletes Tand B-lymphocytes but retains stem cells. Five selection procedures were performed using the combination of 2 devices: first, a cell washer (CytoMate, Baxter) was used, i) to remove platelets, wash and concentrate the cells, ii) to incubate them with CD3-and CD19-labelled MicroBeads (Miltenyi Biotec), iii) to wash and remove excess MicroBeads, and iv) to concentrate again (75-120 min). The product bag was then transferred to a CliniMACS device (Miltenyi Biotec) equipped with a novel program (Depletion 3.1) and a newly developed depletion tubing set in which labelled cells were removed by magnetic separation (75-90 min). The eluted product was transferred to the CytoMate to achieve final concentration (15 min). All preparation steps were monitored by routine 4-color FACS analysis. T-and B-lymphocytes were efficiently depleted while non-T-and B-cells were recovered with reasonable numbers (Table) . Values for log depletion, yield, viability and time required were comparable to CD34 positive selection. All products were sterile. The described depletion procedure is equivalent to the conventional CD34 positive selection procedure. It may be of particular value when addressing the infusion of CD34 negative stem cells or allo-reactive NK cells.

Washing thawed autologous aphereses with the Cytomate biomedical device results in diminished toxicity following reinfusion in cancer patients undergoing high-dose chemotherapy C. Lemarié, B. Calmels, C. Malenfant, V. Arnéodo, D. Blaise, F. Viret, R. Bouabdallah, P. Ladaique, P. Viens, C. Chabannon, Institut Paoli Calmettes (Marseille, F) Introduction: Cryopreservation of autologous hematopoietic grafts requires addition of a cryoprotectant, usually dimethylsufoxyde (DMSO). Infusion of thawed cell therapy products that are not washed and still contain residual DMSO may result in adverse effects in graft recipients; in addition, biological controls are not easily carried out on samples obtained from unwashed cell products. Methods: We here report our experience with an automated biomedical device: Cytomate™ (Baxter Oncology), that we used to eliminate cell debris and DMSO, following thawing of autologous aphereses, and before infusion into recipients. Following preclinical validation (Calmels et al, Bone Marrow Transpl, 2003) we processed 198 apheresis bags from 56 patients, for whom three or more bags were to be re-infused; during the same period, 331 other patients underwent autologous transplantation, and received cell products that were extemporaneously thawed at the bedside. Results: This study demonstrates: 1) mean CD34+ cells recovery for the complete process thawing / washing, was estimated to be 76% +/-34, 2) depletion in DMSO quantities was superior and more reproducible than results observed with standard centrifugation (97.3% +/-1.3 versus 89.6% +/-10), 3) time to neutrophil recovery was comparable in both groups, with a standard deviation that was smaller in the group of patients who received washed cell products (12.6 d +/-7.7 for washed products versus 11.6 d +/-2.3 for non washed ones), 4) a reduction in the incidence and severity of adverse events in recipients of washed cell products, when compared with patients who received cell products thawed at the bedside (incidence of adverse events: 17% in patients who received washed cell products versus 47% in patients who received cell products thawed at the bedside), 5) an improved satisfaction and reduced workload for nurses that are responsible for autologous cell reinfusion to recipients on transplant wards, when washed cell products were delivered. Conclusion: These preliminary data suggest a potential clinical benefit associated with the introduction of the Cytomate™ biomedical device to wash thawed aphereses, and deplete these products of DMSO, prior to reinfusion.

A different but cost-effective schedule for mobilisation of CDd34+ cells for transplantation N. Novitzky, V. Thomas, G. Davison, J. Thomson, L. Abrahams, UCT Medical School (Cape Town, ZA) Aim: We prospectively studied a mobilization schedule in combination with large volume apheresis (LVA) for the collection of blood progenitor cells (PBPC) for transplantation. Methods: Allogeneic donors received G-CSF 5 ug/kg daily for 4 days and on day 5, increased to 10 ug/kg, followed 4 hours later by 30 litres LVA. The target CD34+ cell collected was of 2-x10 6 /kg recipients weight. In those not reaching target values G-CSF at 10 ug/kg and daily apheresis were continued, until this target numbers. CD34+ scores were determined following ISHAGE from the donor on day 4 and 5 of mobilisation and from the collection bag at 15 litres and the end of apheresis. Results were correlated with clinical and laboratory parameters. Results: On day 4 of cytokine therapy 27 donors with a median age of 38 years (range 10-60) had a blood CD34+ number of 16.37 (2.6-85.62) 

Prospective characterisation of short-term repopulating cells in haematopoietic stem cell allografts K Raaschou-Jensen, N. Buza-Vidas, K. Theilgaard-Mönch, E. Dickmeiss, S.E. Jacobsen, Rigshospitalet, Lund Stem Cell Center (Copenhagen, DK; Lund, S) Objectives: The prediction of safe haematopoietic reconstitution following allogeneic SCT is of great importance. Measuring the yield of CD34+ cells in mobilized peripheral blood (mPB) allografts and of total nucleated cells (TNCs) in bone marrow (BM) allografts are currently the best predictors. However, neither of these parameter measures the direct content of shortterm reconstituting cells (STRCs) in the allografts. Characterization of the STRCs is of great interest as these cells provide the early neutrophil and platelet recovery following allogeneic SCT and thereby shorten the time of the devastating cytopenias seen within the first 4 weeks post transplantation. Materials and Methods: Three populations of myeloid STRCs: Common myeloid progenitors CMPs: CD34+CD38+IL-3RaloCD45RA-lin-, granulocyte/macrophage progenitors GMPs: CD34+CD38+IL-3RaloCD45RA+lin-, megakaryocyte/ erythroid progenitors MEPs: CD34+CD38+IL-3Ra-CD45RA-lin-is defined by immunophenotyping and sorted out. The distinct cell fate outcome of the sorted STRCs will be tested in vitro and in vivo using the NOD/SCID-b2m-/-mouse transplantation model. This mouse model will be chosen due to the low NK-activity favouring a highly efficient engraftment of myeloid STRCs. The final aim of our study is to correlate the numbers of distinct progenitor subsets in BM and mPB allografts with time to granulocyte and platelet engraftment in recipients of the same allografts to evaluate the predictive value of progenitor subset analysis for clinical engraftment. Results: Our preliminary analysis of 3 BM samples from healthy individuals indicated an average of 21.2% CMPs, 15.0% GMPs and 4.4% MEPs within the human bone marrow CD34+ cell compartment. According to these percentages the calculated total numbers of STRCs in 12 BM allografts were as follows: CMPs 48.2+7.3 x10 6 , GMPs 34.1+5.2 x10 6 and MEPs 10.0+ 1.5 x10 6 (Mean+SEM). Conclusion: We here state the numbers of myeloid STRCs within BM allografts and present the numbers of CMPs, GMPs and MEPs within the CD34+ compartment. Additional studies in mice are needed to confirm the reconstituting ability of immunophenotypically defined STRCs. Finally the predictive value of progenitor subset analysis on clinical engraftment has to be evaluated.

Childhood pre-B-ALL blasts are killed by alloreactive NK-cell clones, independent of a HLA-Cw mismatch T. Feuchtinger, A. Pfäffle, H.-M. Telschik, M. Pfeiffer, C. Weinstock, D. Niethammer, P. Lang, Children´s Hospital (Tuebingen, D) Natural killer cell mediated alloreactivity in HLA-C mismatched hematopoietic stem cell transplantation has been shown against adult AML blasts, where as in lymphoblastic leukemias it was so fare not possible to confirm an effect of NK-mediated killing. Nevertheless NK-cells remain a promising effector for a therapeutic graft versus leukemia reaction post allogeneic stem cell transplantation. This study investigated the ability of alloreactive NK cells to lyse childhood pre B-ALL blasts and, specifically, the role of the three major NK specificities, ie, those for HLA-C group 1, HLA-C group 2, and HLA-Bw4 alleles. NK cell clones from healthy donors were obtained by limiting dilution techniqe and tested for lysis of recipient's cryopreserved preB-ALL blasts and K562 cells as a positive control. A total number of >150 NK-klones were tested from 2 groups of 3 healthy donors expressing exclusively either HLA-Cw3 or HLA-Cw4 related alleles. NK-cell clones showed significant (p=0,002) HLA-Cw (KIR-ligand) specific killing of B-LCL, homozygous for either HLA-Cw3 or HLA-Cw4 related alleles, thus confirming alloreactivity according to KIR-ligand mismatch. 32,7% of NKklones showed a specific lysis of cryopreserved preB-ALL blasts higher than 60%. Suprisingly the specific killing of the preB-ALL blasts was independent of the HLA-Cw expression: 37,1% of NKklones expressing HLA-Cw3 group alleles exceeded 60% specific lysis of ALL blasts expressing the same HLA-Cw group. NK-klones were analysed for surface expression of major KIR (CD158a, 158b and DX9), activating receptors and adhesion molecules. Phenotypic analysis of the NK-klones revealed a significant difference in KIR expression between NK-klones with high specific lysis and those klones with low specific lysis of ALL blasts. In conclusion we show that a significant amount of NK cells is capable of killing childhood pre-B-ALL blasts and that this killing is independent of the KIR-ligand (HLA-C) expression of effector and target cells. NK-cells lacking the expression of the three major KIRs show a significant better killing of lymphoid leukemia cells. These cells can be preferential targets for a graft versus leukemia reaktion against lymphoid leukemia cells.

Haploidentical transplant in adult advanced leukaemia: a 3step phase I study of growth factors and DLI post-transplant P. Lewalle, A. Delforge, V. De Wilde, I. Ahmad, B. Nowak, K. Theunissen, D. Bron, P. Martiat, Institut Jules Bordet (Brussels, B) Haplo-identical transplant is now a procedure of choice for patients who lack a compatible donor. However, they are still referred too late, heavily pre-treated, at very advanced stages. We initiated a three-step phase I study to improve both relapse rate and immunity: G-CSF + DLI, GM-CSF + DLI, patient and disease adapted strategy. Thirty consecutive leukemia patients, aged 18-55, were investigated (27 very poor risk: primary refractory or refratory relapses and poor risk: early relapse in CR2). GvH type NK alloreactivity was chosen when possible (14/30) and balanced across the 3 groups. In the first 9 patients, G-CSF was used. Prophylactic DLI started at month 1 (3x10 4 CD3/kg) in the 2 first patients. This resulted in grade II aGVHD. We next gave 1x10 4 /kg monthly for 3 times: only 1 grade I GVHD. Overall, 6 patients relapsed and were given therapeutic DLI, starting at 1x10 5 CD3/kg with escalation every 2 weeks if no GVHD. This led to CR in 1. We next gave escalated (1,3 and S97 10x10 4 ) doses. This produced aGVHD grade 2-3 in all. Thus G-CSF and 1 DLI is safe. It results in faster CD4 recovery and a low rate of infections. However, it remains insufficient to induce a GVL effect. In the next 12 patients, GM-CSF was used plus 1 DLI at day 30 unless aGVHD (3 pts) . In these patients, the mean CD4 count was 183 at day 56. The comparison between the 2 first groups can be summarized as follows: G-CSF + DLI: TRM at day 100: 0, RR: 6/9, severe aGVHD:0. GM-CS + 1 DLI group: RR: 1/12, TRM at day 100: 3, aGVHD > 1: 9/12, price to pay: GVHD resulting in 5 deaths in total.

Step 3 consists of a patient adapted strategy: myeloid disorders with NK allo-reactivity in the GvH direction: no GF and 1 single DLI at day 30 (to improve immune recovery). In the other patients, GM-CSF without DLI (to decrease the rate of severe GVHD). The follow-up of these patients, although promising is currently short and results will be updated in March 2004. Overall, TRM at day 100 is 5/30, reflecting the good tolerance of the conditioning in a population (median 40), heavily pre-treated. NRR-mortality was greater in the GM-CSF group at 1 year, reflecting the impact of severe aGVHD. We conclude that the third strategy might improve the outcome without exposing patients to unnnecessary GVHD. In the future good risk patients (SCT as first line treatment, our strategy will be no GF + 1 DLI if proven non toxic).

Possible graft versus neuroblastoma effect after partially matched related haematopoietic transplantation L. Lacitignola, V. Tintori, F. Bambi, M. Mariani, C. Marchi, B. Porfirio, M. Gelli, A. Tamburini, F. Tucci, A. Lippi, G. Bernini, L. Faulkner, Oncoematologia-Reparto Interdisciplinare di Terap (Florence, I) High risk neuroblastoma (NB) relapsing after dose-intesive chemiotherapy and autologous trasplantation has a dismal prognosis. We treated 2 cases of relapsed stage IV NB (age 6 and 7 years) with a stem cell transplant from a haploidentical family donor. Both patients lacked a matched sibling. Conditioning consisted of thiotepa 400 mg/m 2 , fludarabine 90 mg/m 2 , ATG and melphan 140 mg/m 2 followed by purified CD34+ peripheral blood stem cells (CliniMACS, Miltenyi Biotec, Bergish Gladbach, Germany) . No further GVHD prophylaxis was administered. Hematological recovery was prompt in both cases with >98% persistent donor engraftment. Post-trasplant serial marrow (BM) and blood (PB) minimal residual disease (MRD) assessment was performed by anti-GD2 immunocytology on large-size cytospins (sensitivity 1 NB cell in 105-106 MNCs). Patient n.1: evaluable disease was limited to BM. On hematological recovery form haploidentical transplantation MRD evaluation showed persistent BM positivity and detectable circulating NB cells. This finding prompted the administration on day +26 of 6 x10 4 donor CD3+ cells/kg followed by a second infusion of 12 x 10 4 donor CD3+ cells/kg on day +57 since non GVHD developed after the first DLI, he developed grade III intestinal GVHD on day +71. Figure 1 summarizes the time course of GVHD and blood/marrow residual disease (NB cells are expressed per 106 bone marrow mononuclear cells). This patient died 5 months post trasplant with decreasing MRD for a deep-sited fungal infection. Patient n.2: relapse sites were both local (mediastinum) and bone marrow. He was treated with an initial course of thiotepa and melphalan (same dose as the conditioning regimen) and autologous stem cell rescue with a partial response. Marrow disease became undetectable following haploidentical transplantation while local mediastinal disease regressed 5 months post transplant. This patient is alive and well with no evidence of disease, complete chimerism, and no GVHD at 8 months from trasplant. Both cases lacked an NK alloreactivity setup. In conclusion haploidentical transplantation may provide a graft vs. neuroblastoma effect.

Reduced-intensity conditioning prior to allogeneic transplantation of haematopoetic stem cells: the need for Tcells early after transplantation to induce a graft-versuslymphoma effect B. Glass, M. Nickelsen, P. Dreger, J. Hasenkamp, G. Wulf, L. Trümper, N. Schmitz, University of Goettingen, Universitätsklinikum Schleswig-Holstein, AK St. Georg (Goettingen, Kiel, Hamburg, D) Reduced conditioning followed by allogeneic SCT is a promising concept in treatment of lymphoma. In patients with primary progressive disease or poor-risk relapse this approach may have its limitations because of rapid regrowth of the tumor. We tried to address this problem by intermediate intensity conditioning followed by allo SCT. Twenty one patients received Fludarabine 125 mg/m 2 , Busulfan 9.6 to 12 mg/kg and cyclophosphamide 120 mg/kg prior to allogeneic PBSCT. In a first group of patients, rigorous GVHD prophylaxis by CD34+ selection of grafts, pretransplant ALG and short course CSA post transplant (d 1-28) was employed. The next 11 pts received non-manipulated grafts and mycophenolat mofetil plus CSA as GVHD prophylaxis. 12 patients had diffuse large cell NHL, 3 patients blastic mantle cell lymphoma, 3 patients hodgkins disease, 1 patient NK cell lymphoma, 1 patient acute lymphoblastic leukemia and one patient a rapidly progressing follicular lymphoma. There were no apparent differences between both groups concerning the diagnosis or other known risk factors like time from primary diagnosis, number of prior lines of therapy, patients with relapse after HDT, chemo-resistant relapse or patients with an unrelated donor transplant. In both groups engraftment was rapid (median 10 days, range 6-13) and all patients achieved complete (> 95%) donor type chimerism after alloSCT. In group I, no GVHD was observed prior to DLI. In contrast, patients in group II had a significant risk of acute GVHD (6 patients with grade II-IV aGVHD). TRM was moderate with 2 cases in group I (Pneumonitis, GVHD post DLI) and 2 cases in group II (VOD, viral pneumonia). However, after CD34+ selected alloSCT, all surviving patient progressed within 9 months after alloSCT and all except 1 pt. died due to relapse. DLI was given in 6 pt. of group I with limited success (1 stable diesease). In contrast, 8 of 9 surviving patients receiving in group II remained in remission after a median observation time of 10.5 months (range 4 -22 months). Survival differs significantly between both groups (p = 0.004). Multivariate analysis identified intense GVHD prophylaxis as the most important risk factor in our patients. These results support the existence of a graft-versus-lymphoma effect in aggressive lymphoma. T cell depletion (or CD34 selection) of grafts even if followed by T cell add back with DLI is not recommended in patients with poor-risk aggressive NHL

E. Calistri, F. Patriarca, C. Filì, A. Sperotto, A. Geromin, F. Zaja, A. Candoni, S. Prosdocimo, M. Tiribelli, D. Damiani, R. Fanin, Division of Haematology (Udine, I) Donor leukocyte infusion (DLI) is a therapeutic option for patients with acute leukaemia (AL) relapsing after allogeneic stem cell transplantation (allo-SCT), but optimal schedule and clinical response are still unclear. We reported efficacy and toxicity of this form of immunotherapy in 14 AL in relapse after allo-BMT. Median age was 28 years (range 26-66). Nine patients had acute myeloid leukemia (AML); five patients had acute lymphocytic leukemia (ALL). Ten patients received their transplant in complete remission (CR) and the remainder four had resistant disease. Eight patients received peripheral blood stem cells, six bone marrow cells. Donors were HLA identical sibling (10) or matched unrelated donors (MUD) (4). Conditioning consisted of TBI and cyclophosphamide for MUD allo-SCTs, and busulfan and cyclophosphamide for sibling allo-SCTs. Graft-versus-host disease (GVHD) prophylaxis was based on methotrexate and cyclosporine A. The median interval between allo-SCT and relapse was 5 months (range 2-21). All the patients had an overt haematological relapse with involvement of extrahaematological sites in 2 cases. Ten out 14 patients, received chemotherapy before DLI, which consisted of Ara-C and liposomialdaunorubicin (6) or Ara-C and idarubicin (4). The median time between relapse and first DLI was 61 days (range 7-247). We administered an average number of 2 infusions at a median interval of 43 days (range 6-125). The median total number of CD3+ cells was 1 x 10 7 /kg of recipient body (range 0.5-2.5) after MUD allo-SCT and 2.5 (range 0.4-18) after sibling allo-SCT. Nine out of 14 patients (64%) achieved CR after DLI; 5 out of 9 these responsive patients had already obtained their CR after standard chemotherapy. Eight out 9 CR patients but no refractory case developed GVHD, with a clinical picture of grade III-IV acute GVHD (3) or extensive chronic GVHD (5) requiring treatment. At a median follow-up of 8 months (range 2-25) after DLI, 5 out 9 responsive patients were in continuous CR with signs of extensive chronic GVHD, while the other 4 had a relapse within 6 months and died for leukaemia. Three of 5 patients in continuous CR had ALL and 1 of them had an extramedullary relapse. In conclusion, DLI ± chemotherapy achieved CR in 9 of 14 patients (64%) relapsing after allo-SCT. Response was strongly associated with the development of GVHD and could be achieved in 3 ALL relapses.

Evidence of ''graft-versus-tumour'' effect following haploidentical transplantation in a patient with metastatic relapse of Ewing's sarcoma I. Dolgopolov, R. Protsenko, L. Andreeva, V. Boyarshinov, R. Ravshanova, G. Mentkevich, Institute of Pediatric Oncology Bone Marrow Transplant (Moscow, RUS) A 16-years old girl was admitted with a both side metastatic lung relapse of Ewing's sarcoma six months after completion of multimodal therapy receiving for localized left shoulder tumor. She received second line ICE chemotherapy and no decrease in lung tumor size was seen. Lung surgery was performed and 2 and 3 metastases were removed from right and left lung, respectively. No histological response was seen. Within 2 following weeks an appearance of 1 new focus and an increase in size of the remaining metastasis in right lung was observed. She received a reduced-intensity conditioning including Fludarabine 180 mg/m 2 , Busulfan 8 mg/kg and ATG 40 mg/kg. She also received 10.8 Gy of hole lung irradiation as a part of conditioning. Donor was 4/6 HLA mismatched mother. She was transplanted on d0 and +1 with 2.2x10 6 CD34/kg and 6.7x10 8 CD3/kg after 30' incubation of graft with vincristine and methylprednisolone. GvHD prophylaxis consisted of short methotrexate and cyclosporine A. The level of WBC >1.0x10 9 /l was reached on day +12. The patient required no PLT transfusion. A complete donor chimerisme was observed on d+90. In early posttransplant period grade I skin GvHD was seen. A cyclosporine A related encephalopathy occurred on d+60 and the GvHD consisted of MMF and methylprednisolone so far. The recovery of immunity was prompt and fast. The decrease in the size of metastases was observed starting from d+30 and they disappeared by day +90 She is disease-free and well, with no evidence of GvHD 150 days after transplantation. According to our knowledge this is the first report of a successful family mismatched transplantation with evidence of "graft-versustumor" effect in patients with relapsed Ewing's sarcoma refractory to second-line therapy. Supported by Mr. J.Watkins donation.

S. Matthes-Martin, A. Velardi, G. Fritsch, T. Lion, O.A. Haas, C. Peters, H. Gadner, St. Anna Kinderspital, Policlinico Monteluce, Children´ s Cancer Research Institute (Vienna, A; Perugia, I) Relapse rate following allogeneic stem cell transplantation (SCT) for AML varies between 10-20%. Outcome following a second SCT is poor due to a high transplant related mortality or subsequent relapse.

Transplant related mortality following second SCT can be reduced by the use of fludarabine based reduced intensity conditioning regimens. Velardi et al have shown that KIR incompatible NK-cells are able to eliminate recipient hematopoietic cells and thus facilitate engraftment together with graft versus leukemia effect. A prospective study combining SCT from KIR incompatible donors with reduced intensity conditioning for children relapsing after allogeneic SCT was initiated in the St Anna Children's Hospital, Vienna. Two children underwent allogeneic bone marrow transplantation for AML from an HLA-identical sibling donor and HLA-identical unrelated donor respectively. Both children relapsed after bone marrow transplantation (interval 8 and 11 month). Following chemotherapy according to BFM-AML-relapse protocol one child was in complete third remission, one child had persistent blasts in bone marrow. For both children one antigen mismatch KIRincompatible unrelated donors could be identified. (HLAmismatch: Patient 1: Cw *0202*1502; donor 1: Cw*0704*1502. Patient 2: Cw*0701*0702; donor 2: Cw*0602*0702.) Conditioning regimen consisted of Fludarabine 180 mg/m², Melphalan 140 mg/m², ATG and total lymphoid irradiation with 2 Gy. GvHD and rejection prophylaxis consisted of CyA and mmf. The conditioning regimen was well tolerated an toxic side effects did not exceed WHO grade II. Patient 1 received unmanipulated PBSC with a total of 9x10 6 /kg CD34+ , 150x10 7 /kg CD3+ and 3 x 10 7 /kg CD3-/CD56+ cells. The Patient 2 received PBSC following CD3+/CD19+ depletion with 12,3x10 6 /kg CD34+, 0,5x10 7 /kg CD3+, 5x10 7 /kg CD3-/CD56+ cells. In both children engraftment occurred on day +10 with 100% donor chimerism in all cell-lines. Patient 1 experienced GvHD grade III. No GvHD was observed in patient 2. Both children are alive and well in complete remission on day +370 and +355 respectively post second SCT. In patients with relapse of AML following allogeneic SCT a second transplant from KIR incompatible donors with reduced intensity conditioning regimen is feasible. By CD3+/CD19+ depletion instead of CD34+ positive selection (thus infusing a high number of KIR mismatched NK-cells) GvHD can be effectively be prevented and graft versus leukemia effect seems to be preserved.

Efficacy of donor lymphocyte infusion following cytoreductive chemotherapy for relapsed acute leukaemia after allogeneic stem cell transplantation D.-Y. Kim, K.-W. Lee, T. Yun, S.-I. Kim, S.R. Park, J.Y. Paik, I.S. Choi, D.-Y Oh, S.-S. Yoon, J.-S. Lee, S. Park, B.K. Kim, Seoul National University College of Medicine (Seoul, KOR) Objective: Donor lymphocyte infusion (DLI) is used for graftversus-leukemia in the treatment of relapsed hematologic malignancies after allogeneic stem cell transplantation (alloSCT). We investigated the efficacy and toxicity of DLI for relapsed acute leukemia. Methods: From 1998 to 2002, patients with relapsed or persistent disease after alloSCT from HLA-full-matched-donor for acute leukemia were enrolled. Patients were given cytarabine i.v. (100mg/m²/day x7) and daunorubicin i.v. (30mg/m²/day x3) for acute myeloid leukemia (AML); vincristine i.v. (1.4mg/m²/week x2), prednisone p.o. (60mg/m²/day until 3 days prior to DLI), and daunorubicin i.v. (30mg/m²/day x3) for acute lymphoid leukemia (ALL) for cytoreductive chemotherapy. Donor lymphocyte was collected from the same alloSCT donor with a target lymphocyte count of 1x10^8/kg. Immunosuppression after DLI was allowed in case of GVHD. Results: 7 AML and 8 ALL patients were enrolled. Three patients who received non-myeloablative stem cell transplantation (NST) were included. The median age was 36 (17-50). The status of disease at the time of DLI was 'hematologic relapse' in 12, 'molecular relapse' in 1, and 'remission failure' in 2 patients. Except 2 patients, performance status was ECOG grade 1 or 2. Blasts were persistent in peripheral blood in 4 patients after cytoreductive chemotherapy. The median number of total DLI was 2 (1-4) and the median number of CD3+ cells was 1.4x10 8 /kg. Six patients (40%) achieved remission (1 hematologic, 3 cytogenetic, and 2 molecular) and the median duration of remission was 6.3 months (2.5-11.6). After median follow up of 18.1 months (4.5-56.9), 3 patients who achieved remission relapsed and 1 patient died of sepsis during recovery. One AML patient survived for 11.6 months, and one ALL patient, for 15.5 months. The median duration of survival after salvage therapy was 7.0 months (0. 2-15.5 ) in all patients, 2.3 months in ALL, and 7.6 months in AML. Toxicity included 6 acute GVHD (grade I:II:III=3:2:1) which was easily controlled with moderate doses of corticosteroid and cyclosporin. Conclusion: Although the efficacy of DLI needs improvements as a salvage therapy for relapsed acute leukemia after alloSCT, a few patients could be salvaged to become long term survivors who otherwise were in persistent disease.

A. Ackerstein, S. Morecki, Y. Gelfand, L. Dray, R. Or, S. Samuel, C. Nabet, S. Slavin, Hadassah University Hospital (Jerusalem, IL) Graft-versus-leukemia to induce immunologically mediated disease response following allogeneic hematopoietic stem cell transplantation led to the development of non-myeloablative allogeneic transplantation (NST). The success of NST prompted the development of other immunotherapies for patients with relapse of retractory malignancies. The goal of the present study was to evaluate feasibility and possible benefit of mismatched alloreactive lymphocytes for cancer patients resistant to conventional therapies. Twenty-one patients were enrolled: 9 females; 12 males; 1.5-74 (median 52) years. Breast (7), glioblastoma (3), colon (4), thyroid (1), ovary (1), multiple myeloma (MM) (2), carcinoid (1), non-Hogdkin's (1), melanoma (1) . In order to induce transient chimerism in the host, immunosuppressive treatment with Fludarabine was given at a dose of 25mg/m 2 from day-7 to day-3 followed by 2 subcutaneous (SC) injections of IFN alpha in a dose of 3x10 6 IU/day from day-2 to day-1 in an attempt to upregulate immunogenic cell surface antigens. On day 0, lymphocytes obtained from partially HLA matched donor were infused after in vitro activation with rIL-2 for 4 days. On day 1, rIL-2 was administered for 5 consecutive days SC in a dose of 6x10 6 IU/m2 daily.

For patients (5) with localized liver metastases, lymphocytes were injected selectively into the hepatic artery. Median mononuclear cell dose infused 1.4 (range 2-25.5)x10 7 /Kg. Patients were monitored for treatment-related toxicity, chimerism and signs of graft-versus-host disease (GVHD). No major side effects were observed, treatment was well tolerated.

One patient with metastatic colon cancer demonstrated transient engraftment of donor cells and partial response for 16 months. Second patient with metastatic breast cancer had cutaneous GVHD with elevation of liver enzymes and showed no evidence of chimerism 1 month post cell therapy. She was with stable disease for 23 months. Two patients with MM with no evidence of disease for 57, 42 months, respectively. Seventeen patients had progressive disease. We conclude that mismatched allogeneic cell therapy is feasible on an outpatient procedure, may induce transient parking of cells. This approach should be further developed.

G. Pricolo, L. Stani, A. Prudenzano, G. Pisapia, G. Palazzo, B. Amurri, A. Maggi, P. Mazza, P.O."S.G.Moscati" (Taranto, I) Multiparameter immunophenotypic analysis of neoplastic cells has proven to be of great help for the investigation of minimal residual disease (MRD ) in acute leukemias; however, its utility has not been systematically explored in B cell chronic lymphoproliferative disorders. The aim of the present study was to investigate the incidence of the phenotype of B cell chronic lymphoproliferative disorders through the comparison of the phenotypic vs the molecular characteristics of tumor in order to explore the applicability of this strategy for minimal residual disease monitoring. From the 143 patients analyzed 42 corresponded to B-CCL/SLL, 32 to LPL, 37 to FL, 16 to MM, 5 to MCL, 7 to DBCL, and 4 to HCL. The following four-color stainings were systematically applied to leukemic samples: FMC7/CD22/CD45/CD19, CD5/CD23/CD45/CD19, CD103/CD25/CD45/CD19, CD10/CD11c/CD45/CD19, CD56/CD38/CD45/CD19, CD138/CD38/CD45/CD19, CD20/CD23/CD45/CD19 and sKappa/sLambdal/CD45/CD19. The molecular techniques used for practical patient management were largely based on BCL2-IgH rearrangements and IgH oligoclonality identified by consensus PCR strategies . Overall, 80% of the leukemic B cell chronic lymphoproliferative disorders cases displayed aberrant phenotypes at diagnosis with no significant differences between peripheral blood vs bone marrow samples. The most common types of aberrant criteria detected included asynchronous antigen expression (90%) and antigen over-expression (45%); abnormal light scatters were found in 44% of the cases. Only 57% of the patients were characterized by BCL2-IgH rearrangements and IgH oligoclonality ( 19% and 38% respectively ). At the follow-up the detection of the tumor associated phenotype correlated with the molecular data in 86% of patients PCR positive for the IgH oligoclonality, and in 59% of patients PCR positive for the BCL2-IgH rearrangements. In summary, this study shows that immunophenotypic analysis of neoplastic cells is of great help for the investigation of MRD mainly due to the fact that flow S100 cytometry immunophenotyping is more sensitive than morphology in detecting residual leukemic cells and, at the same time,

it is applicable to a high proportion of diseases. Additionally, immunophenotyping has proved to be of utility for evaluating the effectiveness of high-dose chemotherapy and for predicting relapses.

Disease eradication in patients with poor prognosis non-Hodgkin lymphoma by non-myeloablative verus conventional haematopoietic stem cell transplantation M. Mitterbauer, P. Kalhs, U. Jaeger, C. Mannhalter, F. Keil, N. Worel, W. Rabitsch, G. Fischer, K. Dieckmann, I. Simonitsch-Klupp, P. Hoecker, A. Rosenmayr, W. Hinterberger, K. Lechner, G. Mitterbauer, H. Greinix, University Hospital of Vienna (Vienna, A) Allogeneic hematopoietic stem cell transplantation (HSCT) has a curative potential for poor risk non-Hodgkin´s lymphoma (NHL), but high treatment-related morbidity and mortality with conventional high-dose conditioning has remained a major limitation for its application. We report the results of our single center study with 49 consecutive NHL patients with a median age of 39 (range, 29-62), 36 males and 13 females, who underwent myeloablative (n=39) or nonmyeloablative (NMA) (n=10) HSCT between 1987 and 2002. Eleven patients had indolent and 38 aggressive lymphoma. All patients were extensively pretreated with a median of 3 (range, 1-7) chemotherapy regimens and local radiotherapy (n=21). Eleven patients had autologous stem cell transplants prior to HSCT. Twenty-seven patients received bone marrow (BM) and 22 peripheral blood stem cells (PBSC) from sibling (n=38) and unrelated donors (n=11). Myeloablative conditioning consisted mainly of total body irradiation (TBI) of 12-13.2 Gy and cyclophosphamide, NMA of fludarabine and TBI of 2 Gy. Status at transplantation was chemosensitive disease (n=21), chemoresistant disease (n=24) or untreated relapse (n=4). For myeloablative HSCT graft-verus-host disease (GVHD) prophylaxis consisted mainly of cyclosporine (CsA) and methotrexate, for NMA of CsA and mycophenolate mofetyl. All patients surviving 4 weeks had durable donor engraftment. Thirty-five of evaluable 43 patients (81%) attained complete clinical remission 1 (range, 1-5) month after myeloablative (n=28) and 4 (range, 1-9) months after NMA (n=7) HSCT. Minimal residual disease monitoring of the BCL2/IgH translocation (n=6), the immunoglobulin heavy chain (n=8) and the immunoglobulin light chain (n=2) gene rearrangements by PCR in 7 patients after NMA and in 9 after myeloablative HSCT resulted in molecular remission (MR) in 14 patients, in 9 of them, MR was attained after development of GVHD. Progression-free survival (PFS) was 69% after NMA and 34% after conventional HSCT. The actuarial treatment-related mortality (TRM) was 40% for myeloablative and 10% for NMA grafting. In summary, HSCT resulted in long-term clinical and molecular disease eradication. Nonmyeloablative HSCT was associated with lower TRM and higher PFS compared to conventional HSCT and thus, should be evaluated in studies with larger patient numbers and longer follow-ups.

Molecular monitoring of minimal residual disease after reduced-intensity conditioning and allogeneic stem cell transplantation in relapsed chronic lymphocytic leukaemia L. Farina, D. Soligo, E. Rizzo, A. Dodero, A. Locasciulli, R. Scimè, A. Santoro, A.M. Gianni, C. Tarella, M. Lucesole, A. Olivieri, P. Corradini, Istituto Nazionale Tumori, Ospedale Maggiore, Ospedale San Camillo, Ospedale Cervello, University of Turin, Ospedale Le Torrette (Milan, Rome, Palermo, Turin, Ancona, I) Graft-versus-leukemia effect (GVL) after allogeneic stem cell transplantation (allo-SCT) has been already described in chronic lymphocytic leukemia (CLL). Clinical responses can be achieved after cyclosporin withdrawal, donor lymphocyte infusions (DLI), and with the onset of graft-versus-host-disease (GVHD). The slow clearance of minimal residual disease (MRD) after allo-SCT also supports the hypothesis of the presence of an ongoing GVL effect. In order to reduce treatment related mortality (TRM), we devised a RIC program for CLL or small lymphocytic lymphoma patients (CLL=18; SLL=4) having a HLA identical sibling donor. Median age was 55 years (range 41-69); all patients were heavily pretreated and 4 of them had failed a previous autologous transplantation. Before transplant, 13 patients were in partial or complete remission (CR=3; PR=10); the remaining patients were chemorefractory or minimal responders. The conditioning regimens consisted of thiotepa 5-10 mg/kg, fludarabine 60 mg/ms and cyclophosphamide 60 mg/kg (18 patients), and fludarabine-TBI2Gy (4 patients). GVHD prophylaxis consisted of short course of methotrexate and cyclosporin (CSA) for the first group, and CSA plus mycophenolate mofetil for the second one. All patients engrafted; 9 patients were in CR at day + 30 after allo-SCT. The incidence of acute GVHD grade II-IV was 36%; 10 of 20 evaluable patients developed chronic GVHD (limited=7, extensive=3), one was after DLI. At a median follow up of 522 days, 10 patients were alive in CR; 7 patients died, 4 of them of TRM (18%). In 15 of 22 patients a molecular marker based on the rearrangement of IgH genes was generated. Ten patients attaining CR were monitored for MRD by means of nested PCR with patient-specific primers. At a median follow up of 504 days, 5 of 10 patients are alive in clinical and molecular remission and 3 patients died: 2 of them for infections, while they were still in molecular remission. Two patients in CR relapsed after one year, however, they never attained a post-transplant PCR negativity. All the patients attaining molecular remission experienced acute or chronic GVHD. We conclude that RIC allo-SCT can produce clinical and molecular remissions even in chemorefractory patients; further studies using quantitative PCR are ongoing to modulate posttransplantation immunesuppressive therapy on MRD status.

Minimal residual disease in chronic lymphocytic leukaemia after stem cell transplantation. Comparison of ASO-PCR and flow cytometry C. Moreno, N. Villamor, D. Colomer, J. Esteve, F. Bosch, E. Campo, E. Montserrat, Hospital Clinic (Barcelona, E) Stem-cell transplants (SCT) are increasingly used in the treatment of patients with chronic lymphocytic leukemia (CLL). Some studies have shown that minimal residual disease (MRD) status after transplantation correlates with clinical outcome. However, the method of choice to evaluate MRD in patients with CLL undergoing transplantation has not been established. Clonal IgH rearrangement by PCR, that provides a qualitative result, and flow cytometry (FC) are the methods commonly employed for MRD analysis. The development of real time PCR of IgH region using allele-specific oligonucleotides (ASO-PCR) constitutes a highly sensitive and quantitative method for MRD analysis. The primary objective of this study was to compare S101 ASO-PCR and FC in the assessment of MRD in patients with CLL receiving SCT; a secondary objective was to correlate MRD status with clinical outcome. VH sequence of CLL IgH clones from 12 allogeneic-SCT (allo) and 16 autologous-SCT (auto) were amplified and sequenced using family-specific primers of FR1, FR2 and FR3 region. Clone specific primers designed at CDRIII region were obtained in 25/28 patients. 308 DNA samples obtained at fixed intervals after transplant from these 25 patients were analysed by real time ASO-PCR using the Taqman system. In 166 of these samples, MRD was also quantified by FC using triple (e.g., CD20/CD5/CD19) or quadruple (e.g., CD22/CD23/CD19/CD5) antigenic combinations. Sensitivity of ASO-PCR, as determined in three CLL patients, was 1 tumoral cell in 105 mononuclear cells. A very good agreement was obtained in MRD values obtained by . At three months after SCT, MRD was detected by ASO-PCR in all but 3 patients (2 allo,1 auto) with values ranging from 10-1 to 10-5. In follow up samples, increasing levels of MRD were observed in patients submitted to auto-SCT, whereas this pattern was only observed in 1/12 allo-SCT patient. After a median follow-up of 4.65 years (range: 0.5-12), clinical relapse was observed in 8/13 auto-SCT (median of 1.2 years; range: 0.7-6) and in the allografted patient in whom increasing MRD values were detected (4.1 years). In conclusion, ASO-PCR and FC are both useful to monitor MRD in patients with CLL receiving SCT, although FC is slightly less sensitive (10-4) than ASO-PCR (10-5). On the other hand, this study confirms that increasing values of MRD precedes clinical relapse.

CD34+ cell dose, reduced-intensity conditioning and prior chemotherapy: factors with significant impact on the Kinetics of donor chimerism after allogeneic haematopoietic cell transplantation B. Mohr, R. Koch, C. Thiede, F. Kroschinsky, G. Ehninger, M. Bornhäuser, Universitätsklinikum Dresden (Dresden, D) Regular analysis of donor chimerism has been established as a routine method to monitor engraftment of allogeneic hematopoietic cells. The slope of donor cell engraftment in the early phase after transplantation varies between individual patients and transplant procedures. The aim of this study was to define factors which significantly influence the kinetics of donor chimerism. In a retrospective study, the percentage of donor chimerism in peripheral blood measured with sex-chromosome specific probes and fluorescence-in-situ hybridization was analyzed in 184 recipients of allogeneic hematopoietic cells between day 1 and 30. Using a generalized linear model for longitudinal observations, the dose of CD34+ cells infused had a significant impact on the slope of donor chimerism. In multivariate analysis, cell doses of 2-8 x 10 6 /kg (p=0.003) and < 2 x 10 6 /kg (p < 0.0001) were associated with slower increase of donor chimerism compared to > 8.0 x 10 6 CD34+ cells/kg. In addition, fludarabine-based reduced intensity conditioning (p<0.0001) resulted in a significant delay of donor cell increase compared to standard conditioning therapy (p<0.0001). The application of chemotherapy before the start of conditioning (p=0.003) and the use of ATG were associated with a faster increase of donor chimerism (p=0.003). The factors which showed to have some impact on the engraftment kinetics in this analysis may be useful to assess whether the increase of donor chimerism is within the expected range for an individual patient.

Sensitive and quantitative chimerism analysis in ALL patients with real-time PCR M. Uzunel, U. Sundin, J. Mattsson, M. Schaffer, D. Hauzenberger, O. Ringden, Huddinge University Hospital (Stockholm, S) Introduction: Today, the most widely used methods for chimerism analysis are based on PCR analysis of Short Tandem Repeats (STRs) and Variable Number of Tandem Repeats (VNTRs). However, the sensitivity limit of these methods to detect the minor cell population is usually 1-5%, which may not be sensitive enough to predict relapse after SCT in patients with ALL. Therefore, we wanted to evaluate if a more sensitive and quantitative chimerism method could be more useful for predicting relapse. Patients and Methods: Chimerism analysis was performed with Realtime PCR (ABI 7000 SDS) using Single Nucleotide Polymorphisms (SNPs) as polymorphic markers. Eleven biallelic genetic systems were used for screening to find an informative marker. Quantification was performed with the delta-CT method using GAPDH as a reference gene and the pre-SCT patient sample as a calibrator. Peripheral blood (PB) and bone marrow (BM) samples from 20 ALL patients were analyzed at different time points after allogeneic SCT. For comparison, the samples were also analyzed with the VNTR method. Results: Using dilution series, one patient cell could be detected among 10 000 donor cells. In paired BM and PB samples, the level of recipient cells was a median of 17 times (range 1,14-3400) higher in BM as compared to PB. A high degree of mixed chimerism was found after SCT. In patients without relapse, low level (<0.1%) of patient cells could be detected in PB up to one year after transplant. In patients with relapse, however, high (>0.1%) or increasing levels of patient cells preceded the hematological relapse with a median of 2 (range 0-6,7) months. This was earlier than detected with the VNTR method, median 0 (range 0-3,4) months. Conclusion: Using a new sensitive chimerism method, we could detect low level (<0.1%) of persistent patient cells in ALL patients in remission for almost one year after transplant. These levels were not detected by the conventional PCR method using VNTRs as polymorphic markers. The Realtime PCR based chimerism method is associated with high sensitivity and accurate quantification and makes it possible to predict relapse earlier than before.

Cell lineage specific chimerism in post-haematopoietic stem cell transplant patients S. Adams, P. Veys, J. Hollifield, B. Gaspar, Great Ormond Street Hospital for Children (London, UK) Monitoring chimerism levels post haematopoietic stem cell transplantation (HSCT) has rapidly become a standard test for many centres over the past few years. Methods for providing measurement of donor engraftment levels have become more sophisticated during this time, with genotyping based on fluorescent detection of short tandem repeats (STRs) of DNA the preferred method for many laboratories. STRs contained in a number of alleles are PCR amplified and are then genotyped to generate donor and recipient peaks that can be analysed using computer software. The level of discrimination between donor and recipient genotype profiles depends on the number of informative STR loci used and thus the greater the number of STR loci amplified the greater the chance of obtaining donor/recipient specific peaks. The test requires only a few nanograms of DNA for multiplex PCR amplification leading to the possibility of separating out specific cell fractions from the whole blood samples prior to analysis and providing answers to many scientific and clinical questions regarding HSCT. For example S102 the use of low intensity conditioning (LIC) HSCT for higher risk patients at our centre (both for haematological malignancy and congenital immunodeficiencies) has increased over the last few years. The use of LIC HSCT often results in mixed chimerism of donor and recipient cells existing in a state of host and graft tolerance. However what is unclear is the relative contribution of donor cells to the different T, B and myeloid cell lineages. This has important implications for certain congenital immunodeficiencies where engraftment of donor cells in multiple cell lineages may be required for disease correction long term. Examples of the use of cell-separated chimerism analysis in our centre are provided in table 1. In P1 and P2 -who received unconditioned HSCT -there is no evidence of B cell engraftment. In P3 and P4 (who had undefined SCID) conditioned HSCT have led to functional T cell recovery but decreased engraftment in other lineages. However this is sufficient for correction of the clinical phenotype. In P5, who had Wiskott-Aldrich syndrome (WAS), decreased engraftment in the myeloid cells may have functional consequences given the multi-lineage defects in WAS. In P6, who has a neutrophil disorder, conditioned HSCT has led to total engraftment in the neutrophil fraction but decreased engraftment in other cell lineages.

ChimerTrack: quantification of STR-based chimeric status using an interactive software tool D. Kristt, J. Stein, I. Yaniv, R. Narinsky, T. Klein, Rabin Medical Center, Schneider Children´s Medical Centre of Israel (Petach-Tikva, IL) Chimerism analysis using short tandem DNA repeat (STR) analysis is a powerful tool for post-hematopoietic stem cell transplant (HSCT) monitoring of engraftment. To help resolve issues relating to the appropriate application and standardization of this technology in a clinical setting, we have reviewed our experience with computer-assisted chimerism estimates based on STR analysis. Twenty children received HSCT's for malignant (11) or non-malignant (9) diseases. DNA was extracted from peripheral blood lymphocytes and STR's were amplified using a commercially available multiplex PCR forensic kit. Electrophoresed PCR products were analyzed using the Genescan® program. Our ChimerTrack software computed % donor chimerism from data interactively transferred from the Genescan program on an allele-by-allele basis for each informative locus. The software generated graphic and tabular readouts of the average levels of donor signal as measured at three informative loci, reflecting the current and previous assays performed in each patient. Initial evaluations were often performed in the very early stages of engraftment, when the patient's total white blood cell count was less than 500/ul. Complete donor chimerism was found in 16 patients at some time following transplant and was sustained for 1-2 yrs posttransplant in 11 patients. Five patients had more variable courses, with 4 patients experiencing stable mixed chimerism of between 50 -100% donor signal. Mixing experiments showed that the sensitivity of this assay is approximately 5%; accuracy levels were more variable, reflecting allelic and technical factors. The modest sensitivity and accuracy levels of this assay suggest that this semi-quantitative technique provides clinically relevant data regarding long-term engraftment trends following HSCT, but that other assays should be utilized to detect very small amounts of residual malignant disease during follow up. This STR assay as analyzed using ChimerTrack software is an effective tool for serial semi-quantitative evaluation of chimeric status following HSCT, and is recommended for laboratories involved in post-transplantation monitoring.

Chimerism analysis using quantitative real-time PCR. A new method for clinical use? M. Schaffer, M. Uzunel, D. Hauzenberger, U. Sundin, Karolinska Institutet (Stockholm, S) Hematopoetic stem cell transplantation is a well-established method to treat patients with several malignant or non-malignant hematological disorders. The most common treatment failures are GVHD, graft rejection and relapse. The aim of post transplantation monitoring is to try to predict these negative events. During the last years, chimerism analysis has become a sensitive and important method to investigate if the patients cells post transplantation is of donor stem cell origin. Careful monitoring of post transplant samples makes it possible to predict relapse. Conventional methods for chimerism analysis includes Fluorescent in situ hybridisation (FISH) and polymerase chain reaction (PCR) amplification of Variable Numbers of Tandem Repeats (VNTRs) or Short Tandem Repeats (STRs). The drawback of these methods is the level of sensitivity. It is relatively low, usually between 1-5%, which might be too insensitive for accurate detection of relapse. For that reason we wanted to evaluate if a new and more sensitive quantitative technique could be used for chimerism analysis. We performed chimerism analysis with Realtime PCR (ABI 7000 SDS). Single Nucleotide Polymorphisms (SNPs) were used as polymorphic markers. In order to find an informative marker, patient and donor samples were screened with 19 primers and probes for 11 biallelic loci. Quantification was performed using the delta-CT method with GAPDH as a reference gene. DNA samples from patients and donors were taken before transplantation. Post transplantation samples were separated into three cell populations (CD 19, CD 3, CD 33) by the use of immunomagnetic beads and thereafter DNA was extracted. So far in this ongoing study eight patients have been monitored with the Realtime PCR method and the results are compared to our routinely performed STR-PCR method. In addition serial dilutions of mixed DNAs were performed. In the mixed DNAs a level of ‹0,1% could be detected. In the patient samples we detected low level of recipient cells previously not detected by the STR-PCR method. In conclusion, Realtime PCR gives a more accurate quantification than the STR and VNTR methods, is less labour intensive and also a very reliable and sensitive method for chimerism analysis and therefore well suited for clinical use.

In vivo purging by campath 1H: molecular quantitative evaluation of minimal residual disease in CLL patients resistant to fludarabine S. Galimberti, N. Cecconi, G. Cervetti, S. Pacini, F. Guerrini, C. Manetti, R. Riccioni, A. Azzara, G. Carulli, M. Petrini, Ospedale S.Chiara (Pisa, I) Notwithstanding the novel adopted therapies for CLL resulted in higher hematological response rates, the complete eradication of disease rarely occurs. Campath 1H seems to be extremely effective also in pre-treated patients, resistant to fludarabine, and has been reported to induce 33% of PCR-negativities. In this study, we employed a molecular semi-quantitative PCR method to assess the capacity of Campath 1H to induce PCRnegativities in eight patients already resistant to fludarabine. IgH rearrangement was co-amplified with a housekeeping gene and fluorescent PCR products were analyzed on a DNA automatic sequencer. Each patient was evaluated at diagnosis, after S103 fludarabine and after Campath 1H. The median interval between the last therapy course with fludarabine and the start of Campath 1H was 14 weeks. Enrolled patients received subcutaneous doses up to 10 mg, three times at week for six weeks, with a median dose of 180 mg. For each patient it was possible to find a specific clonal IgH rearrangement at diagnosis. After 6 cycles with fludarabine, only one patient (12.5%) achieved molecular remission and in other two cases IgH levels decreased by <1 log. At the beginning of Campath 1H administration, all patients were PCR positive, including the one previously found to be negative. At the end of treatment, 5 patients achieved molecular remission (62.5%), four of them within 3 months. Moreover, in the other three PCR-positive cases, minimal residual disease remained stable during the entire molecular follow-up. Three patients were mobilized with Ara-C: in one of them the harvested CD34+ precursors were PCR-negative. The third patient did not mobilize, but its bone marrow appeared PCR-negative. With a median follow-up of 36 months, we observed 72% of hematological responses, with 43% of CR. A significant reduction of lymph node and spleen diameters was noted in the 40% of patients. All cases were evaluable for toxicity: a grade-2 skin reaction in the site of the subcutaneous injection and fever were observed in four cases. Two patients developed grade 3 neutropenia and two cases hemolytic episodes. Three patients showed CMV and one VZV reactivation. These results show that Campath 1H is a quite safe therapy for CLL patients. Interestingly, it resulted active also in patients resistant to fludarabine and seems to represent an efficacious in vivo purging tool, either by significantly reducing the MRD or improving molecular remission rates.

Real-time PCR SNP method for the monitoring of chimerism after allogeneic stem cell transplantation R. Eshel, O. Vainas, E. Naparstek, Tel Aviv Sourasky Medical Center (Tel Aviv, IL) Chimerism analysis after allogeneic stem cell transplantation (SCT) is an important diagnostic tool for the documentation of engraftment, early detection of graft failure and recurrence of the basic disease. It is particularly important when using reduced intensity preparatory regimens. Mixed chimerism may reflect graft failure or the presence of residual disease and indicate imminent relapse. Such findings often require therapeutic intervention, such as the administration of donor lymphocytes. The currently used techniques for documentation of chimerism utilize polymorphic DNA sequences, such as the short tandem repeats (STRs), that allow semi-quantitative or quantitative analysis of engraftment. However, its relatively low sensitivity of about 5% is inadequate for early diagnosis of relapse, when medical intervention is urgently needed. Therefore more accurate methods for detecting chimerism and particularly minimal residual disease are required. We have developed a quantitative, real-time PCR method based on the detection of allele specific, single nucleotide polymorphism (SNP). We identified distinguishable allele specific SNP loci for donor/recipient pairs, and designed primers that can selectively detect the recipient specific allele using real time PCR. To assess the sensitivity of this method, we used serial dilution of mixed DNAs and showed that the recipient's DNA could be detected at a concentration as low as 0.6-0.7%, thus exceeding by far the sensitivity of the currently used STR assay. Moreover, using patient-specific SNP on archived DNAs, we were able to demonstrate mixed chimerism on samples that previously showed only donor DNA. Thus, our preliminary results indicate that patient specific SNP real-time PCR offers a reliable and highly sensitive method not only for the monitoring of hematopoietic chimerism in patients after allogeneic SCT, but also for the early detection of minimal residual disease, undetected by the current STR method.

Long-term follow-up of PCR status after autologous transplantation during first remission in patients with Bcl-2/IgH positive follicular lymphoma and bone marrow infiltration at diagnosis T. Papajík, S. Vlachová, R. Kubaláková, L. Raida, J. Vondráková, S. Skranc, M. Jarosová, I. Skoumalová, K. Indrák, University Hospital Olomouc (Olomouc, CZ) Although a lot of patients with advanced follicular lymphoma achieve a complete remission (CR) with conventional treatment, most of patients ultimately relapse. Recurrent disease is the result of residual lymphoma cells persistence and their further multiplication in a patient´s organism. Now, due to sensitive PCR detection of hybrid bcl-2/IgH(JH) sequence we can better describe residual disease status in patients with initial bone marrow (BM) involvement and thus we usually detect residual lymphoma cells in BM of all patients treated with standard chemotherapy. Recently, some authors confirmed that patients who achieved and sustained CR at a molecular level with some newer therapeutic approaches have better failure free survival (FFS) and may have better overall survival (OS) than those who never achieved such a response. We analyzed 12 patients with advanced high-risk follicular lymphoma and BM involvement detected by the use of PCR assay at the start of treatment. After first line chemotherapy only 2 patient had CR with complete molecular response, both received intensive initial treatment combined with infusions of rituximab. 11 patients harvested adequate number of peripheral blood stem cells (PBSC) after chemotherapy and G-CSF administration. In 2 patients were PBSC harvests PCR bcl-2/IgH negative, whereas in other 4 patients were PBSC harvests PCR positive. All 11 patients underwent high-dose chemotherapy (BEAM) with PBSC support. 8 patients achieved clinical and molecular CR, 2 patients had residual BM infiltration documented by PCR assay and 1 patient is short after autologous transplantation and will be eligible for PCR detection later. The two patients with residual disease attained clinical and molecular CR after the administration of rituximab. 3 patients relapsed 24, 25 and 61 months from diagnosis. The median follow-up of the 7 remaining patients who are still in molecular CR after autologous transplantation is 27 months (range 13 -81) from diagnosis. The molecular response is an important factor for the FFS in patients with advance high-risk FL and BM infiltration. High-dose therapy even with the reinfusion of PCR positive PBSC harvests can lead to long-lasting molecular CR. The administration of anti-CD20 antibody before PBSC harvesting or after autologous transplantation in patients with BM residual disease becomes standard therapeutic option. hematological toxicity profile, dose-escalated intravenous (i.v.) treosulfan combined with fludarabine was evaluated in 3 cohorts of > 15 pts with hematological malignancies at high-risk of organ toxicity and otherwise not amenable to standard conditioning. Preparative regimen consisted of treosulfan (10 g/m²: 20 pts; 12 g/m²: 18 pts; 14 g/m²: 17 pts) given on day -6, -5, -4 and fludarabine (30 mg/m² i.v.) day -6 to -2. No prophylactic anticonvulsive treatment was given. Pts with MUD/1misMRD (28/1) received rabbit-ATG (2 mg/kg) on day -3, -2, -1. Unmanipulated BM or PBSC was transplanted on day 0. GvHD prophylaxis consisted of short course MTX and standard dose CsA. A total number of 55 pts were included (19 AML, 10 MM, 8 NHL, 7 MDS, 6 CML, 3 CLL, 1 ALL, 1 MH), median follow-up: 8.9 months (range 2.5 -19.9). Leukocyte engraftment was reached at a median of 13 d (range 10 -24), platelet engraftment at a median of 13 d (range 9 -36). No primary, but 1 secondary graft failure was reported (CML, 2. CP with rapid disease progression shortly after transplantation). Complete donor chimerism was reached by day 28 in 77 % of 55 pts. So far (September 01, 2003) , 15 pts died (disease progression: 5, sepsis: 4, aGvHD: 3, myocardial infarction: 1, EBV infection: 1, cerebral bleeding: 1 (TRM rate: 27 %)). 11 pts (20 %) relapsed (5 AML, 2 CML, 2 NHL, 1 MDS, 1 CLL). Reported CTC °III/IV adverse events (preliminary data) included increased liver transaminases, but no VOD, fever/infection, mucositis (only 4 pts), diarrhea, hyper-/hypotension, renal failure (3 reversible, 3 in context with multiorgan failure). Acute GvHD (°II -IV) occurred in 45 % (° II to IV) and chronic GvHD in 57 % of pts so far. 12 months Kaplan-Meier estimates for TRM, overall and progression-free survival were 21%, 69% and 52%. Considering the poor prognosis of the study population, treosulfan-based conditioning was demonstrated to be safe and efficient in all 3 dose levels tested. Due to these promising results, new phase II protocols in pts with AML and MDS will be initiated.

Chemo-based (reduced intensity or busulphan-based myeloablative) conditioning preserves the in vitro growth capacity of bone marrow stroma, which remains of host origin, compared to TBI based conditioning A. Spyridonidis, T. Küttler, E. Sammek, M. Waterhouse, K. Potthoff, M. Egger, H. Bertz, J. Finke, University of Freiburg (Freiburg, D) The ability of BM samples to generate confluent stromal layers in long-term BM cultures (LTBMC) was used as surrogat assay in order to determine the in vivo toxic effects of different conditionings on stroma. Here, 32 pts receiving a fludarabinebased RIC regimen were compared to those in a control group of 23 pts treated with standard TBI-based (12 Gy fractionated) or busulphan based myeloablative (BuCy) conditioning. BM was aspirated before conditioning (17 samples, 58% pos cultures(+)), and at d30 (47%+) and/or at d100 (25% +). RIC or BuCy did not alter the ability of BM to generate stromal layers both early (d30, 75%+ vs 50%+, ns) or late (d100, 80%+ vs 50%+, ns) after HSCT as compared to pre-HSCT studies (66,6%+ / 40%+). In contrast, TBI-based HSCT resulted in a significant impairment of BM to generate stromal layers as evaluated up to day 30 post-HSCT (14%) and compared with pre-HSCT studies (66%) or when compared with the d30 RIC (75% vs 14%, p<0,05) or d30 BuCy (50% vs 14%, p<0,05). In univariate analysis, TBI conditioning was the only factor found to predict stroma growth pattern up to day 100 post transplant (p<0,05). Age, remission status before HSCT, underlying disease, sibling vs unrelated donor, severe acute GvHD or CMV reactivation did not correlate with the results of the stroma cultures. Neither within the RIC group nor within the BuCy or TBI group could stroma growth predict the tempo of neutrophil or platelet recovery. Mean time to confluence cell composition of the stromal layers was the same in all cultures studied. We analysed the ex vivo generated stromal layers from 5 female, RIC treated, sex-mismatched PBSC transplanted pts for the presence of stromal cells of donor origin by double FISH-Y / Vimentin (Vm) or von Willebrandt (vW) fluorescence stain. BM was aspirated at d30 (1 case), d100 (2), d200 (1) or d300 (1) post-HSCT. Stromal layers were highly depleted from CD14+ macrophages or other CD45+ hematopoietic cells as evidenced by immunofluorescence studies. All Vm+ or vW+ stromal cells were found to be Ychromosome negative. We conclude that chemotherapy based conditioning preserves stroma damage as compared to TBI treatment. The novel, donor derived, hematopoiesis in the allogeneic RIC transplanted pts is supported and maintained by a host derived BM stromal microenviroment.

Reduced-intensity allogeneic transplants: an update report from the Australasian Bone Marrow Transplant Recipient Registry I. Nivison-Smith, D.D Ma, K. Bradstock, J. Szer, A. Dodds, Australasian BM Transplant Recipient Registry, St. Vincent´s Hospital Sydney, Westmead Hosptial, Royal Melbourne Hospital (Sydney, Melbourne, AUS) We at the Australasian Bone Marrow Transplant Recipient Registry (ABMTRR), have recorded an increase in the number of reduced intensity allogeneic haemopoietic stem cell transplants (RITx) in Australia and New Zealand in recent years, rising steadily from 24 registrations in 1999 to 88 in 2002. 151 of the total 210 adults cases were first transplants and 59 were second or subsequent transplants. Indications for transplant were myeloma (42), non-Hodgkin's lymphoma (37), AML in first remission (23) and beyond (35), CLL (16), CML in first chronic phase (5) and beyond (12), ALL in first remission (3) and beyond (8), and renal cell carcinoma (7). The median age of recipients was older than full intensity allotranaplants (FITx) carried out over the same period (50 versus 40, n = 1030). 82.5% (173) of the cases were 6/6 HLA-identical siblings with smaller numbers being HLA-identical unrelated volunteers, 5/6 matched relatives, HLA-identical relatives and 5/6 matched unrelated volunteers (12, 2.5, 2 and 1% respectively). There were 60 deaths in first year post transplant. 21 were due to relapse or persistent disease, 39 were transplant related including 12 from infection and 10 from GVHD. Thus, the estimated one-year transplant-related mortality rate was 19% as compared to 18% for FITx. The causes of death were relapse (19), persistent disease (13), infection (18), acute GVHD (10), graft failure or rejection (4), organ failure (4) and other causes (5). Disease free survival probability at three years was 56% for the whole group with a median follow-up of 16 months for surviving patients. Three-year DFS was higher for first transplants (59%) than for second or subsequent (47%) and higher for recipients from 6/6 HLA-matched siblings (57%) than for other donor types (48%). Compared to myeloablative allografts carried out over the same period, three-year DFS was higher for non-myeloablative procedures for myeloma (53% vs 38%), NHL (72% vs 63%), AML past CR1 (52% vs 38%), and equivalent for AML in CR1 (67% for both).

In conclusion, the Australasian registry data shows a trend towards increasing numbers of RITx being performed and, in spite of the older patient population, the estimated mortality rates are comparable or better than standard myeloablative Tx for the same period, however, longer follow-up is needed.

Reduced-dose conditioning followed by allogeneic stem cell transplantation induces stable long-term remission in highrisk patients suffering from acute myeloid leukaemia L.O. Mügge, K. Weiß, K. Wirth, K. Schilling, S. Scholl, M. Charbel-Issa, K. Höffken, H.G. Sayer, Friedrich-Schiller-Universität (Jena, D) It has been been shown that reduced dose conditioning regimens reduce transplant related mortality and are effective in remission induction. But until the long term effectiveness of such protocols is proven, the use of reduced dose conditioning has been restricted to patients at increased risk of therapy related complications.

Here we present the long term follow up data of 31 AML patients including 3 MDS/RAEB patients not eligible for conventional conditioning due to age, impaired organ function or severe infections. 24 of these patients had an advanced disease status ( 2 pt. 2nd CR, 15 pt. PR, 7 pt. refractory). The mean observation time has been 26 months (4-50). In all patients conditioning consisted of fludarabine (30 mg/m 2 per day, i.v.) day -10 to -5, busulfan (4 mg/kg body weight per day, p.o.) day -6 to -5 and anti-lymphocyte-immunoglobuline (10 mg/kg bw per day, i.v.) day -4 to -1. A mean of 7.6x10 6 CD34+ unmanipulated stem cells (range 1.93-14.26x10 6 ) was transplanted at day 0. Ten of the patients received stem cells from HLA-matched related donors, 21 patients from HLA-matched unrelated donors (MUD). GVHD prophylaxis consisted of Cyclosporine A in 31 patients plus mycophenolate mofetil (MMF) in 12 MUD-patients. G-CSF was given to all patients to promote engraftment. After a mean observation time of 26 months (4-50) an overall survival (OS) of 74.2 % (23/31), a relapse rate (RR) of 25.8% (8/31) and a toxicity related mortality (TRM) of 6.5% (2/31) can be noted. Patients in first complete remission showed an OS of 85.7% (6/7) and a RR of 28.6 % (2/7). OS of patients in an advanced status was 70.8% (17/24), the RR 25% (6/24). With regards to the donor source, OS and EFS of patients with HLA-identical sibling donors was 100% (10/10). Patients with HLA-identical unrelated donors showed an OS of 61.9 % (13/21) and a RR of 38.1% (8/21). 9.5 % (2/21) of them died due to TRM and 28.6 % (6/21) as a result of their relapse. Long-term observation showed that relapse occurs within the first 15 months after transplantation. After this period OS and EFS have not decreased further so far. In conclusion, reduced dose conditioning with fludarabine, busulfan and ATG is a promising alternative for patients who are not eligible for conventional conditioning even in patients with an advanced status of AML. This holds especially true if a HLAidentical sibling donor is available.

H.J. Kolb, B. Simoes, I. Bigalke, C. Schmid, M. Schleuning, J. Tischer, G. Ledderose, University of Munich, GSF-National Research Center f. Environment/Health (Munich, D) Stem cell transplants from family members sharing one HLAhaplotype and differing in 0 -3 HLA-antigens (A, B, DR) of the second haplotype carry a high risk of rejection and graft-versushost disease. Pretransplant conditioning consisted of total body irradiation (TBI), buffy coat transfusion from the prospective marrow donor, antithymocyte globulin and cyclophos-phamide. Post-transplant cyclosporine A and methotrexate was given routinely for prevention of graft-versus-host disease (GvHD). Here we report on the results of reduced intensity (RIC) of TBI instead of 12 Gy (SIC) and the use of CD6-negative mobilized blood cells on day 6 in addition to a marrow graft on engraftment and GVHD. 55 patients were grafted with marrow and CD6-negative MBC for advanced leukemia (refractory AML 27 patients, ALL 14 pats., CLL 2 pts., MDS 1 pt., advanced CML 2pt., refractory NHL 9 pt.). HLA-antigen differences of the donor (host-versus-graft direction) involved 3 antigens in 19, 2 antigens in 20, one an-tigen in 10 and no antigen in 3 pts. In the GVH direction 3 antigens were involved in 15, 2 antigens in 16, one antigen in 15 pts. and no antigen in 5 pts. 36 patients were male and 19 female, the median age was 37 years (range 17 -61). Four pts. died early and were excluded from further evaluation. 19 patients were given 4 Gy TBI, 36 patients 12 Gy TBI. All patients showed full engraftment. GvHD occurred in 40 patients, it was mild in 10, moderate (grade 2) in 10, moderately severe (grade 3) in 11 and severe in 4 patients. The incidence of GVHD did not differ after reduced intensity conditioning from full intensity conditioning. Most remark-able was the absence of GVHD in two patients with a better depletion of CD6-positive cells using directly coated immunomagnetic beads. The 2 year actuarial survival of the whole group is 22 %, the one year survival of patients with reduced intensity conditioning 21 % , that of full intensity conditioning 34%. Of the 19 patients given RIC 2 died early, 5 of pro-gression or relapse of the disease, 2 with infection, 2 with cerebral hemorrhage and EBV-lymphoma resp. and 8 are alive, two patients with recurrent disease. We conclude from this study that reduced, non-myeloablative conditioning is sufficient for full engraftment of hap-loidentical transplants, if marrow is supplemented with CD6-negative mobilized blood cells. Better depletion of CD6-positive T cells prevents GVHD more efficiently. Survival may be improved by transplantation at an earlier disease state and reinforcing immune recovery.

A reduced-intensity conditioning regimen for elder patients with myelodysplastic syndrome or acute leukaemia employing radioimmunotherapy with an Yttrium-90 or Rhenium-188 labelled monoclonal CD 66-antibody M. Ringhoffer, M. Wiesneth, S. von Harsdorf, T. Zenz, R. Schlenk, S.N. Reske, H. Doehner, D. Bunjes, University of Ulm (Ulm, D) Objective: The high treatment related mortality due to the organ toxicity of the conditioning regimen and the significantly higher incidence of graft versus host disease (GVHD) is a major obstacle to allogeneic stem cell or bone marrow transplantation in elder patients. We employed radioimmunotherapy with an anti-CD 66 (a,b,c,e)-antibody labelled either with Rhenium-188 or with Yttrium-90 as part of a reduced intensity conditioning regimen, which was followed by a T cell depleted stem cell graft. Patients and Protocol: We treated 17 patients (pts) (13 males and 4 females) with a median age of 64 years (56-67y). The pts were suffering from AML>CR1 (n=7), AML in CR1 (n=7), T-ALL CR1 (n=2) and MDS (RCMD, n =1). The median follow up is 16 months. 10 pts were transplanted from a HLA-identical sibling donor and 7 pts from a matched unrelated donor (MUD). We used radioimmunotherapy with the isotope labelled monoclonal anti-CD 66 antibody in every patient. The pts transplanted from a HLA identical sibling were additionally conditioned with 180 mg/m(2) fludarabine plus 40 mg/kg ATG, the pts transplanted from a MUD received 150 mg/m(2) fludarabine plus 140 mg/m(2) melphalan. GVHD prophylaxis was performed by T cell depletion (TCD) with Campath-1H alone in the pts transplanted from a sibling donor, whereas additional Cyclosporine A was in pts transplanted from a MUD . Results: We achieved a favourable dosimetry in all pts. The antibody provided a mean dose of 20.6 Gy to the bone marrow, 5.6 Gy to the kidney and 1.9 Gy to the whole body. All pts engrafted. Grade II -IV acute GVHD occurred in 2 pts (12%) and chronic GVHD in 1 pt (6 %). 3/17 pts died from transplant related mortality (TRM, 18%). The relapse rate was 29%, most relapsed patients were suffering from advanced disease (AML > CR1, n=4, AML CR1, n=1 and MDS n=1). 3 pts received donor lymphocytes for the treatment of relapse. After the median follow up of 16 months 11/17 pts (65%) are alive and 9/17 are free of disease (53%). Conclusion: Reduced intensity conditioning by the more selective irradiation approach of radioimmunotherapy is feasible in elder patients suffering from acute leukemia or MDS. While it is too early to finally assess the therapeutic efficacy, the preliminary data show encouraging results in terms of organ toxicity, engraftment, incidence of GVHD and TRM.

High dose of in-vitro T-cell depleted CD34+ donor peripheral stem cells after non-myeloablative conditioning accelerates platelet recovery but does not establish complete donor chimerism R.M.Y. Barge, W.A.F. Marijt, S. Osanto, C.W.J. Starrenburg, M.R. Schaafsma, W.M. Smit, W.E. Fibbe, J.H.F. Falkenburg, R. Willemze, Leiden University Medical Center, Medisch Spectrum Twente (Leiden, Enschede, NL) In-vitro T-cell depleted alloSCT using Campath 1-H incubation following nonmyeloablative conditioning with fludarabine (50 mg/m 2 orally, 6 days), ATG (10 mg/kg, 4 days) and busulphan (3.2 mg/kg i.v., 2 days) appears to be a feasible treatment modality with a low toxicity profile (Exp Hemat 2003,31:865-872) . We investigated whether transplantation with a CD34+ dose of > 10 x 10 6 cells/kg was associated with faster platelet recovery and with establishment of complete donor chimerism. Presently 34 patients with leukemia (15), lymphoma (10), multiple myeloma (1), severe aplastic anemia (1) and metastatic cancer (7) were transplanted with high dose T-cell depleted peripheral stem cells (target of 15 x 10 6 CD34+ cells/kg) from an HLA-identical sibling (27) or unrelated donor (7). The median CD34+ dose administered in these patients was 14.1 x 10 6 cells/kg (range: 5.8 -24.9 x 10 6 ). All patients had rapid engraftment of donor cells. We compared neutrophil and platelet engraftment rates in 25 HLA sibling transplant patients receiving < 10 x 10 6 CD34+ cells/kg (n=6) with those receiving doses of > 10 x 10 6 CD34+ cells/kg (high dose group, n=19). Patients receiving the high dose of allogeneic stem cells showed significant faster platelet recovery to > 20 x 10 9 /l than the patients receiving < 10 x 10 6 CD34+ cells/kg (5 versus 14 days; p=0.03). In the high dose group a non-significant faster recovery was observed regarding the neutrophil recovery to > 0.1 x 10 9 /l (9 versus 5.4 days; p=0.124). Sequential chimerism studies showed persistent donor chimerism with a median of 95% donor cells (range 74-100%) at 3 months after alloSCT. Complete donor chimerism was accomplished only in 4 patients. No correlation was observed between CD34+ dose and percentage of donor chimerism at 3 months. Our data show that HLA-identical sibling transplantation with a high dose (> 10 x 10 6 ) of in-vitro T-cell depleted CD34+ cells/kg following nonmyeloablative conditioning is associated with faster platelet but not granulocyte recovery. The establishment of complete donor chimerism was not enhanced by transplantion a dose of > 10 x 10 6 CD34+ cells/kg. We conclude that the clinical benefit of administration of a high dose in-vitro T-cell depleted stem cell dose following nonmyeloablative conditioning is limited.

Thrombotic microangiopathy after non-myeloablative stem cell transplantation N. Worel, H.T. Greinix, C. Buchta, M. Mitterbauer, F. Keil, P. Hoecker, P. Kalhs, University Hospital (Vienna, A) Thrombotic microangiopathy (TMA) is a well known complication after allogeneic stem cell transplantation (SCT), especially in heavily pretreated patients and patients receiving unrelated donor grafts and cyclosporin A and steroids for GvHD prophylaxis. In an effort to reduce the transplant-related mortality associated with allogeneic SCT, dose-reduced conditioning regimens have been developed and are currently being explored in patients not eligible for conventional SCT due to age or comorbidity. We analyzed the influence of ABO-incompatibility on development of TMA in patients undergoing nonmyeloablative SCT. Between April 1999 and August 2003, 52 consecutive patients (ABO-identical n=30, ABO-mismatch n=22) underwent nonmyeloablative SCT from HLA-identical siblings (n=31) or unrelated donors (n=21). The preparative regimen consisted of fludarabine (30mg/m²) and 2 Gy total body irradiation, CsA and mycophenolate mofetil were used for GvHD prophylaxis. TMA was defined as haemolytic anemia with occurrence of fragmented red cells, renewed thrombocytopenia, increase in LDH levels with or without renal impairment and/or fluctuating central nervous system abnormalities. Seven of 52 (13%) patients developed TMA, 6/22 (32%) patients of the ABO-mm group and 1/30 (3%) with an ABO-identical donor. TMA was associated with GvHD (n=1), infections (n=3), infections and GvHD (n=3). The median time of onset was 101 days (range 9-906) post transplantation and resolved after CsA discontinuation and plasma exchange in 4 (57%) patients, whereas 3 died on days 35, 113 and 320, respectively. Probability of TMA projected at 2.5 years after SCT was 27% for the whole cohort, 17% for patients with an ABO-identical and 34% for patients with an ABO-mismatch donor (p=0.02). Significant risk factors for the development of TMA were ABO-mismatch (p=0.012) and an unrelated donor (p=0.008). TMA after allogeneic SCT following dose-reduced conditioning regimens is a severe clinical problem. Whether besides the known risk factors heavy pretreatment, unrelated donor SCT, GvHD, CsA and viral infections ABOincompatibility negatively influences the development of TMA remains speculative and has to be studied in a larger series of patients.

Mixed chimerism can be associated with circulating autoantibodies and may favour chronic graft-versus-host disease occurrence S. Perruche, F. Kleinclauss, R. Angonin, D. Paris, P. Tiberghien, P. Saas, INSERM E0119/MEN EA2284, CHU Jean-Minjoz (Besançon, F) Use of a reduced intensity conditioning regimen before allogeneic hematopoietic cell transplantation is frequently associated with an early state of mixed hematopoietic chimerism. Such co-existence of both host and donor hematopoietic cells may influence post-transplant alloreactivity and may affect the occurrence and severity of acute and chronic graft-versus-host disease as well as the intensity of the graft-versus leukemia effect. We investigated the relation between chimerism state after reduced intensity conditioning transplantation (RICT), autoantibody production and chronic GvHD (cGvHD)-related pathology. Chimerism state, circulating anti-cardiolipin and antidouble stranded DNA auto-antibody titers as well as occurrence of cGvHD-like lesions were investigated in a murine RICT model. We observed, 9 weeks post-BMT, that recipients with a mixed chimerism state had higher levels of circulating pathogenic IgG auto-antibodies (anti-dsDNA auto-Ab: 193 U/mL [6-1716] (median [range]), n=41; AcL auto-Ab: 834 U/mL , n=70) than non-(anti-dsDNA Ab: 69 U/mL , n=90, P<.01; AcL Auto-Ab: 111 U/mL [10-4398], n=117, P<.01) or full-donor chimeric mice (anti-dsDNA Ab: 73 U/mL , n=15, P<.01; AcL auto-Ab: 164 U/mL , n=24, P<.01). The high auto-Ab median level observed in mixed chimeric mice resulted from the presence of high levels (> mean + 2 SD of Ab levels found in naïve mice) of auto-immune IgGs in nearly half of the mixed chimeric mice (16/41 for anti-DNA Abs; 31/70 for AcL Abs). In 4/8 mixed chimeric randomly tested mice, increased levels of pathogenic IgG auto-Abs were associated with a decrease of natural IgM auto-Abs, suggesting self-immunization. Moreover, mixed chimeric mice presenting higher levels of pathogenic IgG auto-Abs developed cGvHD-like lesions (8/10 mixed chimeric mice tested vs no lesions in 10 full-donor or 19 non-chimeric mice, P<.001). Thus, we observed a novel association between mixed chimerism state, high levels of pathogenic IgG autoantibodies and subsequent development of cGvHD-like lesions. Furthermore, we found that the persistence of host B cells (for 5 out of 11 mixed chimeric mice), but not of dendritic cell origin or subset, may be a factor influencing the occurrence of cGvHD-like lesions. Recipient B cell persistence may therefore contribute to the frequency and/or severity of cGvHD after RICT. B-cell chimerism and auto-immunity monitoring might prove useful in such a setting.

Non-myeloablative stem cell transplantation in beta thalassaemia major patients A. Ghavamzadeh, A. Mousavi, M. Iravani, B. Bahar, S. Gholibeikian, M. Moradiseresht, P. Heydari, Dr.Shariati Hospital (Tehran, IR) Objectives: Allogeneic blood and marrow transplantation (BMT) can be a curative treatment for beta thalassemia major. Conventional transplantation with myeloablative conditioning regimen is associated with high morbidity and mortality in advanced disease or class III beta Thalassemia major. Non myeloablative stem cell transplantation (NMSCT) with less toxic and more immunosuppressive conditioning regimen is an alternative for conventional transplantation. In order to evaluate the success of this kind of transplantation, we studied this method in our class III beta thalassemia major patients. Methods and patients: We studied 13 patients (7 male and 6 female). The mean age of patients was 13 (5 to 24 years old). The conditioning regimen was Fludarabine 30 mg/m 2 daily for 5 days and Busulfan 4mg/kg for 2 days and ATG 10 mg/kg daily for 4 days. GVHD prophylaxis regimen was Cyclosporin A, 3 mg/kg/IV (-2 to +5) then 12.5 mg/kg orally (from +5 to +30) then tapered off. After transplantation, donor lymphocyte infusions (DLI) was performed if needed to improve mixed chimerism. Chimerism assessment (by VNTR and STR-PCR) was in +28, +56, +84 and +112. Results: In our follow up from 79 to 801 days (median follow up= 336 days), 11 patients are alive (84.61%), 3 are transfusion independent (23.1%). All of them had mixed chimerism after transplantation, and then DLI was given to 9 pts. Mixed chimerism was converted to full chimerism in 3 patients. Rejection was observed in ten pts. (76.9%). 2 pts. died (16.6%), one due to infection and another due to acute GVHD. After transplantation or DLI, 5 patients (38.5%) developed acute GVHD (grade II=3, grade III= 1, grade IV= 1), and 3 patient (23.1%) developed chronic GVHD (Extensive). Conclusion: It seems non-myeloablative blood and marrow transplantation is feasible and tolerable for class III beta thalassemia major patients but the more accurate assessment of chimerism and dose adjustment of cyclosporine and appropriate time of DLI may improve the results.

Impact of graft composition on the outcome after nonmyeloablative haematopoietic stem cell transplantation F. Baron, P. Frere, N. Schaaf-Lafontaine, G. Vanstraelen, E. Baudoux, C. Herens, V. Bours, G. Fillet, Y. Beguin, University of Liege (Liege, B) We analyzed the impact of cell doses on the outcome after nonmyeloablative hematopoietic stem cell transplantation. Fortyseven patients, aged 22-65 years, were analyzed. Underlying diseases were NHL (N=13), multiple myeloma (N=7), renal cell carcinoma (N=7), AML (N=5), MDS (N=4), CML (N=4), CLL (N=2), myelofibrosis (N=2), HD (N=1) and ALL (N=1).

Nonmyeloablative conditioning regimen consisted in 2 Gy TBI alone (N=16), 2 Gy TBI and fludarabine (N=23) or cyclophosphamide and fludarabine (N=8). Stem cell source was unmanipulated PBSC (N=20), CD8-depleted PBSC (N=20), CD34-selected PBSC (N=6) or unmanipulated BMT (N=1). Donors were HLA-identical siblings (N=20), 1-mismatch related (N=6) or HLA-identical unrelated (N=21). Postgrafting immunosuppression was carried out with CSP and MMF. For each cell subset, cell dose was categorized in 2 groups (less or more than percentile 50). Events analyzed were calculated from the time of transplantation using Kaplan-Meier product-limit estimates. The impact of cell doses on CD3 chimerism was analyzed by the two-way ANOVA test using cell doses and time as variables. A median (range) of 5.7 (1.8-14.9) x 10 6 CD34+ cells/kg, 136 (0.04-426) x 10 8 CD3+ cells/kg, 87 (0.02-273) CD4+ x 10 8 cells/kg, 6.3 (0.01-178) x 10 8 CD8+ cells/kg and 34 (0.04-140) x 10 8 CD56+ cells/kg were infused. The 180-day incidence of grade II-IV acute GVHD was 35%. Median CD3 chimerism on days 28, 60, 100 and 180 was 78%, 74%, 89% and 85%, respectively. The 1-yr probabilities of extensive chronic GVHD, graft rejection and overall survival were 30%, 15% and 64%, respectively. Higher dose of CD3 (p=0.071), CD8 (p=0.0045) and CD56 (p=0.036) but not higher dose of CD4 or CD34 injected were associated with a higher incidence of grade II-IV acute GVHD. Higher doses of CD3 (p=0.004), CD4 (p<0.001), CD8 (p<0.001), CD56 (p<0.001) and CD34 (p<0.001) were all associated with an increased T cell chimerism from day 28 to day 180. There is a trend for a higher 1-yr probability of graft rejection in patients who received lower doses of CD34 (26% vs 5%, p=0.16) and CD3 (24% vs 6%, p=0.18) cells. On the other hand, none subset cell dose did significantly influence the probability of extensive chronic GVHD nor overall survival. To conclude, higher doses of CD3 and CD8 cells but not CD4 cells increased the incidence of acute GVHD. This observation underlined the potential role of CD8 depletion of the graft to reduce the incidence of GVHD after NMSCT.

Increased post-transplant morbidity associated with a greater cumulative in-patient stay in recipients of reduced intensity conditioning regimens (RIC-ALLO) compared to myeloablative conditioning regimens (CC-ALLO) in allostem cell transplantation (1) and Sezary Syndrome (1) . 16/27 pts (59%) had received 3 previous treatment courses and 18/27 pts (67%) underwent a previous transplantation . At transplant 9pts had refractory disease (33%), 10(37%) were in partial remission (PR) and 8pts (30%)in complete remission (CR). Median age was 47(17-60) yr. NMR was Campath-1H 20mg/die (from day-8 to-5), Melphalan 30mg/m 2 (day-8), Fludarabine 30mg/m 2 (from day-4 to-2)and TBI single fraction 200cGy (day-1). All pts received GVHD prophylaxis with CSA (3mg/Kgpo) and mycophenolate mofetil (15mg/Kg po) respectively from -1 to+120 and from 0 to+35. Ten pts received donor peripheral blood progenitor cells (PBPCs) collected after mobilization with G-CSF and 17 pts received bone marrow (BM). In PBPCs group median number of infused TNC and CD34+ cells were respectively 9.0x10 8 /Kg (5.3-14.6) and 5.5x10 6 /Kg (3.3-15.4 (1), IP (1), liver and kidney failure (1) and acute GVHD +infection (1). Acute GVHD was observed in 8 patients (in 1pt post DLI). 4/13 evaluable pts developed cGVHD. Four pts received DLI for relapse. After a median follow-up of 133 d. (29-425) overall survival was 66%. Out of 17 pts evaluable at 3 months, 11 pts are in CR, 3 pts PR and 3 pts show stable disease. These preliminary results suggest that this regimen in unrelated transplants is well tolerated with low TRM also in pts with advanced disease or age. Longer follow-up is needed to evaluate overall response.

Haematopoietic stem cell transplantation with nonmyeloablative conditioning in the out-patient setting. Results, complications and admission requirements in a single-centre S. L. Petersen, H. O. Madsen, L. P. Ryder, A. Svejgaard, H. Sengelov, C. Heilmann, E. Dickmeiss, L. L. Vindelov, Rigshospitalet (Copenhagen, DK) Patients: 30 patients with hematologic malignancies received peripheral blood stem cells from their human leukocyte antigen identical sibling donors after non-myeloablative conditioning with fludarabine and 2 Gy of total body irradiation. The median age was 51 (range 27-63) years and the patients had received a median of 4 (range 1-10) chemotherapy regimens prior to the transplant. At the time of transplant 6 patients were in complete remission, 19 were in partial remission, 4 had progressive disease and 1 patient was in aplasia after induction therapy for acute myeloid leukemia. In 27 patients the transplant was planned as an out-patient procedure. Results: All patients engrafted. The median time needed to reach complete donor chimerism was 42 (range 14-104) days for the granulocytes, 123 (range 14-378) days for the CD8+ T cells and 147 (range 14-378) days for the CD4+ T cells. The median granulocyte and thrombocyte nadirs were 0.2 (range 0-0.9) x 10 9 /l and 30 (range 1-88) x 10 9 /l respectively. The absolute neutrophile count was below 0.5 x 10 9 /l at a median of 12 (range 0-23) days. A median number of 3 (range 0-92) red blood cell transfusions and 0 (range 0-60) platelet transfusions were given during the first 60 days post transplant. The probability of acute graft versus host disease (GVHD) grades II-IV and extensive chronic GVHD was 57 % and 80 % respectively. The risk of cytomegalovirus reactivation was 18 % on day +100 and 32 % on day +365. During the first 60 days 6 patients did not require hospitalization and the remaining patients were hospitalized a median of 8 (range 1-61) days. However, a median of 44 (range 4-151) days of hospitalization were observed during the first year in patients alive on day +365. During the first year the entire cohort was hospitalized for 1596 days. GVHD accounted for 22 %, infections for 18 %, thrombotic thrombocytopenic purpura for 16 %, engraftment syndrome for 14 %, fever of unknown etiology for 8 % and pneumonia of unknown etiology for 7 % of these 1596 days. With a median follow up of 602 days in patients alive, the 2 year overall and progression free survival estimates were 68 % (95 % CI 48-88%) and 43 % (95 % CI 20-66%). Conclusion: this transplantation regimen is feasible and can induce long term remissions in heavily pre-treated patients. The transplant can be performed in the out-patient setting, but complications result in a substantial number of admissions during the first year. Allogeneic HSCT in CML is one of the standard therapeutic approaches. In comparison to myeloablative regimen, the reduction in regimen related toxicity and mortality forced transplant physicians to use more frequently the promising reduced intensity stem cell transplantation (RIST) modality. The feasibility of this approach has been reported in many high-risk patients with different diagnoses but there is still lack of experience at standard risk population concerning efficacy and toxicity. The aim of this retrospective case-control single center study is to compare ablative HSCT and RIST in standard risk CML patients. We have matched 12 RIST recipients with 12 CML patients, selected among eligible 116 patients according to their Gratwohl (Lancet 1998; 352: 1087) risk score. All the patients received HLA id. sibling PBSC. All the patients were in early CP1 of CML. Ablative group received Bu-Cy, whereas RIST patients received Flu based regimen. Median age and M/F ratio of the RIST and ablative group were 40 (24-47) and 37.5 (29-46), and 4/8 and 5/7, respectively. Transplant characteristics and complications are shown in table. The median duration of ANC<500 was significantly shorter in RIST recipients (p=0.004). The need for SDP was very low in RIST group in comparison to ablative group (p=0.004). RIST recipients experienced less mucositis and diarrhea, which correlated into significantly reduced need for TPN. Median follow-up period in ablative and RIST group was significantly different; 36.5 mo.s (range, 4-64) and 14.25 mo.s (range, 1-48), respectively (p=0.029). We observed almost always a delay in the establishment of donor chimerism in RIST group, which caused relatively frequent use of DLI and later on STI571 in patients with mixed chimerism and rising bcr-abl load. The incidence of GVHD was not statistically different between two groups. But we observed remarkably higher de novo onset extensive cGVHD in RIST. Transplant related early mortality was similar. Two-year's probability of disease free survival in RIST and ablative group was 72.2% and 75% (log-rank, p=0.67), respectively. In addition, two-year's probability of overall survival in RIST and ablative group was 67.9% and 75% (p=0.78), respectively. As expected RIST recipients showed less regimen related toxicity and myelosupression, but there is no difference in cumulative incidence of early transplant related mortality and GVHD.

Reduced-intensity cord blood transplantation for adult T-cell leukaemia / lymphoma D. Kato, E. Kusumi, S. Takagi, S. Koyama, K. Yuji, J. Ueyama, S. Miyakoshi, S. Morinaga, S. Taniguchi, Toranomon Hospital (Tokyo, J) Background: The prognosis of acute and lymphoma types of adult T-cell leukemia/lymphoma (ATL) remains poor. Although allogeneic hematopoietic stem cell transplantation is reported to be effective for hematologic diseases, patients who have HLAmatched sibling donor are only 30%. Cord blood transplantation is a novel therapeutic strategy for patients with hematologic diseases who lack HLA-matched donors. We performed a feasibility study to test the role of reduced-intensity cord blood transplantation (RICBT) for ATL. Patients and method: Between July 2002 and November 2003, eleven patients with ATL (median age, 56 y; range, 27-79: median body weight, 53kg; range 38-75: 3 acute, and 8 lymphoma type) underwent RICBT. The grade of performance status at transplantation was 0 in two patients, 1 in two, 2 in four, 3 in three. Median interval from diagnosis to transplantation was 8 months (range, 1-24). Chemotherapy preceding RICBT had induced CR in 1, SD in 1, PR in 2 and PD in 7. Serological HLA matching was 6 of 6 in 1 patient, 5 of 6 in 5, and 4 of 6 in the other 5. Median number of infused all nucleated cells was 2.78 x 10 7 /kg (range, 1.99-4.31). The conditioning regimen consisted of 25 mg/m 2 fludarabine for 5 days, 80 mg/m 2 melphalan for 1 day, and 4 Gy total body irradiation (TBI). Graft-versus-host disease (GVHD) prophylaxis was cyclosporine alone. All patients received filgrastim 300 microgram/ m 2 starting on day 1 until engraftment. Results: Median follow up period is 32 days (range, 7-217) . Engraftment was evaluated in seven patients. Six achieved stable engraftment, while secondary graft failure occurred in one. Median time of neutrophil engraftment was 16 days (range, 10-18) . Other four patients died before engraftment. Five patients died of transplant-related toxicity (sepsis=3, encephalitis=1, and multiple organ failure=1), and one died of relapse. Regarding the clinical response, CR was observed in eight patients. Estimated 6-months DFS is 24% and OS is 48%. Conclusion: Although the number of patients is too small and the follow-up period is short, our results suggest that a strategy that incorporates RICBT for ATL may be worth considering for further intense evaluation. More effective prophylaxis for opportunistic infection is needed. The control of management of transplantrelated toxicities will be the focus of future investigation. Recently, in an effort to reduce the transplant related mortality, reduced intensity stem cell transplantation (RIST) methods have been developed. It has been reported that patients receiving RIST from an ABO-mismatched donor had more transplantassociated complications compared to standard ablative AHCT (BMT 2001; 28:315, Transfusion 2002; 42:1293 42: , Transfusion 2003 43:1153) . In this single center, case-control design study, we aimed to analyze the influence of ABO-incompatibility on transplant-related morbidity and mortality between ablative and RIC retrospectively. Between 1988 and 2003, 39 patients underwent RIST and only 14 were ABO-incompatible. Ten patients with ABO-incompatibility and having a regular follow-up for blood group typing were evaluated for immuno-hematological complications, such as acute or delayed-type hemolysis (DTH), pure red cell aplasia (PRCA), thrombotic thrombocytopenic purpura (TTP) and early transplant-related complications, were compared to 20 case control recipients having matched pretransplant characteristics and similar follow up, but myeloablative regimen, either bone marrow (BM) or peripheral blood (PB) stem cells infused. Patients' characteristics are shown in table below. All the recipients and donors underwent a detailed pretransplant work up and all the recipients were followed twice a week post transplant by a transfusion specialist according to guidelines (BMT, 2001; 28:315) . Median follow-up was 195 days (range, 51-538d). We did not observe any acute hemolysis, but 11 experienced DTH. No significant differences were encountered among the three groups in terms of DTH (p=0.356). In all recipients having a major ABO incompatibility, the blood group switched to donor type, but 50% of the patients with minor ABO-incompatibility still had either their antigen persistency or the appearance of donor-derived isoagglutinins. We observed mild (n=1,BM group) and severe pure red cell aplasia (n=1,RIC group) in two patients having a major ABOincompatibility. TTP was developed in one patient with major ABO-incompatibility. In conclusion, we did not observe any difference between ablative AHCT and RIST in ABO incompatible patients in terms of immunohematological complications in contrast to published case series. In addition, we could not show any negative impact of ABO-incompatibility on the severity of acute GVHD and the rate of early transplant related mortality.

Reduced-intensity conditioning allogeneic transplant in patients with aggressive non-Hodgkin lymphoma: results of a prospective multicentre trial R. Martino, M. Mateos, A. Urbano, A. León, J. Odriozola, J. Ribera, D. Valcarcel, J. Sarrá, L. Vázquez, A. Sureda, J. Sierra, J. San Miguel, Hospital de la Santa Creu i Sant Pau, Hospital Clínico, Hospital SAS de Jerez de la Frontera, Hospital Ramon y Cajalñ, Hospital Germans Trials i Pujol, ICO (Barcelona, Salamanca, Jerez de la Frontera, Madrid, Badalona, E) In September 1999, we started a prospective multicenter trial with RIC allogeneic transplant in hematological malignancies. Up to 30 september 2003, 18 had aggressive histology-DLBCL 7 patients( 39%),Mantle Cell Lymphoma (MZL), 5 patients(28%), Burkitt(BL)1and Peripheral T-cell Lymphoma(PTCL) 5 (28%). The conditionig regimen consisted of fludarabine followed by melphalan. Filgrastim-stimulated peripheral blood stem cells from related HLA identical donors were infused on day 0. GVHD prophylaxis consisted of cyclosporine A (CsA) plus short-course methotrexate (MTX), followed by folinic acid rescue. Median age at transplant was 46 years, (range: 27-61).According status at transplant, only 2 patients(11%)-one MZL and 1 PTCL were to the transplant on first CR after one line-;the other patients went to the transplant in later stages-First PR (3 patients with MZL)(17%) , 3 patients(17%) in more than 1 rst CR and 12 patients (64%) with active disease , 3 of them with progressive disease..In fact , excluding 4 patients with MZL and one PTCL who have received only one line , most of the patients (72%) had received a median of 3 lines(2-6) before the transplant, and 9 out 13 non MZL(70%) and one MZL had failed a previous transplant. All except one patient died on day plus 10 engrafted. Acute GVHD grade I-IV developed in 12 patients (67%),grade II-IV in 9 (50%) and grade IV in 3 (17%) .From 12 patients at risk, 8 (67%)developed chronic GVHD, extensive in 7 (58%) patients at risk. With a median follow-up of 21months (range: 8.4-40.4)), 10 patients are alive disease free and 2 have stable disease. 8 patients died (44%): 4 (22%) due to progression and 4 (22%) due to transplant related mortality. Estimated Overall survival (OS) and EFS at 30 months are 55 and 47% respectively. Considering histology 60%,50% and 20% of MZL, other B cell Lymphoma and PTCL are disease free. Concerning relationship between GVHD and follow-up,OS and EFS are significantly better for patients developing acute and or chronic GVH (36 vs 85 % and 30vs71%)(p<0.05). When a Cox regression analysis is performed ,patients developing acute GVH had a significantly lower risk of death-HR=5.68,IC 95%(1.07-30)(p=0.04)-. Comments: This RIC program associated with good engraftment and it seem to be effective in these high risk patients and although numbers are small our results suggest that an early GVL effect is necessary to control the disease in NHL patients with aggressive histology.

Reduced Intensity conditioning in patients with B-chronic lymphocytic leukaemia (B-CLL): results of a prospective multicentre trial D. Caballero, R. Martino, M. Mateos, A. León, A. Urbano, G. Sanz, J. Sarrá, J. Perez Simón, D. Valcarcel, A. Sureda, J. Sierra, J. San Miguel, Hospital Clínico, Hospital de la Santa Creu i Sant Pau, Hospital SAS de Jerez de la Frontera, Hospital La Fe, ICO (Salamanca, Barcelona, Jerez de la Frontera, Valencia, E) With the following button you have the possibility to add graphics to your abstract. The graphics must be in GIF or JPG format. Please sIn September 1999, we started a prospective multicentre trial in non-myeloablative allogeneic transplant in hematological malignancies. Up to september 2003 16 with a diagnosis of B-CLL have been registered:. The conditioning regimen consisted of fludarabine 30 mg /m 2 intravenously (IV) on days -8 to -4 followed by melphalan 70 mg/m 2 IV on days -3 and -2. Filgrastim-stimulated peripheral blood stem cells from a HLA related identical donor were infused on day 0. GVHD prophylaxis consisted of cyclosporine A (CsA) from day -7 plus short-course methotrexate (MTX) (10mg/m 2 , days +1, +3, +6) , followed by folinic acid rescue. Median age at transplant was 54 years, (range: 35-67).At the transplant 11 patients (68 %) had sensitive disease (12 % were in CR and 56% were in PR) and 5 (32%) had refractory/progressive disease. Median number of CD34 cells infused was 4.6 x106 (1.9-8.4). All 15 evaluable patients engrafted. Regarding graft versus host disease(GVH), at a median of day +32 days(range 17-40), 8 patients(52%) developed acuteGVH grade I,1 grade II and 3 grade III-.From 14 patients at risk at a median of 178 days(102 to 391),10 patients (71%) developed chronic graft versus host disease( cGVH) -5 limited and five extensive c-GVH-. Evaluation was performed at day +100 and at that moment 9 patients(56%) were in CR, 4(25) in PR,1 had stable disease and 1 was not evaluable With a median follow-up of 5.3 months, 11 out of the 15 evaluable patients(69 %) are alive ,all of them in CR and 5 patients (31%) have died-1 due to progression and 4 due to a toxic cause with a transplant related mortality of 25%-. Overall survival (OS) and event free survival (EFS) are 64% and 77% respectively. Regarding the potential beneficial effect of the GVL in LLC-B EFS is significantly better for patients developing cGVH as compared to those without GVH(83% vs 33%) (HR=5,47 (95%CI: 0.89-33.62))p: 0,06. Comments: although numbers are small these encouraging results suggest a clear role of nonmyeloablative allogeneic transplant in curing bad prognosis B-CLL patients .

Plasma pharmacokinetics of low-dose oral busulfan in Fanconi 's anaemia patients undergoing haematopoietic stem cell transplantation J. Kühl, A. Jenke, E. Schleier, J. Freiberg-Richter, U. Schuler, H. Neitzel, W. Ebell, Charité-Virchow Klinikum, Universitätsklinik (Berlin, Dresden, D) Objectives: Toxicity of conditioning regimens is a major obstacle of HSCT in Fanconi anemia, a rare bone marrow failure syndrome characterized by hypersensitivity to DNA cross-linking agents. We therefore analysed specifically the pharmacokinetics of low-dose busulfan in the context of a fludarabine/T-cellantibodies containing, non-irradiation protocol (GEFA protocol). Methods: 7 Fanconi anemia patients (median age 12 years, range 7-19 years) received an oral busulfan dose of 0,25 mg/kg (n=2) or 0,5 mg/kg (n=5) twice daily for two consecutive days (days -4 to -3 before transplant). Prior to treatment 3 patients had significantly increased liver enzymes, 2 an impaired renal function. Busulfan plasma levels were measured after the first oral dose by HPLC. Pharmacokinetic parameters were derived by modeling the raw data to fit first-order single compartment kinetics. Results: With a wide inter-patient variability the median peak levels (Cmax) and area under the curve (AUC) values were as follows: Cmax 648 ng/ml (range 506-755 ng/ml) and AUC 3220 ng*h/ml (range 1750-6400 ng*h/ml). Compared to children with other diseases receiving higher doses of busulfan Fanconi anemia patients demonstrated an increased exposure to busulfan due to a lower clearance as well as lower volume of distribution. Conclusion: According to these results a cautious dosing of busulfan is indicated in Fanconi anemia if added into reduced intensity conditioning regimens, not only because of the known hypersensitivity on a cellular level, but also the apparently impaired metabolism of the drug in this specific disease.

Optimisation of recombinant human erythropoietin therapy after non-myeloablative allogeneic haematopoietic stem cell transplantation F. Baron, G. Vanstraelen, P. Frere, G. Fillet, Y. Beguin, University of Liege (Liege, B) Purpose: Nonmyeloablative stem cell transplantation (NMSCT) is associated with prolonged anemia and most of the patients require red blood cell transfusions (RBC Tx). We enrolled 45 patients with advanced hematological malignancies or renal cell carcinoma in 2 pilot trials to determine the optimal utilization of recombinant human erythropoietin (rHuEpo) therapy in this setting. Thirteen NMSCT recipients who did not receive rHuEpo served as controls (control group). Patients and Methods: Twenty-one patients were scheduled to start rHuEpo (NeorecormonR) on day 0 (Epo group 1) and 24 other patients were scheduled to start rHuEpo 1 month after the transplant (Epo group 2). RHuEpo was administered qw at a dose of 500 u/kg/wk with the aim of achieving Hb levels of 13 to 14 g/dL. Nommyeloablative conditioning regimen were 2 Gy TBI (N=23), 2 Gy TBI and fludarabine (N=27) or 2 Gy TBI and cyclophosphamide (N=8). The three groups were well balanced concerning underlying disease, donor type, age and ABO compatibility. Results: Mean + SD number of RBC Tx was 2.2+2.9 for the 37 pts who did not receive rHuEpo therapy the first month after the transplant versus 0.8+1.5 for the 21 patients who did receive rHuEpo therapy the first month after the transplant (p=0.02). 0/13 patients in the control group, versus 13/24 (p=0.0009) patients in Epo group 2 achieved Hb value of 13 g/dL before day 150 after the transplant. Hb levels between days 50 and 180 after HSCT were significantly higher in the 2 Epo groups combined compared with the control group (p=0.01 with the 2-way Anova test). However, the mean+SD number of RBC tx between day 30 and 100 after the transplant were similar in patients who received rHuEpo and in the control group (1.2+1.9 vs 1.1+1.9 (NS), respectively). The evolution of the soluble transferrin receptor (sTFR) and of the reticulocyte counts in the 3 groups will be presented. Conclusions: Anemia after NMSCT is sensitive to rHuEpo therapy. Contrarily to that observed after myeloablative allogeneic HSCT where the benefit of rHuEpo therapy seems maximal when the drug is started 1 month after the transplant (Baron et al., Exp Hematol 2002) the benefit after NMSCT seems maximal when rHuEpo therapy is started on day 0 after the transplant. These data provide the basis on which to conduct a prospective, randomized, placebo-controlled trial of rHuEpo therapy after allogeneic HSCT.

Follow-up of patients with non-acute lymphoid malignancies after reduced-intensity conditioning regimen with allogeneic haematopoietic stem cell transplantation: results from a single centre T. Guillaume, B. Gueglio, P. Moreau, N. Morineau, B. Mahé, P. Chevallier, V. Dubruille, M.J. Rapp, J.L. Harousseau, N. Milpied, Hotel Dieu (Nantes, F) Allogeneic hematopoietic stem cell transplantation (HSCT) may provide a potentially curative graft-versus-tumor effect in patients with relapsed or high risk lymphoma and chronic lymphocytic leukaemia (CLL). Since these patients have usually received several lines of chemotherapy including autologous HSCT, non myeloablative preparative regimen is administered to reduce conditioning-related toxicity. We summarize results in 22 patients (median age, 51 years; range 20-65) with advanced non-Hodgkin's lymphoma (NHL, n=12) Hodgkin's disease (HD, 1 relapsed Stage III and 2 pulmonary stage IV) or CLL (n=7) treated at a single institution and who received allo-HSCT following reduced intensity (RI) regimen. NHL included 2 mantle cell lymphomas, 6 transformed lymphomas, 2 high-grade lymphomas and 2 follicular lymphomas. Sixteen patients had previously received autologous HSCT. Pretreatment of CLL patients was heterogeneous with 3 to 7 chemotherapy regimens. Prior to allogeneic transplantation, 2 patients had refractory disease, 10 patients were in CR, while 10 achieved PR. Following RI conditioning regimen combining fludarabine and an alkylating agent (busulfan (21) or melphalan (1)) with antithymocyte globulin, patients received G-CSF-mobilized blood stem cells from related donors (n=21) or unrelated donor (n=1). Neutrophil and platelet engraftment occurred following a median of 14.5 and 10.5 days respectively. Acute GVHD developed in six patients (27%): 4 developed grade I-II cutaneous GVHD, 1 had grade II gastrointestinal involvement and 1 extensive disease. Chronic GVHD was observed in seven patients (32%): 3 limited to the skin, 1 oral, 1 hepatic, 2 extensive disease. After a median follow-up of 372 days (range 47-1933) , 15 pts are alive and 7 have died. Causes of death were disease progression in 5 patients (2 mantle, 1 transformed, 1 follicular, 1 HD) and treatment-related complications in 2 patients. The Kaplan-Meier estimate for 1 year survival is 75%. Allo-HSCT following RI regimen for high-risk non acute lymphoid malignancies in our experience is complicated by a relatively low transplant-related mortality and provide long-term complete remission.

Reduced-intensity conditioning followed by allogeneic transplantation: poor outcome in secondary versus de novo acute myeloid leukaemias P. Corradini, R. Milani, A. Dodero, C. Tarella, G. Bandini, A. Locasciulli, M. Bernardi, M. Lucesole, A. Busca, D. Soligo, A.M. Gianni, M. Bregni, A. Olivieri, Istituto Nazionale Tumori, University of Turin, University of Bologna, H. San Camillo, H. San Raffaele, University of Ancona, Policlinico (Milan, Turin, Bologna, Rome, Ancona, I) Patients with secondary AML (sAML) usually have a worse prognosis then de-novo AML. After myeloablative conditioning, the transplant-related-mortality (TRM) in sAML is higher than in patients with de-novo AML. We report the results of a phase II study employing reduced-intensity conditioning (RIC) followed by allogeneic peripheral blood stem cell (PBSC) or bone marrow (BM) transplantation in 31 patients with de-novo (n=14), or secondary to MDS (n=6), or therapy-related AML (n=11). Median age was 53 years (range:22 -67). Disease status at transplant was categorized as low risk (n=15 CR1 or CR2), high risk (n=5 PR, n=8 PD) and up-front (n=3). The median time from diagnosis to allograft was 6 months (range: 1-80 months). After conditioning with thiotepa (10 mg/kg), fludarabine (60 mg/ms) and cyclophosphamide (60 mg/kg), pts received BM (n= 10) or lenogastrim-mobilized PBSC (n= 21) from HLA-matched/ 1ag mismatched siblings (n=21), or HLA-matched unrelated doors (n=3). GVHD prophylaxis consisted of cyclosporine A and short course methotrexate. All patients engrafted. Acute GVHD grade II-IV occurred in 48% of the pts (n=2 post-DLIs, n=1 for early CSA tapering), chronic GVHD developed in 36% of pts (n=2 post-DLIs). Overall non-relapse mortality was 20% (n=6); nonrelapse mortality was higher in sAML(35%) than in de-novo AML (0%) (P<0.02). The 2-year event-free survival (EFS) was 45% (95%CI, 12% to 78%) in patients with de novo-AML versus 11% (95%CI, 1% to 26%) in patients with sAML (p<0.01). Patients with de novo AML in CR at the time of transplantation had a favorable outcome with an EFS of 71% (95%CI, 36% to 99%). At a median follow-up of 24 months (range: 8 to 50 months), 11 pts are alive:9 in CR, and 2 with PD. In conclusion, RIC is an effective strategy for de-novo but not sAML. Results are particularly disappointing in patients with therapy-related leukemias.

Molecular monitoring of chimerism and minimal residual disease in multiple myeloma patients after nonmyeloablative stem cell transplantation S. Galimberti, E. Benedetti, R. Fazzi, F. Papineschi, F. Caracciolo, E. Orciuolo, F. Andreazzoli, F. Morabito, M. Cuzzola, M. Martino, P. Iacopino, M. Petrini, Ospedale S.Chiara, Transplant Unit -Haematology (Pisa, Reggio Calabria, I) Allogeneic transplantation has been shown to offer significant advantage for eradication of minimal residual disease in multiple myeloma (MM) patients. Non-myeloablative transplant (NMT) offer this opportunity even to patients not elegible for standard myeloablative procedures. Chimerism percentage was tested by a fluorescent multiplex PCR method and IgH rearrangement monitored in 22 MM cases after NMT. Twelve male and ten female, from 35 to 66 years (median = 49), were transplanted from HLA-identical sibling. Conditioning regimen included fludarabine and TBI (200 cGy) in 21 cases; one patient received busulfan. The post-grafting immunosuppression was performed by a combination of mycofenolate mofetil and cyclosporine. A median of 7.2 x 10 6 CD34 cells/Kg were infused. In 13 cases the monoclonal component was IgG, in 5 IgA and 4 patients showed increased light chains only. Fifteen cases were in the stage III of disease. Before NMT, all patients have been treated with tandem autologous transplantations. Before NMT, 6 cases were in disease progression and 11 in PR. All patients engrafted; 16 of 22 (73%) are still alive, with 4 cases in CR. Three-year PFS was 38%. aGVHD occurred in the 50% of cases and cGVHD in the 54%. PFS was significantly influenced by the cGVHD, with a median of 12 months for cases without cCVHD versus not reached for those patients with cGVHD (p=0.03). The chimeric status and IgH rearrangement have been assessed in serial samples every 3 months, starting on day +30. Six patients (27%) achieved a complete donor chimerism (CC), three of them within the first month after transplant; only one resulted "full recipient" at +30 days (RR), but she converted to a mixed chimerism lately. There was no significant difference between patients who showed and who did not show MC in terms of clinical characteristics or disease status at transplantation. Median chimerism values ranged from 75% at +30 days to 90% after 12 months. Six of the 15 cases serially evaluated for minimal residual disease (40%) resulted IgH-negative, 4 achieving molecular remission within 6 months; four of them simultaneously presented a mixed chimerism. These result show that non-myeloablative allogeneic transplant is frequently associated with a mixed chimerism and, in MM patients, it is able to offer a good percentage of molecular remissions, even to patients without a full donor pattern.

Cytomegalovirus infection and disease in allogeneic nonmyeloablative transplant patients: a single-centre study M. Uzunov, O. Adam, E. Chachaty, F. Griscelli, P. Arnaud, C. Boccaccio, J. Bourhis, Institute Gustave Roussy (Villejuif, F) CMV is an important cause of morbidity and mortality in patients receiving a reduced intensity allogeneic blood or marrow transplant. The aim of this study was to evaluate the relationship between lymphocyte reconstitution and CMV reactivation in patients at risk(either donor or recipient CMV+). We review data on 50 pts that received an allogeneic NMA transplant from Nov 1999 until Oct 2003 for various hematological or solid malignancies .The conditioning regimen included Fludarabine-47 pts (94 %)+TBI2Gy, Busulfan and ATG, Endoxan, Ida and Ara-C or Melphalan; 3 pts (6%) received TBI 6Gy and Endoxan. Post transplant immunosuppression consisted in CSA and/or MMF or short course MTX. The CMVpp65 antigenemia assay was used to monitor the pts, at least once weekly till day 100, and the pts at risk had a BAL (day30-35). At a median of follow-up of 6 months , CMV reactivation occurred in 15 pts (30%) and CMV disease in 1pt(2%). First episode of CMV infection occurred at day 38 (median27 -210). The 15 pts who developed CMV infection or disease had received a graft containing a median of 2.77x10 8 CD3/kg (0.23 -5.55). The median lymphocyte count at day 30 was 0.8 x10 9 /L and at day 100 was 0.6x10 9 /L. Only five of these pts developed acute GVHD, grade II or I and none of them severe GVHD. 1 of these 15 pts died because of CMV pulmonary disease. 2pts developed recurrent activation of CMV. At day 100 and before the 2nd episode both of these pts were lymphopenic, 0.5x10 9 /L and 0.6x10 9 /L lymphocytes. 26 of the recipients (52 %), were at risk and did not reactivate CMV. They had received a graft containing median 3.02x10 8 CD3/kg (0.16-8.54). Their median lymphocyte count at day 30 was 0.8x10 9 /L at day 100 was 1x10 9 /L. Only 4 of them developed acute GVHD, 3 pts had grade I-II GVHD and 1 patient died of grade IV gastrointestinal GVHD. We noted a trend to a lower lymphocyte count at day 30 as well as at day 100 in pts reactivating CMV compared to the pts at risk that did not reactivate de CMV. Pts who survived at one year had a similar lymphocyte count in both groups (the absolute lymphocyte count was 2.2x109/L (300-5100) for 8 pts at risk and 2.3x109/L (200-400) for 4 pts that had activated the CMV). In conclusion, CMV infection seems to be less frequent in pts receiving an enriched CD3 graft. Pts at risk who received DLI did not reactivate CMV. Future studies will evaluate immune reconstitution following NMA regimens and incidence of reactivation of CMV infection.

Reconstitution of T-cell receptor CDR3 repertoire after myeloablative and reduced-intensity conditioning allogeneic heamatopoietic stem cell transplantation P. Hentschke, B. Omazic, J. Mattsson, I. Näsman-Björk, D. Gigliotti, L. Barkholt, O. Ringdén, M. Remberger, Huddinge University Hospital, Arvid Wretlind Laboratory, Biovitrum, Microbiology and Tumour Biology Centre (Stockholm, S) Background: T-cells play an important role in the adaptive immune system. After hematopoietic stem cell transplantation Tcell function is impaired. This is reflected by different opportunistic infections that are often difficult to treat due to the incomplete immune function. Methods: TcR reconstitution was measured by CDR3 spectratyping to analyze the diversity of the T-cell repertoire at 3, 6 and 12 months after myeloablative and reduced intensity conditioning (RIC) hematopoietic stem cell transplantation (HSCT). Immune function in vitro was tested by activation by mitogens and antigens at 3, 6 and 12 months after HSCT. 23 patients with AML, CML, renal cancer, MDS, NHL, trombocytopenia, colon cancer and CLL were analysed.

Results: Lower diversity in the CDR3 repertoire was seen in CD4+ cells after RIC HSCT at 3 and 6 months and in CD8+ cells at 3 months compared to healthy donors. After myeloablative HSCT lower diversity was seen at 3, 6 and 12 months in CD4+ cells and at 6 and 12 months in CD8+ cells after HSCT. Responses to phytohemaggluttinin (PHA), Concanavalin (Con-A) and Staph. aureus protein A (SpA) were significantly lowered compared to healthy donors during the first 6 months after RIC HSCT. After myeloablative HSCT response was significant lower to Con-A at 3 months. Decreased responses to CMV and VZV antigens were only significant in patients suffering from GVHD grade II-IV. Conclusion: The T-cell repertoire is skewed under the first year after HSCT and immune reconstitution after HSCT with myeloablative and RIC conditioning seem to be comparable. Factors as GVHD and age are probably of greater importance than type of conditioning.

Reduced-intensity conditioning followed by allografting in patients with advanced haematological malignancies older than 60 years A. Olivieri, M. Lucesole, S. Cecchi, A. Dodero, F. Ciceri, M. Bregni, C. Tarella, M. Falda, A. Locasciulli, R. Scimè, F. Patriarca, M. Casini, A. Bacigalupo, P. Corradini, University of Ancona, Istituto Nazionale Tumori, Ist. Scientifico H.S. Raffaele, University of Turin, S. Camillo Hospital, Ospedale Cervello, University of Udine, Bolzano Hospital, S. Martino Hospital (Torrette di Ancona, Milan, Turin, Rome, Palermo, Udine, Bolzano, Genoa, I) Allogeneic transplantation has been mainly used in people younger than 60 years, and the recent development of RIC has allowed a quite significant reduction of morbidity and mortality. Published series have reported patients with a median age ranging from 45 to 52 years, and little is known about the toxicity and the outcome of patients older>60 yrs. We have enrolled in a multicenter italian protocol 23 patients aged from 60 to 69 years (median 62 yrs): 15 male, 8 female; 13 indolent (7 CLL, 3 FCL, 2 MCL, 1 marginal cell lymphoma) and 6 aggressive lymphomas (3 PTCL, 3 DLCL), 2 myeloma, and 2 AREB. The RIC regimen consisted on Fludarabine (60 mg/m 2 ), Thiotepa (5 mg/kg) and Cytoxan (60 mg/Kg); the GVHD prophylaxis consisted of cyclosporine and short course methotrexate. The median number of previous chemotherapy regimens was 2.5 (range 1-4) and 26% of patients had already failed a previous autologous transplant. Forty percent of patient had a chemosensitive disease, but only 13% were in CR before allogeneic transplant; the median interval between diagnosis and allotransplant was 1180 days (range 193-4769) . Nineteen patients received PBSC and 4 BM; the median dose of CD34+ cell and CD3+ cell was 4.9 x10 6 /Kg (range 1.4-8.5), and 2.3 x10 8 /Kg (range 0.3-5.9) respectively. All patients engrafted with 14 days to achieve ANC>500 and 14 days to achieve PLT>20000; 69% of patients showed complete donor chimerism (CC) in bone marrow (BM), and 60% in CD3+ subset at day +30; at day +90 85% had CC in the BM and 80% on CD3+ subset. The incidence of grade 1-2 and 3-4 acute GVHD was 22% and 25% respectively (median onset at day 17); 26% of patients are evaluable for chronic GHVD: 2 limited and 3 extensive (median onset at day 139). Fifty-six percent of patients reactivated CMV antigen and 3 developed a microangiopathy (TTP). We observed 4 toxic deaths with a median follow up of 255 days (range 55-1214): the nonrelapse mortality rate is 17%. Twenty patients were evaluable at day +90 for disease response with 70% CR, 15% PR, 5% SD, 15% PD; 27% of responding patients already relapsed, and 4 of them died of disease progression. Fifteen patients are currently alive: 13 in CR and 2 in PR. In conclusion, this preliminary experience shows that this RIC regimen is feasible also in people older 60 yrs, with a rather low non-relapse mortality; the incidence of severe acute GVHD seems higher than in younger patients.

The outcome of allogenic stem cell transplantation for myeloid leukaemias following reduced-intensity conditioning regimens is highly dependent on the pretransplant status of disease and the occurrence of a graftversus-host disease M. Varvenne, T. Winkler, H. Diedrich, E. Dammann, H. Kamal, M. Eder, A. Ganser, B. Hertenstein, Hannover Medical School (Hannover, D) High toxicity rates of myeloablative therapies as well as the GvHD-associated graft versus leukemia (GvL) effect have led to the development of reduced intensity conditioning regimens (RIC) for allogenic hematopoietic stem cell transplantation (HSCT). We assessed the outcome of 35 patients (median age 56 ys., range 24-62, female 15, male 20) who received related (n=9) and unrelated HLA-identical transplants (n=26) in our center for the treatment of myeloid malignancies (AML n=27, CML n=8). RIC was chosen because of age or comorbidity. The conditioning regimens consisted of fludarabin in combination with either busulfan (n=19), melphalan (n=11) or 2 Gy TBI (n=4). Twelve patients received additional radioimmunotherapy. The source of stem cells was peripheral blood in 29 patients and bone marrow in 10 patients. Transplants were unmanipulated in all cases. GvHD prophylaxis consisted of cyclosporin A+/methotrexate or mycophenolic acid. No prophylactic donor lymphocyte infusions were given. 34 of the 35 patients engrafted within 18 days and one graft failure was observed. The overall transplant-related mortality was 9% on day 100 and 16% at 1 year. Acute GvHD was seen in 12/34 (35%) patients whereas chronic GvHD occurred in 16/30 (53%) cases. Low risk patients (1. CR or 1. CP, n=11) had a substantially better outcome with a disease free survival (DFS) of 88+/-12% after a median followup of 22 months compared to patients transplanted in 2. CR or 2 CP ("intermediate risk";DFS 59+/-18%) or "high risk" (>2. CR, 2.CP, no remission; DFS 20+/-10%). The incidence of relapse was 0% in the good risk, 20% +/-18% in the intermediate and 84 +/-10% in the high risk group. Interestingly, all patients with acute GvHD had a significantly better outcome (DFS 74.1 +/-13%) regardless of assigned risk group. These findings demonstrate the importance of GvL effects in treating myeloid leukemia patients with allogenic HSCT. Furthermore, controlling GvHD intensity either by reducing anti-GvHD prophylaxis or by inducing GvHD/GvL with infusion of donor lymphocytes may prove to be beneficial for patients in RIC regimens. In conclusion, our results demonstrate the value of reduced intensity conditioning regimens for allogeneic HSCT especially in good and intermediate risk myeloid leukemia patients.

Reduced-intensity conditioning allogeneic transplant in patients with indolent non-Hodgkin lymphoma: result of a prospective multicentre trial D. Caballero, R. Martino, M. Mateos, A. Urbano, V. Rubio, J. Odriozola, J. Ribera, J. Sarrá, D. Valcarcel, M. Cañizo, J.M. Moraleda, A. Sureda, J. Sierra, J. San Miguel, Hospital Clínico, Hospital Santa Creu i Sant Pau, Hospital SAS de Jerez de la Frontera, Hospital Ramon y Cajal, Hospital Germans Trials i Pujol, ICO, Hospital Morales Meseguer, Institute Catala d'Oncologia, Hospital Universitario Salamanca (Salamanca, Barcelona, Jerez de la Frontera, Madrid, Badalona, Murcia, E) With the following button you have the possibility to add graphics to your abstract. The graphics must be in GIF or JPG format. Please spIn September 1999, we started a prospective multicenter trial with RIC allogeneic transplant in hematological malignancies. Up to 30 september 2003, 25 patients had indolent lymphoma-20 follicular (FL) (80%), 2 marginal lymphoma (ML) and 2 Waldenstrom macroglobulinemia(WM) The conditionig regimen consisted of fludarabine 30 mg /m 2 intravenously (IV) on days -8 to -4 followed by melphalan 70 mg/m 2 IV on days -3 and -2. Filgrastim-stimulated peripheral blood stem cells from related HLA identical donors were infused on day 0. GVHD prophylaxis consisted of cyclosporine A (CsA) from day -7 plus short-course methotrexate (MTX) (10mg/m 2 , days +1, +3, +6) , followed by folinic acid rescue. Median age at transplant was 50 years, (range: 33-67).According to the status at transplant,only 2 patients with FL(8%) were in CR1, 5 in CR2(20%) and 18(72%) had active disease-12 sensitive and 6(24%) progressive disease-.At transplant 8 out of 18 patients(44%) had bone marrow involvement. Median number of treatment lines before the transplant was 3(1 to 4), moreover 8(31%) patients had failed a previous autologous transplant.. All except one patient died on day plus 10 engrafted. Acute GVHD grade I-IV developed in 10 patients (40%),grade II-IV in 6 (24%) and grade IV in 4 (16%) .From 15 patients at risk, 11 (73%)developed chronic GVHD, extensive in 6 (40%) patients at risk. With a median follow-up of 22months (range: 3-47.8)), 15 patients(60%) are alive and disease free and 10 patients have died due to transplant related mortality. TRM is significantly higher for patients older than 50 years (17% vs 60%; p:0,01). TRM is not influenced significantly by other factors. Estimated Overall survival (OS) and EFS at 48 months are 58 and 53 % respectively. No patient have relapsed after de transplant. Comments: This RIC program associated with good engraftment and very good control of the disease; however, TRM is still high for patients older than 50 and new strategies should be designed for those patients.

Allogeneic haematopoietic stem cell transplantation after reduced-intensity regimen containing fludarabine, busulfan, and antihuman T-lymphocyte globulin (ATG) M. Doubek, J. Mayer, Z. Koristek, M. Krejci, Y. Brychtova, M. Navratil, I. Vasova, M. Tomiska, I. Buchtova, University Hospital Brno (Brno, CZ) Background. Reduced-intensity regimens offer the possibility to perform allogeneic transplantation also in patients who are not eligible for conventional conditioning regimens. Aims and methods. A nonmyeloablative preparative regimen was retrospectively evaluated in all 44 patients transplanted in the University Hospital Brno using fludarabine (30 mg/m 2 /d; 5 days), busulfan (total dose 8 mg/kg), and ATG (10 mg/kg/d; 4 days). Results. The patients of a median age of 53 years, with a variety of haematologic malignancies (14 AML, 13 CML, 5 NHL, 4 CLL, 3 HD, 2 MM, 2 MDS, 1 AA), were not considered for conventional myeloablative regimens because of age, comorbidity, or complications of a previous therapy. Bone marrow from 1 HLA-matched unrelated donor, peripheral blood stem cells (PBSC) from 1 HLA-mismatched sibling, PBSC from 1 HLA-mismatched unrelated donor, and PBSC from 41 HLAidentical siblings were used. Graft-versus-host disease (GvHD) prophylaxis consisted of cyclosporine in 42 patients. Cyclosporine with methotrexate was used in 2 patients. The median number of transplanted CD34+ cells and CFU-GM was 7.5x10 6 /kg, and 108.0x10 4 /kg, respectively. 43 patients engrafted. Leukocyte count >1x10 9 /L was reached in median on day +19. Thirty-two patients developed febrile neutropenia. The incidence of acute GvHD was 33%, severe acute GvHD was seen in 5%. Limited chronic GvHD developed in 13 patients, extensive chronic GvHD in 2 patients. Four patients were retransplanted using the same regimen or reduced BuCy regimen for disease relapse. One patient with AML developed graft rejection 4 months after transplantation. Transplant related non-hematologic toxicity was seen in 8 patients. Severe nonhematologic toxicity was observed in only 3 patients. Venoocclusive disease was not observed. On day +100, complete chimerism was achieved in 58% of patients. CMV disease was observed in 1 patient. During a follow up, 32% of patients experienced relapse or disease progression after a median of 209 days. 14 patients have died. Causes of death were relapse or disease progression (n=7), infection (n=5), GvHD (n=1) and severe bleeding (n=1). Transplant-related mortality was 7%. At present 66% of patients survive disease free. Conclusion.

Reduced-intensity conditioning containing fludarabine, busulfan, and ATG is well tolerated. It causes manageable hematological toxicity; non-hematological toxicity is limited.

Haematopoietic chimerism monitoring is useful in evaluation of clinical outcome in patients with haematological malignancies treated with non-myeloablative allogeneic transplantation J. Gozdzik, M. Iwola, K. Lewandowski, J. Hansz, M. Komarnicki, University of Medical Sciences (Poznan, PL) To evaluate the clinical outcome of the disease of patients (pts) with hematological malignancies treated with non-myeloablative allogeneic stem cell transplantation, the hematopoietic chimerysm screening is necessary. We have analyzed the hematopoietic chimerysm status in 67 pts transplanted using non-myeloablative regimens. The study group (44 males and 23 females; mean age 50, range 30-63 years) consisted of patients with chronic myeloid leukemia (17), acute myeloid leukemia (13), chronic lymphocytic leukemia (11), multiple myeloma (9), non-Hodgkin lymphomas (7), myelofibrosis with agnogenic myeloid metaplasia (4), myelodysplastic syndromes (3), chronic eozynophilic leukemia (2) and paroxysmal nocturial haemoglobinuria (1) . The conditioning regimens consisted of fludarabine (150 mg/m 2 ) and busulfan (8 mg/kg) in 44, fludarabine and melphalan (140 mg/m 2 ) in 15, and fludarabine with cyclophosphamide (2250 mg/m 2 ) in 4 pts. Another 4 pts were conditioned with low-dose total body irradiation (200cGy). All pts were grafted using peripheral blood stem cells collected from donors after mobilization with a help of G-CSF. Hematopoietic chimerysm was assessed on day +30, +60 and +90 after engraftment using a DNA obtained for peripheral blood leukocytes and PCR methods described by Rapanotti and Bader. Full donor chimerysm was documented on day +30 in 42% and mixed in 54% of the pts. Autologous engraftment was found in 4% of pts. Similar analysis performed on day +60 and +90 showed complete chimerysm in 42% and 28%, mixed chimerysm in 48 and 43%; and autologous engraftment in 8% and 13% of pts, respectively. On the basis of the chimerysm results on days +60 and +90 adoptive immunotherapy was used in 11 (16%) pts who showed tendency to autologous hematopoesis. In 3 pts immunotherapy produced complete hematopoietic chimerism. Conclusions: allogeneic non-myeloablative stem cells transplantation is effective method of the treatment of poor prognosis pts with myeloid and lymphoid malignancies. This is also true in a case of older patients, with coexisting disorders routinely excluded form myeloablative therapy. Monitoring of chimerysm by PCR allowed to rationale use donor lymphocyte infusion in the prevention of disease relapse after nonmyeloablative transplantation.

Successful engraftment after second allogeneic haemtopoietic cell transplantation following conditioning with 3 Gy total body irradiation in patients who rejected after conditioning with 2 Gy TBI U. Hegenbart, J. Shizuru, M. Stuart, L. Uharek, T. Lange, W. Pönisch, M. Raida, S. Leiblein, H. Al-Ali, L. Grommisch, E. Edel, U. Schönfelder, S. Heyn, M. Schwittay, F. Kamprad, R.F. Storb, K. Blume, D. Niederwieser, University Leipzig, Stanford University, FHCRC (Leipzig, D; Stanford, Seattle, USA) Graft rejection remains an important issue in patients who had been treated with allogeneic HCT from unrelated donors using conditioning with 2 Gy total body irradiation (TBI) + fludarabine (FLU) continued with mycophenolate mofetil (MMF) and cyclosporine (CSP). A phase-I-study at the Stanford University showed that patients who rejected their grafts after conditioning with 2 Gy TBI could be rescued by a second allogeneic HCT after dose-increased immunosuppressive conditioning with 3 Gy TBI/FLU. We report on 3 patients (Ph-neg CML, AP=1, secondary AML, CR1=1, MM, PR=1) given HCT from unrelated donors who rejected their grafts using the 2 Gy approach. Patients were 38, 55 and 58 years old. A median of 83 (range 71-418) days after the first HCT, a second HCT from a different unrelated donor has been performed. 2 patients received grafts from 1-Ag-mm donors. Conditioning included 3 Gy TBI on day 0, FLU (30 mg/m2 /day on days -4, -3, and -2), MMF 3g/day started day 0 to be tapered from day +40 until day +130 and CSP started day -1 to be tapered from day +100 until day +180, dependent on occurrence or presence of GvHD. The median follow-up is 127 days (range 29 to 205) after second HCT. Major non-hematological side effects included nausea and vomiting (CDC grade III). Two patients showed FUO in the neutropenic phase after HCT. Two patients were neutropenic at time of transplant from prior graft rejection or chemotherapy. Hematological recovery was fast with ANC < 0,5/nl of 14 (range 7-16) days and platelets < 20/nl of 11 (range 0-12) days in median. Chimerism analysis on day +28 showed a median of 100% donor cells in all sorted fractions (T-cells, granulocytes, CD34+ cells), in peripheral blood and bone marrow as well. Acute GvHD grade II of the skin occurred in 2 patients and was successfully treated with prednisolone in 1 patient and with topic steroids and PUVA in the second patient. One patient developed limited chronic GvHD. All 3 patients are alive and in partial or complete remission of their diseases. We conclude that allogeneic HCT after conditioning with 3 Gy TBI + FLU is safe and can rescue patients with hematological malignancies who rejected their graft after condtioning with 2 Gy TBI/FLU.

Economic analysis on reduced-intensity cord blood transplantation D. Kato, K. Yuji, S. Takagi, E. Kusumi, S. Miyakoshi, J. Ueyama, S. Morinaga, S. Taniguchi, Toranomon Hospital (Minato-ku Tokyo, J) Non-myeloablative regimens have been proven to allow engraftment following allogeneic stem cells transplantation with minimal procedure-related toxicity and lower costs. Cord blood has emerged as an appealing alternative source of hematopoietic stem cells for unrelated donor transplantation, but delayed engraftment and frequent transfusion were reported. Few studies have formally evaluated the cost of reduced-intensity cord blood transplantation. We performed an economic analysis of data from 36 patients in a clinical trial of reduced-intensity cord blood transplantation for hematologic diseases at a single institution between February 1, 2002, and August 31, 2003 . Data on resource use, including hospitalizations, medical procedures, medications, and diagnostic tests, were abstracted from subjects' clinical trial records. Resources were valued using the Japanese national insurance reimbursement system for inpatient costs at one hospital and average wholesale prices for medications. Monthly costs were calculated and stratified by treatment group and clinical phase. Mean follow-up was 77 days. The median initial inpatient cost in euros was 70,011 (range, 36,018 to 132,850). When baseline variables were considered, disease status was significant predictor of costs. When clinical events were considered, in-hospital death was associated with higher costs. The association between mortality and higher costs suggest that prevention of clinical complication may have significant economic benefits. Interventions that decrease these complications may have favorable cost-benefit ratios, and will be the focus of future investigation.

Importance of disease stage and C-GvHD for curative potential of allo-PBSCT after a reduced-intensitiy regimen using fludarabine (125mg/m 2 ) and melphalan( 80mg/m 2 ) J.F. Tomas, J. de la Serna, A. Alegre, I. Buño, A. Roman, J. Lahuerta, Fundación Jimenez Díaz, H 12 Octubre, HU La Princesa, HUGM, H La Paz (Madrid, E) The use of nonmyeloablative regimens in patients not suitable for allogeneic SCT because of poor performance status or advanced age has become a field of clinical research in the last few years. The optimum low-intensity allogeneic SCT technique, which maximizes engraftment and mantains GVT effects while minimizing transplant-related complications, has yet to be defined. We started a pilot study in March 1999 in order to explore whether the use of fludarabine (25/m 2 /day/5 days) and melphalan (80mg/m 2 ) followed by non-manipulated G-CSF mobilized PBSC was able to obtain a sustained stable donor engraftment and also to exert a GVT effect, with a minimum toxicity. Intravenous CsA alone (3mg/kg/day) started on day -1 was used to prevent GVHD and graft rejection in the first 15 patients, and CsA with methotrexate (days +1,3,6,11) in the last 33 patients. So far 48 cases has been transplanted. Median age was 52 (24-68). Median number of CD34+ infused cells were 4,8 x 10 6 / kg. Underlaying diseases (12 in CR) were: NHL (18), HD (4), MM (7), AML (9), CML (3), MDS(4), ALL (1), CLL (1). In 22 cases (47%) the procedure was a second SCT. Regimen related toxic death ocurred in 2 cases (4%) and non-relapse mortality (GVHD ± infection) in 7 patients (15%). No one patient rejected the transplant and chimerism assesed at day 30 showed complete donor chimerism in 50% of evaluated patients. Acute GVHD ocurred in 66% of those that received CsA alone vs 38% of tose that treated with CsA/Mtx (p<0,05).. Antitumor response after SCT was evaluated in 34 patients, beeing complete in 17, partial in 8 and absent in 9. With a median follow-up of 26 months (6-56) 24 patients survived after transplant, and 21 of them are in continous complete remision. Overall survival and DFS at 4 years for the whole series were 54±8% and 41±8% respectively. No one patient with MM mantained CR after SCT. Both absence of C-GVHD and active disease at SCT were associated with a worse DFS and selected in a Cox regression analysis (RR: 1,25 and 8,21 respectively).

Allogenic peripheral blood stem cell transplantation following a non-myeloablative (NMST) conditioning with fludarabine and low dose TBI in patients with haematological malignancies and metastatic melanoma: a single-center experience E. Benedetti, F. Papineschi, F. Caracciolo, S. Galimberti, A. Azzarà, G. Carulli, M. Petrini, University of Pisa (Pisa, I) NMST is considered an option worth to be explored for patients with hematologic malignancies not deemed candidates for conventional allografts because of age or medical controindications. Twenty patients median age56.5(range 26.1-67.7) have been transplanted, 8/20 patients received a previous ASCT eighther as a disease debulky or as salvage post relapse, 1/20 received MEL100mg/m 2 as part of the conditioning, and 55% were in advanced phase (relapse/refractory) at transplant.The basic conditioning regimen included fludarabine 30 mg/m 2 for 3 days and low-dose TBI (200cGy) followed by GCSF mobilized PBSC from an HLA identical sibling; postgrafting immunosuppression was with a combination of mycofenolate mofetil (MMF) and cyclosporine (CSA). Diagnosis included 10 multiple myelomas (MM), 1 Myelofibrosis (MF), 1 acute lymphoblastic leukemia (ALL), 1 acute myelod leukemia (AML) 1st CR, and 3 beyond 1st CR and 1 chronic lymhpcitic leukemia (CLL). A median of 6.4 x 10 6 CD34cells/kg (range 5.0-9.5) were infused. Median donor T cell chimerism was by day 28 and by day 84 was 86.5% and 95% respectively. Overall aGVHD developped in 57.8% of patients and was grade I in 15.7%, grade II in 36.8%, grade III 0% and grade IV in 5.2%. There was 1 rejection and was non fatal. Six out of 10 assessable patients developped cGVHD, in 4/6 was clinical extensive; 3/ 4 had quiescent and 1/ 4 had de novo cGVHD. All patients responded to treatment with improvement of symptoms and signs and 2/ 4 are off immunosupprssion at 1125 and 500 days of follow-up.

Overall response rate was in 13/20 patients (65%), of responding patients PR were 76.9% and CR 23%. Responses (5MM 1 MF 3 AML) were mostly observed if status at transplant was PR or CR although 4 PRs were seen in relapsed/refractory MM (3) and in progressive MF (1) . Disease recurrence/progression was the most frequent cause of death (36.8%) while overall TRM was 20%. DLI was performed in 3/20 patients for recurrence or progression but no CR was achieved, and in 2/20 because of rejection: unsuccessfull in 1 patient withdowing immunosuppresion , and successfull in 1 patient treated with immunosuppression and one preceding dose of Fludarabine. In conclusion responses were encouraging mostly occurring patients with stable disease pre transplant with acceptable TRM considering that these patients were not considered elegible for a conventioal transplant and that more than 50% were in advanced phase pre-transplant. Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment for patients with hematological malignancies but its use is limited to young patients without comorbidities. The aim of present study was to evaluate the efficacy of a reduced-intensity conditioning (RIC) consisting of fludarabine and TBI in older patients aged 60 to 70 years. Methods: between March 2000 and October 2003, 18 patients (median age 62 years; range 59-70) with hematological malignancies (n=12 MDS; n=3 acute leukemia; n=1 CLL; n=1 myelofibrosis) and solid tumor (n=1 renal cell carcinoma) were treated with a RIC regimen based on fludarabine (30 mg/m 2 x 3-5 days) and 200 cCy TBI followed by alloHSCT from a matchedsibling donor. GVHD prophylaxis consisted of cyclosporine and mycophenolate. The source of stem cell was blood in all patients. The median CD34+ and CD3+ contents of the grafts were 6.1 x 10 6 /kg and 2.7 x 10 8 /kg respectively. Three patients had received a prior allograft. Eight patients had active disease at transplantation, 5 patients were untreated and 5 were allografted in complete remission (CR). Results: neutrophil recovery occurred in 13 patients (72%) at a median time of 16 days (range 10-33). One patient who died early after transplantation (day +5) and 3 patients who experienced an autologous reconstitution, failed to reach this threshold; 1 patient did not become granulocytopenic. Eleven of 15 evaluable patients (73%) developed acute GVHD (n=5 grade I; n=6 grade II), whereas chronic GVHD was observed in 9 of 11 patients (82%) surviving more than 100 days (limited in 7 cases and extensive in 2 cases). Two patients (11%) died of treatment-related complications: 1 patient died of toxic encephalopathy on day 5 after transplantation and 1 patient of thrombotic thrombocytopenic purpura on day 915. Six patients (33%) died as a result of relapse. With a median follow-up of 505 days (range 4-1087) 10 patients are alive (6 with CR and 4 with relapsed disease). The probability of overall survival at 2 years was 83% for patients treated in CR and 28% for patients transplanted in advanced phase of disease (p= 0.03).

Conclusion: these preliminary data indicate that alloHSCT can be successfully performed in patients older than 60 using a RIC regimen. Chronic GVHD was the major late complication whereas the leukemic burden at time of transplant has proven to be the most important risk factor for the outcome.

Fludarabine-melphalan reduced-intensity conditioning matched sibling bone marrow stem cell transplantation is an effective treatment for myeloid disorders R. Malladi, A. Peniket, G. Collins, T. Littlewood, John Radcliffe Hospital, John Radcliffe Hospital Clinical Haematology (Oxford, UK) Reduced intensity conditioning(RIC) followed by matched sibling allogeneic bone marrow stem cell transplantation is showing promise in the treatment of myeloid disorders.We report the results of a fludarabine(150mg/ m2 ) and melphalan(140mg/m 2 ) conditioning regimen without T-cell depletion in 16 patients(11 male,5 female) with predominantly poor risk AML(n=10),AML/MDS(n=2) and MDS(n=4).The disease characteristics of the de novo AML patients were resistance to primary chemotherapy(n=3),relapsed disease(n=3),complex cytogenetics(n=1),standard risk(n=2),good risk(n=1).3 MDS patients were RAEB and 1 was RA.The median age of all patients was 47 years(27-66).There were 6 sex mismatched transplants,of which 5 were from female donors.GVHD prophylaxis was with cyclosporin alone(n=8) or cyclosporin and methotrexate(n=8).Of the AML & AML/MDS patients,10 were in CR and 2 were in PR at transplant.14 received PBSC with a median CD34 positive dose of 3.91 x 10 6 /kg(range 0.88-8.19) and 2 received bone marrow.Median time to neutrophil recovery >1 x 10 9 /l and platelet recovery >50 x 10 9 /l was 15 days(range12-32) and 14 days(range8-26) respectively.1 patient required a 2nd PBSC infusion in order to achieve platelet engraftment.Chimerism by sex chromosome karyotype of bone marrow cells by FISH in the six sex mismatched transplants showed full donor chimerism in all 6 patients when first tested between 12 & 47 days posttransplant.No patients required DLIs.Only 1 patient(with RAEB) relapsed and then subsequently died giving a relapse mortality of 6%.12/16(75%) patients remain alive and in complete remission on completion of the study with a median follow-up of 553 days(range 200-1612).The actuarial 4 year disease-free and overall survival was 75% for both.The nonrelapse mortality was 3/16(19%) .The cause of death in each case was CVA with active aGVHD grade III,myocardial infarction and idiopathic pneumonitis respectively.Overall,aGVHD occurred in 53% (8/15),with aGVHD grade II or above occurring in 47% (7/15).In the 14 evaluable patients,cGVHD occurred in 53%(7/13),with this being extensive in 4 patients.The donor or recipient were CMV seropositive in 12 patients.Of these,CMV reactivation occurred in 2/12(17%).No patient developed CMV disease.This RIC protocol introduces a GvL effect against poor risk myeloid disorders and has a low non-relapse mortality.This is achieved without T cell depletion.Further studies are warranted to investigate this treatment strategy.

Red blood cell and reticulocyte chimerism studies in nonmyeloablative allogeneic peripheral blood haematopoietic stem cell transplantation L. Harritshoj, A. Berkowicz, S.L. Petersen, H.O. Madsen, L.P. Ryder, L. Vindelov, P. Andersen, E. Dickmeiss, Rigshospitalet (Copenhagen, DK) Patients and materials: 17 patients with hematologic malignancies were treated with non-myeloablativ allogeneic peripheral blood stem cell transplantation (NM-PBSCT). They were conditioned with fludarabine and 2 Gy of total body irradiation, and received stem cells from either human leucocyte antigen identical sib-lings or unrelated donors. Peripheral blood (PB) was investigated for red blood cell (RBC) chimerism day 14, 21, 28 after NM-PBSCT to detect onset of donor RBC chimerism, and in addition investigated about day 60, 90 and 120. Moreover some patients were investigated weekly for reticulocyte chimerism until complete donor reticulocyte chimerism was reached. Methods: RBC chimerism was investigated by flowcytometry (FC), by commercially available blood group antibodies and FITC-or PE-conjugated F(ab) 2-anti-immunoglobulin. Reticulocyte chimerism was investigated by two color FC, gating on the 50% brightest thiazol orange stained reticulocytes. In RBC chimerism studies blood transfusions had to be selected to be negative for the blood group antigens used in the studies. Results: Median time for detection of donor derived RBC was 14 days (range 14-21). The course of RBC chimerism in 10 patients until day 120, showed nearly complete donor RBC chimerism (median 97.5 % range 23-100%) in PB about day 120. These data correspond to an increase of approx. 1% donor RBCs per day and disappearance of recipient derived RBCs with a rate of approx. 1% per day. The reticulocyte chimerism studies showed, that the fraction of recipient derived reticulocyte fell abruptly from day 14 with a corresponding increase in donor derived reticulocytes close to 100% day 28-35. These findings correspond closely with the kinetics of the granulocyte chimerism measured from the same patients. We could not detect a significant delay in the appearance of donor derived RBCs in the major ABO mismatched transplantations stud-ied (n=9) as compared with the ABO compatible transplantations (n=5). Conclusion: Our data show that the donor marrow contributes to nearly all of the erythropoiesis within 2-5 weeks after the NM-PBSCT, in parallel with donor contribution to granulopoiesis. Also our data could not show any delay in the donor RBC chimerism among patients receiving a major AB0 incompatible marrow.

Fresh frozen plasma plus heparin has a possible beneficial effect in veno-occlusive disease prophylaxis: a retrospective multivariate analysis E. Yannaki, I. Batsis, P. Kaloyannidis, C. Smias, I. Sakellari, A. Fassas, A. Anagnostopoulos, The George Papanicolaou General Hospital (Thessalonica, GR) It has been shown that significant differences exist in the mean levels of ATIII and Protein C (PrC) between patients who develop VOD and those who do not. Since 1994, we treat the majority of transplanted patients with heparin+2FFP/day during conditioning, as this regimen could potentially protect more effectively from VOD, by replacing to some degree the consumption of endogenous anticoagulants and by augmenting the function of heparin (HEP) due to corrected ATIII levels. We retrospectively analyzed the incidence of hepatic VOD, from 1990 through 2001, in 403 consecutive allogeneic (siblings 138, VUD 18, syngeneic 2) and autologous (245) blood or marrow transplants, comparing two VOD prophylactic regimens and no VOD prophylaxis. Patients underwent BMT for leukemia (158), lymphoma (151), MDS (7), MM (28), solid tumors (4) and non malignant diseases (55). Patients received HEP (27), HEP+2FFP/d (306), and no prophylaxis (70). Overall VOD incidence was 8.7% and VOD by prophylactic regimen, was significantly lower in the HEP+FFP group compared to HEP group and no prophylaxis group (5.9%vs20%vs15.7% respectively, p<0.01). VOD was the primary cause of death in 28.6%. ATIII and PrC on day 8 were significantly lower in the VOD group compared to the non-VOD group (69±26% vs 89±19% and 68±26% vs 91±29% respectively, p=0.001). In univariate analysis, significant associations (p<0.05) were detected between VOD incidence and multichemotherapy, hepatotoxic drugs, liver status, ATIII and PrC day 8, BMT number and CMV reactivation. In multivariate analysis, as independent risk factors raised: the administration of more than 2 hepatotoxic drugs (p<0.001, rr5.0), Hepatitis B or C (p=0.03, rr3.08) and BMT number >1 (p=0.01, rr7.0). ATIII and PrC day 8 were excluded from first analysis because of many missing data. However, when the analysis was conducted in a subset of 198 patients (all having recorded data for ATIII+PrC), factors significantly related to VOD were: low PrC on day 8 (p=0.03, rr3.0) BMT number >1 (p=0.02, rr10) and abdomen radiotherapy (p=0.02, rr5.0). We conclude that FFP during conditioning in addition to HEP has potentially a higher prophylactic effect, presumably by minimizing the drop of natural anticoagulants around day 8. Because of this possible beneficial effect in VOD prophylaxis of HEP+FFP, we plan to initiate a prospective randomized trial in order to confirm these data.

G-CSF after CD34+ immunoselected peripheral blood stem cell transplantation: a novel administration schedule N. Piccirillo, S. De Matteis, P. Chiusolo, L. Laurenti, F. Sorà, R. Putzulu, G. Reddiconto, G. Leone, S. Sica, Universita Cattolica S. Cuore (Rome, I) G-CSF is widely used to accelerate haemopoietic recovery after peripheral blood stem cell transplantation (PBSCT); there is no consensus about its indications and schedules of administration. G-CSF has not been used as a routine procedure after PBSCT in our center, but it has been introduced after immunoselected CD34+PBSCT in small randomized clinical trial. In that study, we demonstrated that early G-CSF administration after autologous CD34+PBSCT accelerates neutrophil recovery, thus off-setting the lack of committed progenitors in the purged graft, while induces a 7-12 fold increase of G-CSF serum levels. These results suggested us to evaluate the feasibilty of an every other day administration schedule in order to reduce costs and drug exposure. From April 1999 to September 2002 we studied 33 consecutive patients submitted to CD34+PBSCT: 12 female, 21 male, median age 48 years (range 17-62), affected by Non Hodgkin Lymphoma (17 pts), Multiple Myeloma (12 pts), Hodgkin Disease (4 pts). The patients were randomized in two groups: A group, who received G-CSF (standard dose 263 microg/die) from day +1; B group, who received G-CSF on alternate day. Groups were comparable for age, sex, diagnosis and conditioning regimen. Median number of CD34+ cell/kg infused was 3.94 x 10 6 and 5.1 x 10 6 /kg in group A and B respectively (p=ns). We evaluated haemopoietic recovery, length of hospitalization, antibiotic therapy, sepsis occurrence, number of G-CSF vials administered and transfusional requirements. We did not find statistical significant differences in all analyzed endpoints. On the other hand, every other day G-CSF administration (B group) allowed a significant reduction in number of G-CSF vials used (A group 11 vials, B group 6 vials, p<0.0001). Every other day G-CSF administration after CD34+PBSCT was safe and efficacious achieving the same results of the usual daily administration, combined to a decrease of G-CSF use. This schedule reduced transplant costs (260€ per patient) and, particularly, drug exposure. Our results showed the safety and feasibility of an every other day G-CSF administration schedule in CD34+PBSCT setting. This schedule, implying a reduction in G-CSF use, induces a decrease of both costs of transplant procedure and drug exposure without any detrimental effect to haemopoietic recovery or other clinical outcomes. Further studies are needed to confirm the optimal dose and schedule of G-CSF administration after CD34+PBSCT.

S. Buchholz, A. Woywodt, J. Scheer, L. Hambach, H. Kamal, A. Ganser, H. Haller, M. Haubitz, B. Hertenstein, Medical School Hannover (Hannover, D) Major complications after allogeneic blood stem cell transplantation (HSCT) like microangiopathic haemolytic anemia, veno-occlusive disease of liver (VOD), capillary leak syndrome and GvHD are pathophysiologically related to endothelial damage. Circulating endothelial cells (CEC) are specific and sensitive markers of endothelial damage. The aim of this study was evaluation of CEC in patients undergoing allogeneic stem cell transplantation as a marker of endothelial toxicity. 39 patients (20 male, 19 female; AML n=21, ALL n=4, CML n=10, others n=4) were studied. 28 patients received conventional conditioning regimens (TBI/cyclophosphamide n=14; busulfan/cyclopshophamide n=14). Non-myeloablative regimens were used in 11 patients (fludarabine/busulfan n=9, melphalane n=2). CEC were measured before (baseline) and after conditioning therapy and 7, 14 and 21 days after transplantation. 22 age-matched healthy controls were also studied. In 13/39 patients, CEC were measured after a longer follow-up (range 181 to 451 days, median 305 days after transplantation). CEC were isolated from peripheral blood by use of Dynabeads coated with antibodies against CD 146, were stained for UEA-1 and counted. Cell numbers were significantly elevated (range 8-64, median 16 cells/ml, p < 0.001) at baseline compared to controls (range 4 -24 cells/ml, median 8 cells/ml). CEC increased after conditioning (range 16 -424 cells/ml, median 44 cells/ml, p < 0.001 compared to baseline). Patients undergoing reduced-intensity conditioning showed significantly lower cell numbers (range 20-52 cells/ml, median 24 cells/ml, p < 0.05) compared to conventional conditioning. There was no difference in cell numbers between patients who were treated with TBI (median 58 cells/ml) or myeloablative chemotherapy only (median 52 cells/ml, n. s.) but patients with TBI had a much earlier increase of cells after conditioning. CEC remained slightly elevated at long term follow up. (range 12 -40 cells/ml, median 20 cells/ml). CEC are elevated before conditioning in patients undergoing HSCT. In patients undergoing TBI, cells increased much earlier than in patients who received only chemotherapy. Reduced intensity regimens showed significantly lower cell numbers due to less toxicity. Further studies are needed to investigate larger numbers of patients and provide longer follow-up. Circulating endothelial cells are a promising new marker to monitor mircrovascular endothelial damage in patients undergoing HSCT.

S. Ladeb, A. Abdelkefi, Y. Benabdennabi, F. Mellouli, T. Benothman, L. Torjman, L. Kammoun, S. Aouf, M. Bejaoui, A. Benabdeladhim, Centre National de Greffe de Moelle Osseuse (Tunis, TN) Objective: Although the equimolecular mixture of oxygen and nitrous oxide (EMONO) seems a good choice to relieve procedure-related pain in children, it has not been evaluated for insertion of central venous catheters in children. To assess the safety and the effectiveness of this gas mixture for insertion of central venous catheters, we conducted a prospective study. Methodology. This study was performed by the « centre national de greffe de moelle osseuse ». Procedure and inhalation characteristics, as well as pain evaluations and side effects, were reported. Results: Forty central venous catheters were inserted in 40 consecutive children. Median age was 7 (4-13) years. Seventy percent of children were younger than 10 years of age. The catheters were placed for the following indications: bone marrow transplantation (n=20), chemotherapy for acute leukemia (n=14), and complications of sickle cell disease (n=6). EMLA cream (Astra) was applied 2 hours before EMONO inhalation. No drug association was used. All catheters (polyurethane, 2 lumen) were inserted by the same experienced physician in the operating theater, into the subclavian vein with use of the Seldinger technique. We did not observe any mechanical complications. The inhalation system included a whistle, a mask, and a non breathing respiratory valve. No initial physical restraint was needed. Median inhalation length was 5 minutes (3-6) minutes before starting catheter's insertion and 12 minutes (9-25) minutes for the total inhalation. Median procedural pain evaluations were 10 (0-30) for children on a 0 to 100 visual analog scale (VAS). Ninety six percent of the 36 children who were able to answer the question said they would accept EMONO analgesia if a new procedure was to be performed. Staff satisfaction regarding EMONO efficacy was as follows : very satisfied (65%), satisfied (32.5%), not satisfied (2.5%), and very unsatisfied (0%). Minor side effects were observed during 8 (20%) inhalations. These side effects were euphoria (15%), deep sedation (10%), nausea and vomiting (2.5%), hallucinations (2.5%). All side effects were transient and vanished within 5 minutes after removing the inhalation device. No serious side effects were noted. Conclusions: To our best knowledge, this is the first study which shows that EMONO is effective for insertion of central venous catheters in children, and can represent a simple and safe alternative to general anesthesia.

Protein-C serum levels as a prognostic indicator of sepsis and related diseases in allogeneic bone marrow transplantation F. Gualandi, D. Occhini, C. Di Grazia, A. Dominietto, A.M. Raiola, A. Bacigalupo, Ospedale San Martino (Genoa, I) Background: numerous studies showed that sepsis and disseminated intravascular coagulopathy (DIC) are associated with a disruption of the normal balance between coagulation and fibrinolysis and that the Protein-C pathway plays a critical role in maintaining hemostasis; on the other hands several reports suggested a role for the Protein-C in the inflammatory response either to endotoxin or bacteria in vivo or in vitro. Material and methods: in the present study we evaluated protein C levels in 27 patients who underwent allogeneic bone marrow transplantation (BMT), before transplant, at the end of the conditioning regimen and during the neutropenic phase at day +7 and +15. 15 patients were transplanted from an HLA identical unrelated donor and 12 from an HLA identical familiar donor. The conditioning regimen was with TBI in 13 and without in 14 patients. The underlying disease were: ALL (2), AML (11), CLL (2), HD (1), LnH (1), Mielof. (2), CGL (2), SAA (2), MM (3), MDS (1) Results: before transplant and after conditioning regimen all patients had normal protein C levels (range 84-156%); on day +7 and/or on day +15 protein C levels were reduced in 14/27 pts: ten of them developed sepsis (p=0,00003); 3 pts fever without documented sepsis (p=0,002) and 1 no complications. All patients with sepsis had signififcantly reduced protein C levels, while 3 pts with FUO showed normal protein C levels. Conclusions: the toxicity of the conditioning regimen doesn't seems to increase the consumption of protein C, but there is a correlation between septic fever and protein C low levels. The identification of decreased protein C in neutropenic patients with high risk of septic shock before the onset of clinical symptoms may have prognostic value in predicting the outcome and its replacement may be beneficial.

Home care in a department of paediatric haematology and oncology. Results of 3 years of activity M. Miano, A. Garaventa, L. Manfredini, S. Fieramosca, C. Savio, R. Tanasini, G. Dini, G. Gaslini Children's Hospital (Genoa, I) The quality of life of children with cancer is deeply affected by frequent and sometimes long periods of hospitalisation for the treatment of infective or toxic complications due to chemotherapy. To reduce the duration of hospitalisation, in April 2000, a program of Home Care (HC) was activated in our Department in order to improve the quality of life of children and their families, to reduce the costs, and to improve the availability of the hospital ward for more critical patients. Children in stable, non critical conditions needing i.v. therapies including chemotherapy, parenteral nutrition administration, blood examinations, transfusional, physiotherapic and psychological support as well as terminally ill children needing palliative therapies were considered eligible to be admitted to the program. The staff included 2 doctors, 3 nurses, 1 psychologist, 1 physiotherapist, and 1 teacher. After 44 months of activity 158 children, aged 1 month-19 years (median 3 years), were assisted in 190 therapeutic phases (133 during front line therapy, 44 after HSCT, 13 for palliative care). The median duration of the assistance for each child was 38 days (range 1-563). A median of 10 patients per week (range 2-21) were assisted for a total of 8119 days. A total of 989 medical visits, 1815 blood withdrawals, 2048 days of i.v. therapies, 314 of total parenteral nutrition, 118 chemotherapies, 408 transfusions (150 CGR and 258 Platelets),and 230 physiotherapic treatments were performed. A teacher of primary schools performed 6 hours per week at home of selected patients. A total of 2440 accesses at home replaced 1617 and 823 out-patient and in-patient days of hospitalisation, respectively. The average cost per patient given HC (2422 Euro, range 150-30450) resulted significantly lower (p<0.001) compared to the average cost per patient hospitalised to undergo the same procedures (7835 Euro, range 350-132250). A questionnaire evaluating the quality of this kind of assistance compared to hospitalisation was administered to the families. The results of 50 questionnaires seem to show an improvement of the quality of the assistance. The opportunity to reduce the frequency and duration of hospitalisation represents an incalculable advantage for these children and their families. This report shows that HC is a feasible kind of assistance for children suffering from cancer, improves the quality of assistance, and reduces the costs as well.

Octreotide shortens duration of toxic diarrh0ea after highdose therapy and autologous stem cell transplantation M. Kauppila, M. Itälä, M. Putkonen, K. Remes, Turku University Central Hospital (Turku, FIN) Most common problems associated with high dose therapy (HDT) are infections and gastrointestinal toxicity. Severe diarrhea often prolongs hospitalization. Octreotide, an analogue of somatostatin, inhibits the release of different gastrointestinal hormones, decreases intestinal motility and reduces intestinal fluid volume. The aims of this prospective study were to evaluate whether octreotide is tolerated by patients undergoing HDT and autologous blood cell transplantation (ABCT), whether it relieves gastrointestinal symptoms, especially diarrhea, and whether it can shorten the time spent at hospital when compared to controls.

A total of 35 patients were included in the study. After the first 7 pilot patients the patients where divided in timely manner into those who received octreotide and those who served as controls. There were 20 patients in the octreotide group and 15 patients in the control group. Octreotide acetate (Sandostatin®) was started when there were more than three diarrhea episodes a day. The initial dose was 100 µg thrice a day s.c. for 3 days, and then a double dose if symptoms continued. The control patients used loperamide when needed. The mean onset of diarrhea was day +5 (range: 4-10) posttransplant in the octreotide group. Mean duration of octreotide therapy was 5 (2-10) days. In 10 of the 20 patients (50 %) diarrhea fully disappeared and in 4 (20 %) was relieved during octreotide treatment. In 3 patients (15 %) octreotide had no effect. There was a tendency (p=0.059) towards a shorter duration of diarrhea in the octreotide group compared to the control group (mean duration of diarrhea 4 versus 8 days, respectively). There were no significant differences in the occurrence of nausea, abdominal pain or fever between the study groups. Discharge from hospital was the same in both groups. There were no side-effects in 12/20 (60 %) patients in the octreotide group. Gastrointestinal side-effects caused discontinuation of the medication in four patients. Recovery from HDT supported by ABCT is usually fast and spontaneous resolution of diarrhea is common. We observed a tendency towards shortening of the diarrhea period in patients receiving octreotide when compared to controls. However, this did not result in shorter time spent in hospital.

M. David, O. Toth, A. Szomor, B. Meng, H. Losonczy, University of Pécs (Pécs, HUN) Hereditary and acquired coagulation abnormalities with endothelial dysfunction can effect the outcome of stem cell transplantations causing severe thrombotic tendency or contributing to the pathogenesis of hepatic veno-occlusive disease (VOD). Forty one patients (mean age:42±14.68 years, male/female ratio:24/17) were analyzed for hereditary and acquired coagulation abnormalities and endothelial dysfunction during their peritransplant period. The indications for autologous transplantation were non-Hodgkin's lymphoma in 20, Hodgkin's disease in 12, and multiple myeloma in 9 patients. Thrombophilia studies (taken prior to the conditioning regimen) included fibrinogen, APC resistance, Antithrombin, Protein C and Protein S activity and antigen, lupus anticoagulant, FV:Q506 (Leiden mutation), FII 20210A allele and MTHFR C677T mutation assays and serum homocystein levels. Tests for endothelial dysfunction were vWF and PAI-1 antigens (before and 10 days after the transplantation). 69% (28/41) of patients had abnormal thrombophilia studies, of which 32% (13/28) showed combined defects. High fibrinogen levels were detected in 21% (9/41), Protein C deficiency in 14% (6/41) and hyperhomocysteinaemia in 41% (16/41) of the patients of whom only 26% (10/41) were heterozygous for the MTHFR C677T mutation. The vWF antigen levels were high (147.5 ± 37%) before the transplantation and increased significantly (180 ± 32.5 %, p< 0.0002) afterwards, the mean of PAI-1 antigen levels was in the normal range. Three patients developed hepatic VOD with elevated PAI-1 antigen levels and two patients had central line thromboses. The marked hyperhomocysteinaemia could be attributed to acquired factors, especially folate deficiency. The prevalence of MTHFR C677T mutation was not higher than in the general population. As a consequence of the endothelial damage caused by hyperhomocysteinaemia, elevated vWF Ag levels (and in case of hepatic VOD elevated PAI-1 antigen levels) were detected which together with the high fibrinogen level and Protein C deficiency can contribute to the development of thromboembolic complications associated with stem cell transplantation.

Successful treatment of cyclophasphomide-induced intractable haemorrhagic cystitis with novoseven (recombinant activated Factor VII ) after allogeneic bone marrow transplantation M. Zakerinia, H. Nourani Khojaste, M. Ramzi, M. Haghshenash, M. Karimi, E. Ahmad, Shiraz University of Medical Sciences (Shiraz, IR) Summary: From may 1993 through September 2003, allogeneic bone marrow transplantation was performed on 182 patients with transfusion dependent thalassemia major (n=113), leukemia (n=55), lymphoma (n=6) and aplastic anemia (n=8). The conditioning regimen for lymphoma patients were fludarabine 150 mg/m 2 , busulfan (BU) 4 mg/kg, and Cyclophosphamide (CY) 120 mg/kg (non-myeloablative therapy), for thalassemic patients were BU 14-15 mg/kg, CY 200 mg/kg, with or without ATG 40-100 mg/kg, for leukemia patients were BU 16 mg/kg, CY 200 mg/kg, and for aplastic anemia patients were CY 200 mg/kg and ATG 90 mg/kg. CY induced severe refractory homorrhagic cystitis (HC) was observed in 8 patients (4%). Continuos bladder irrigation was employed for all patient with HC and in the first 5 patients despite transurethral fulguration of bleeding vessels, intravesicular alum, silver nitrate and formalin instillation, one young man with CML died, one child with thalassemia major needed suprapubic cystotomy, and for 3 adults with leukemia, internal iliac artery ligation was undertaken which was life saving for two of them. From Sept, 2002 up to now (November 6, 2003) recombinant activated factor VII (NovoSeven) 200-1200 microgram/kg, iv, was used in three patients leading to cessation of hematuria in two of them. They were one child with thalassemia major and an adult with CML. The other patient finally needed ileal conduit (a yong man with CML). Conclusion: We tried rFVIIa (NovoSeven) in cyclophasphamid induced intractable hemorrhagic cystitis for the first time in the world and found that the frustrating and clinically difficult problem to manage can be handled very easily with iv Factor VII a.

Renal dysfunction, is a common complication during the early period of hematopoietic cell transplantation (HCT). In the present study, renal complications were prospectively evaluated in patients undergoing HCT for various hematologic diseases. We prospectively evaluated 50 patients receiving HCT (25 allogeneic, 25 autologous) during a hundred day period. The allogeneic HCT patients received the same graft-versus-hostdisease (GVHD) and antimicrobial prophylaxis. Most of them (22/25) received myeloablative regimens. The autologous HCT patients did not receive any antimicrobial prophylaxis and had different conditioning regimens. Renal dysfunction was classified as follows: Grade 0 (Normal renal function: <25% decrement in creatinine clearence); Grade 1 (>25% decrement in creatinine clearence but < a twofold increase in serum creatinine); Grade 2 (>twofold rise in serum creatinine but no need for dialysis); Grade 3 (>twofold rise in serum creatinine and need for dialysis). Baseline characteristics and renal complications of the patients are shown in the table: 31 of 50 patients (62%) developed renal dysfunction which was significantly more common in allogeneic HCT (48%) than autologous HCT (76%) (P=0.042). Severe (grade 2 and 3) renal dysfunction was significantly more common in allogeneic HCT (60%) than autologous HCT (24%) (P=0.009). 5 patients developed veno-occlusive disease (VOD) of the liver with severe renal dysfunction and 4 of them had allogeneic HCT. VOD was significantly associated with severe renal dysfunction (P=0.005). Six patients (4/6 allogeneic HCT and 2/6 autologous HCT) developed sepsis. 5 of these 6 patients developed severe renal dysfunction. Sepsis was significantly associated with severe renal dysfunction (P=0.029). The overall 100 day mortality rate was 24% (32% in allogeneic versus 16% in autologous HCT). The mortality rate among patients with severe renal dysfunction was 24%. Of all patients with severe renal dysfunction, the mortality rate among allogeneic HCT recipients (33%) was significantly higher than autologous HCT recipients (0%) (P=0.009).

In conclusion, renal dysfunction, is more common in allogeneic than autologous HCT and is associated with high risk of mortality. The use of cyclosporine A for GVHD prophylaxis, nephrotoxicity of drugs used for antimicrobial prophylaxis and increased incidence of VOD in allogeneic HCT may have contributed to development of increased incidence of severe renal dysfunction.

The aim of this study was to evaluate the balance between cost and clinical benefit of the use of G-CSF (granocyte®) after autologous peripheral blood stem cell transplantation for lymphoma or myeloma conditioned with BEAM or high dose melphalan. Method : This study compares a prospective accrued cohort of 57 adult patients receiving 150 microg/m 2 /d of granocyte® on post transplant day 5 with a historical cohort of 42 adult patients who did not. Time engraftment, non-prophylactic antibiotic administration, transfusion, parenteral nutrition, G-CSF and post transplant cost were compared. Post transplant cost included : the cost of a day inpatient stay, transfusions, anti-infectious drugs, parenteral nutrition and G-CSF. Results : G-CSF and no G-CSF groups had no significant differences in age, sex, conditioning regimens or transplant CD34+ cell counts. The mean time of G-CSF administration was 7 days (5-11). G-CSF use was associated with significant more rapid recovering of absolute neutrophil count (mean 8 versus 11) (p<0,0001), decreased duration of antibiotic administration (mean 6 versus 9) (p<0,0001), number of hospital days after transplant (mean 13 versus 15) (p<0.04). The means of platelet engraftment (platelets > 20000/mm 3 ), number of platelet and blood cell transfusions and parenteral nutrition were not different between the two cohorts. Post transplant cost was reduced in the G-CSF group (11869 euro versus 13695). Conclusion : This study confirms that G-CSF administration at day 5 is effective on the clinical outcome and reduces post transplant cost. This work was supported by Chugai

Intensive care for haematopoietic stem cell transplant recipients: admission and outcome. Advantage of a ''preemptive'' policy P. Marenco, R. Cairoli, G. Grillo, A. Tedeschi, P. Brasca, A. De Gasperi, E. Morra, Niguarda Hospital (Milan, I) Life threatening complications requiring intensive care in transplant (tx) recipients are a well-known adverse event during the long course of a tx. Conditioning regimen, immunological disparity between donor and recipient and immunosuppressive therapy for tollerance induction lead to specific pathological patterns: VOD, graft failure, GVHD, viral reactivations, idiopathic pneumonia … Among the problems still under debate are score predictability, admission or not to Intensive Care Unit (ICU), futility or not of intensive treatment and resources availability. To update our behaviour in this specific setting we reviewed clinical data of our BMT Unit in which a close cooperation between haematologists and intensivists has progressively extended. Materials: 282 autologous tx and 140 allogeneic tx (including 41 MUD) have been performed in the Unit. We consider very important an extensive donor/recipient pretransplant evaluation and we use high sterility nursing procedures. Standard myeloablative conditioning and GVHD prophylaxis with routinely adjusted CSPA dosage are used. Heparine is used for VOD prophylaxis. Careful fluid balance is strictly recorded , with prompt correction of hypoalbuminemia ( less than 3 g/dl); of every dispnoea unresponsive to diuretic treatment is immediately monitored with a pulse oxymeter and intensivist is alerted to start a sort a "preemptive" evaluation and treatment of complications ( such as non invasive ventilation i.e NIV ). After discharge facilities at the out-pt dpt are rapidly available for every adverse event.

Results: 17/422 tx (4%) required intensive care. 13 were transferred to ICU ( 2 after neurosurgery) and 4 remained in the BMT Unit and were followed together with intensivist. Among the 17 pts : 11 (8%) were allogeneic tx while :6 (2%) were autologous. Clinical conditions requiring intensive care were: respiratory distress in 7, septic shock in 4, cardiac failure in 2, hemorrhage in 2, CNS infection in 2. Outcome: 59% ( 10/17) survived intensive care , and 38% (6/16 ) were alive 30 days after. Conclusions: ICU admission with this "preemptive" policy is very low; outcomes confirms recent reports of non futility in properly selected pts.

A preemptive attitude, non invasive cardiorespiratory monitoring, ultrasound sonography and facilities for NIV could reduce requirement of more invasive procedures and ameliorate outcome. This program is critical in the view of the increasing age of tx .

Introduction: To evaluate nutritional policy for adults during a peripheral stem cell transplantation at the department of hematology, it is important to know the evolution of bodyweight, resting energy expenditure (REE) and energy intake (EI) during admission. Methods: Bodyweight was assessed three times a week on a calibrated weight scale. REE was measured before breakfast by indirect calorimetry and assessed at admittance before conditioning treatment (t=0), before transplantation (t=1), one week after (t=2) and two weeks after transplantation (t=3) and at discharge (t=4). REE measured was compared with the predicted energy expenditure according to Harris Benedict (HB). EI was assessed by means of nutritional intake forms every other day. Infusion forms were used for registration of total parenteral nutrition (TPN). EI was calculated as the mean of three consecutive measurements (one week). Results: Results are based on 20 patients, more data on a total of 30 patients will follow. Patients characteristics were Acute Leukemia (n=5), Non Hodgkin lymphoma (n=6) and Multiple Myeloma (n=9). There were 3 allogeneic and 17 autologous transplantations. The mean loss of weight during admission was 3.9 kg (n=19). If TPV was indicated, it started after transplantation. The presence of infections or fever didn't influence mean REE. The results on REE and EI are presented in the table. Mean REE is given as percentage of HB. Mean EI is given as percentage REE. A comparison is made between patients with and without TPV. Total energy demand required is minimal 125% REE. Conclusions: The mean of the group showed a negative energy balance during the hospital stay, which resulted in a mean loss of weight of 3.9 kg. Without TPV, patients EI did not comply to REE. Remarkeble, REE was lowest during pancytopenia (t=2), but recovered rather quickly after resolving of the pancytopenia (t=3). It is of importance to adjust the EI to the total energy demand required during the whole admission period. Objective: To analyse transfusion (tr.) requirements after allogeneic HSCT. Material and methods: retrospective study of all the first allogeneic HSCT performed in our Centre between January 2000 and December 2002. We considered: patient diagnosis, hematopoietic stem cell source (HSCS), conditioning regimen, HLA and ABO incompatibility and relationship of donor/recipient pair. We analysed patients' tr. requirements during a 100 days period after transplantation. The median of red blood cells (RBC) and platelet (PC) tr. episodes (TE) were considered as an estimative of the tr. requirements. TE are presented as median (range); differences in TE medians are compared with Mann-Withney or Kruskall-Wallis tests as appropriate. Results: 110 patients (58% male, 42% female), median age 31.5 years , were submitted to a first HSCT. Diagnosis: acute myeloid leukemia 26.4% (29), acute lymphoid leukemia 17.3% (19), chronic myeloid leukemia 12,7% (14), others 43,6% (48). HSCS: peripheral blood (PB) 78,2% (86), bone marrow (BM): 17,3% (19), cord blood (CB): 4,5% (5). Conditioning regimen: reduced intensity (RIC) 31% (34), myeloablative (MA): 69% (76). HLA compatibility: identical 89.1% (98) mismatch 11% (12). ABO incompatibility: major 22.7% (25); minor 22.7% (25), compatible 54.5% (60). Relationship: sibling (93), progenitor (6), unrelated (11). According to the stem cell source the RBC TE were: CB: 8 (6-21); BM: 7 (0-69); PB: 2 (0-92) -p<0.05. PC TE were: CB: 16 (13 -49); BM: 9 (0 -55); PB: 2 (0-77)-p<0.005. RIC regimen required less PC tr. than MA regimens: 1 (0 -46) and 4 (0 -77) respectively, (p<0,005). In HLA matched and mismatched transplants the RBC TE were respectively: 3 (0 -92) and 7 (2 -29)-p<0.005; in these groups PC TE were respectively: 3 (0-77) and 15.5 (3-55) -p<0.005. RBC TE in sibling, progenitor and unrelated transplants were respectively: 2 (0-69), 10.5 (2-92), 9 (5-21) -p<0.005; in the same grous the PC TE were respectively: 2 (0-55), 10 (3-77) and 16 (7-49) -p<0.005. The medians of RBC and PC TE were not statistically different in ABO major, minor and compatible transplants. Conclusions: The use of PB as a source of HSC reduces the transfusion requirements. Reduced intensity regimens are associated with a reduced PC transfusion need. In unrelated transplants and HLA mismatches the transfusion episodes after transplantation are higher. ABO compatibility had not a major impact in transfusion requirements. JACIE asks for qualified persons to perform risk procedures in transplant programs. We report the implementation at Institut Gustave Roussy, Saint Louis, Pitié Salpétrière, Henri Mondor hospitals, concerning collection services, laboratories, and clinical units. Methods: We elaborate a 5 step procedure to fulfill standards: 1 Listing of qualification and risk procedure definitions, setting of the training plan. Nurse and physician managerial staffs defined ability of each staff member allowing to perform risk procedures i.e., conditioning regimen administration, graft infusion. These requirements are listed in 3 records: Position Profile: the required skills at the position; Employee Profile: individual skills that might be useful; Responsibility Charts of actions and procedures performed. In the physician chart, degree of responsibilities is shared like this: "is responsible for, and does definite validation, implements through protocols, implements outer protocols, manages subsequent actions, does the first validation". The nurse chart mentions: "is responsible for, and does definite validation, implements, implements under tutor control, controls, manages subsequent actions". It also mentions physician validation tasks. 2 Writing of the welcoming and training process, writing a procedure about the qualification assessment and record, validation by the hospital headquarter in order to guaranty consistency between services. 3 Welcoming and training process deployment: Tutor roles are to facilitate integration, transmit the procedure knowledge and enhance technical ability. 4 Assessment: The tutor evaluates the ability of new employee on risk procedure after a tutoring period. The supervisor collects the tutor agreement and assesses the candidate on his/ her awareness relatively to the risk management. If positive, the new employee receives the authorization to perform the risk procedure on his/her own. 5 Abilities are yearly re-evaluated. Results: Only qualified and awarded persons are performing risk procedures. Conclusion: The continual improvement supposes auditing annually risk procedures to ensure compliancy with the best medical practices. Outputs are discussed in the top management review in order to ensure that the risk stays well defined and managed with the available abilities. Staff qualification and adverse event management processes are strategic to provide the required level of quality and security.

A ''single'' dose of G-CSF after autologous stem cell transplantation in patients with malignant lymphomasecond interim analysis of multicentre randomised trial comparing standard schedule with delayed application and placebo E. Faber, R. Pytlik, M. Trneny, J. Slaby, T. Kozak, L. Raida, T. Papajik, E. Zikesova, J. Zapletalova, I. Maresova, M. Trnkova, K. Indrak, University Hospital (Olomouc, Prague, CZ) The optimal schedule of G-CSF given after autologous peripheral stem cell transplantation (ASCT) has not been defined yet. Here we present results of the second interim analysis of randomized multicentre trial comparing standard application of G-CSF from day +5 (arm A) with delayed dosing (G-CSF was started when WBC reached 0,5x109/l and neutrophil count 0,1x10 9 /l; arm B) and placebo (arm C). In order to eliminate the influence of diagnosis and conditioning and to minimize the impact of graft quality only patients with malignant lymphoma conditioned by BEAM and with graft harvested in maximum 3 aphereses containing at least 5,0x10 6 CD 34+cells/kg were included. 91 patients at median age 45 (range 21 -64) were randomized by November 1st, 2003. There was no difference in age, the use of radiotherapy before transplantation, lymphoma bone marrow involvement at diagnosis and CD 34+ cells content in graft between any two of the three arms. Number of chemotherapy cycles applied before ASCT was significantly higher in arm C (median 8, range 5 -13) comparing with arm A (median 6, range 4 -14) (p=0,02) but no difference was found between arms A and B or B and C. Duration of neutropenia below 0,5x10 9 /l and 1,0x10 9 /l and number of days to neutrophil engraftment over 0,5x10 9 /l and 1,0x10 9 /l are listed in the table. A significant difference in duration of neutropenia and time to neutrophil engraftment was found between the arms A and B (p=0,003 -0,008) and the arms A and C (p<0,001). Duration of neutropenia below 1,0x10 9 /l, time in days to neutrophil engraftment over 0,5x10 9 /l and 1,0x10 9 /l signficantly differed between the arms B and C (p=0,008 -0,0001), however, there was no statistical difference in duration of neutropenia below 0,5x10 9 /l (p=0,08). There was no significant difference in platelet engraftment between any two of the arms, the trend for shorter engraftment in arm C was statistically insignificant. The delayed "single" application of G-CSF (usually with one or two doses sufficient for full neutrofil recovery) appears to be safe and cost effective. Comparing with standard G-CSF schedule only one day delay in median neutrofil engraftment was observed. The accrual of patients has been successful and we hope to achieve our final target -randomization of 40 patients into each arm within next year. Objectives: To analyze risk factors to receive total parenteral nutrition (TPN) among patients undergoing allogeneic stem cell transplantation (ASCT) and evaluate the costs and benefits of a daily food oral intake monitoring program.

Methods: We analyzed 84 consecutive patients undergoing ASCT. Median age was 44 years (18-64). Nineteen had AML; 11 ALL; 9 MDS; 16 cLPD; and 29 other malignancies. Fifty two percent were in PR or progression at transplant while 48% were in CR, 1st CP or untreated. Seven (8.3%) received ASCT from an unrelated donor. Forty eight (58.5%) received reduced intensity regimens (RIR). Twenty nine (34.5%) required TPN. Median of days with TPN was 13 . Daily food intake records were collected. TPN indications were severe malnutrition at admission and oral intake < 70% of daily energy requirements for more than five days For the cost-benefit analysis we evaluated cost as time spent in monitoring the oral food intake and benefits as money saved in TPN not administered to patients with an appropriate intake. Results: Risk factors to receive TPN were: ECOG >1 (p=0.046), acute graft versus host disease (aGVHD) (p<0.001), severe mucositis (p<0.001), vomiting grade>3 (P=0.01), low body mass index (BMI) (p<0.001) and conventional conditioning regimen (p=0.018). Twelve patients receiving RIR required TPN; 7 were already receiving TPN prior to ASCT and 5 received it due to previous poor performance status including malnutrition. On multivariate analysis, aGVHD (HR=21.4; 95% CI=1.8-248, p=0.01) and pretransplant BMI (HR=1.73; 95% CI=1.14-2.64, p=0.04) significantly influenced on TPN requirements. TPN was not associated with an increased toxicity neither with a higher TRM. Regarding the cost-benefit analysis: Cost: (15 min/patient/day) x 33 days (median hospital stay) x 0,4 Euros/min=198 Euros/patient. Benefit: 65,5% patients without TPN x 60,3 Euros/TPN/day x 13 days (median of days receiving TPN)=513 Euros/patient. Benefit-cost: 513-198=315 Euros saved/patient. Conclusions: Low BMI and poor performance status pretransplant identify a subset of patients at high risk of requiring TPN. In addition, aGVHD significantly influences TPN requirements. TPN is not associated with an increase of toxicity in terms of TRM, hepatic toxicity neither time of hospital stay. The daily assessment of oral intake in order to identify the need for TPN provides benefits which make the cost of such an activity worthwhile.

Effect of human growth hormone in haemato-oncology patients receiving intensive chemotherapy. A double-blind randomised, placebo-controlled study R. Powles, B. Sirohi, S. Kulkarni, M. Das, J. Treleaven, C. Dearden, G. Morgan, A. Boast, R. McCormack, C. Marriott, C. Horton, J. Wass, Institute of Cancer Research, Royal Marsden NHS Trust, The Radcliffe Infirmary (Sutton, Oxford, UK) This study was designed to see if human growth hormone (GH) can reduce the problems associated with cytotoxic therapy in haematology patients and expediate the haematopoetic recovery. In a randomized, double blind, placebo-controlled (PL), single centre trial patients were randomized on the day of hospitalisation to receive either GH, 250micgm BD (n =75) or placebo (n =75) for a period of 6wk. Initially drug was administered IV and on platelet recovery, by SC route (500micgm OD). Initial 20 patients received 250micgm BD throughout the study period. From July 2002 to September 2003, a total of 150 courses were randomized on 119 patients [GH 75, M/F:47/28, age: 50yr (18-74); PL 75, M/F:51/24, age: 52yr (19-77)]. 17 patients participated twice and 7 three times in the trial, but only 13 patients were crossed over. Thus 106 were exposed to either GH (54) or PL (52). 4 patients died in the first 3 weeks. Two groups had similar demographic features (Leukaemia:GH/PL 44/48; Myeloma:GH/PL 30/23; Allo:GH/PL 6/7; auto:GH/PL 39/25). The median follow-up is 239 days (71-734days). Survival was not significantly different in two arms (GH: 43/54 vs. Pl: 39/52, p=0.47).There was a trend toward better OS in Chemo+BMT patients who survived beyond 3 weeks (GH 12/15, PL11/18, p=0.086). There was no difference in the time to ANC recovery. Among 150 patients (75 pts vs. 75 pts) platelet recovery to 25 (GH 16d, PL 18d, p=0.043) was faster in GH arm but there was no difference in platelet recovery to 50 (GH 19d, PL 22d, p=0.13) . There was no influence on hospital stay. 21/106 patients relapsed and 2 relapsed within one week. There was no difference in the relapse risk, but excluding patients relapsing within a week, there was a trend towards lower relapse with GH (GH: 6/52 vs. Pl: 13/52, p=0.0523). In Chemo+ BMT patients, there was a trend towards lower relapse in the GH arm (GH 2/18 vs. PL 6/19, p=0.0514). At 6 wks, anthropometric measurements biochemical parameters and percentage of patients who retained the baseline parameters were similar in the two groups. IGF-1 levels were significantly higher in GH arm at 6 weeks (median 411 vs. 284, p<0.001) and there was a significant rise over baseline levels in GH arm (median:102 vs. -15, p<0.001). No SAEs were encountered. This pilot trial provides information that may help rationalize the design of future studies to determine if there is role for GH in the treatment of cancer. Collection of sufficient numbers of hematopoietic stem cells is a prerequisite for high-dose cytotoxic treatment of patients with multiple myeloma. Here, we evaluated single-dose application of polyethylenglycole(PEG)-conjugated G-CSF (Neulasta(R), pegfilgrastim) in comparison to daily application of unconjugated G-CSF. Twenty-four patients with untreated stage II or III multiple myeloma received an initial treatment with 2-4 cycles of idarubicine/dexamethasone (ID) or vincristine/ adriamycine/ dexamethasone (VAD). Following cytotoxic therapy with cyclophosphamide (2 g/m 2 , day 1-2) we administered either a single subcutaneous dose of 12 mg pegfilgrastim (n=12) or daily doses of unconjugated G-CSF (n=12) (median dose 8.5ug/kg/d (4.6-11.9), median duration 11 days (6-15)). Patient characteristics did not differ significantly between the Neulasta and the filgrastim group regarding sex, age, body weight, or stage of disease. Maximum peripheral blood CD34+ cell counts in the pegfilgrastim group ranged between 20 and 1055 (median 78) per microliter and were observed 11-16 days (median 12 days) after cyclophosphamide which was significantly earlier (p<0.05) than in the G-CSF group (median 15 days (12-18), median maximum 132/ul (12-760), n.s.). In total, 4.9 +6 to 38 +6 (median 7.4 +6 ) and 4.2 +6 to 87.0 +6 (median 11.0 +6 ) CD34+ cells per kg body weight were collected in the pegfilgrastim and G-CSF group, respectively (n.s.). Eight patients within the pegfilgrastim group and all 12 patients of the G-CSF group were treated by high-dose melphalan and reinfusion of thawed CD34+ cells. Pegfilgrastim patients received 2.5-19.0 +6 (median 3.4 +6 ) CD34+ cells per kg weight, whereas 2.1-29.0 +6 (median 7.5 +6 ) CD34+ cells per kg body weight were transplanted in the G-CSF group (n.s.). Leukocyte reconstitution was observed 13-24 (median 16) days and 12-22 (median 14) days in the pegfilgrastim and the G-CSF group, respectively (n.s.). Independence from platelet transfusions was achieved after 12 days (median) in both groups. In summary, single dose application of 12 mg pegfilgrastim following cyclophosphamide treatment is capable of mobilizing sufficient numbers of CD34+ cells for autologous transplantation in patients with stage II and III multiple myeloma following induction therapy. The maximum numbers of CD34+ cells in the peripheral blood were not significantly different but were observed three days earlier than in patients treated with daily G-CSF application.

Introduction: Umbilical cord stem cell transplantation (UCSCT) is increasingly successful in treating a variety of haematological conditions. Advantages include ready availability, no donor risk, low rate of viral contamination and lesser graft versus host disease (GvHD) risk. We report a single centre experience of UCSCT to treat children with primary immune deficiency (PID). Methods: Of 134 patients transplanted for PID at our centre since 1987, 10 received UCSCT since 1999. Conditions treated included adenosine deaminase (ADA) deficiency (3), chronic granulomatous disease (1), IL7 receptor alpha deficiency (2), reticular dysgenesis (1), Wiskott-Aldrich syndrome (WAS) (2 cords), MHC II deficiency (1), Omenn's syndrome (1) . Donors included 2 sibling and 9 unrelated donors. Six of the patients received conditioning (4 Busulphan /Cyclophosphamide , 2 Fludarabine/Melphalan ). Ten of the cords were well matched ( 6/6 or 12/12 HLA matched), 1 was a single major antigen (A locus) mismatch ( 9/10 ). Nucleated cell doses varied from 0.16"e10 8 /kg to 3.1"e10 8 /kg. Stem cell (CD34+) doses varied from 0.017"e10 6 /kg to 1.6"e10 6 /kg. Results: All patients engrafted. Mean time to neutrophil engraftment was D+23 (range 12-34).

One died post UCSCT of parainfluenza pneumonitis. Only 1 patient developed skin and gut GvHD grade 3 despite receiving a 6/6 match. Of 3 patients evaluated, all have normal antibody responses to protein vaccination antigens, 1 is currently being assessed. Five remain on replacement immunoglobulin, all < 1 year post UCSCT. One patient (WAS) received 2 URD UCSCT 8 days apart because of low stem cell dose and disseminated adenovirus infection. He initially had both donor 1 and donor 2 allelles but retains mixed trimerism with donor 1 and 2 and recipient alleles in T cells. Three patients with PID conditions that are difficult to engraft, engrafted successfully (MHC II deficiency, reticular dysgenesis, Omenn¡¦s syndrome). Conclusion: UCSCT is an alternative to bone marrow transplantation in PID. In this series full immune reconstitution with correction of the underlying immunodeficiency was achieved in all patients, despite a low stem cell dose in some. In this series of mainly HLA-matched UCSCT, the incidence of GvHD is low.

The use of dimethyl sulfoxide in stem cell transplantation: a survey of EBMT centres

Dimethyl sulfoxide (DMSO) is a cryopreservative used to protect frozen haematopoetic stem cells. In animal models it has been shown to exhibit vasoconstrictive and angiotoxic properties in a dose dependent manner. Case reports also suggest neurotoxicity, cardiotoxicity and pulmonary toxicity closely related to DMSO re-infusion in transplant patients. In order to assess current clinical practice in the use of DMSO a questionnaire was designed, piloted and sent to 500 EBMT centres. 96 centres have responded, all of whom used DMSO as their sole cryopreservative. The final concentration of DMSO was 10% for 78 centres, 5% for 10 centres and other concentrations ranging from 2.2% to 20% in 8 centres. 62 centres reported an upper limit of cell concentration used: 1x10 8 /ml in 21 centres, 2x10 8 /ml in 27 centres and others from 0.8-4x10 8 /ml in 14 centres. 61 centres limited the amount of DMSO administered daily although limits varied widely. Only 3 centres regularly washed cells prior to re-infusion while 12 did so occasionally. 1 centre tested for sulphur sensitivity as a predictor of toxicity. Filtration of preserved cells was carried out by 46 centres. 25 centres gave cells directly by syringe. 57 centres who performed an approximate total of 30,000 procedures reported 530 probable or possible cases of severe DMSO-related toxicity, an overall incidence of 1.8%. These included 36 cardiac, 26 respiratory, 11 renal, 8 CNS and 11 miscellaneous events (e.g. hepatic impairment and anaphylactic shock). There was no apparent relationship between toxicity and final DMSO concentration or amount of DMSO given per day. This survey highlights the lack of consensus between transplant centres regarding the use of DMSO and the need for further studies or policy guidelines in this area. For recipients of haploidentically mismatched stem cell allografts, T cell depletion is mandatory for prevention of lethal graft-versushost disease (GVHD). Prevention of GVHD can be accomplished by negative selection of T cells, or positive selection of stem cells. Recently, a new method for positive selection of stem cells was introduced using monoclonal antibodies against AC133 antigen. We report the first case of successful application of immunomagnetic separation of AC133+ stem cells for haploidentically mismatched allogeneic stem cell transplantation. A 63-year-old male with myelodysplastic syndrome (MDS), refractory anemia with excess of blasts, underwent peripheral blood stem cell transplantation (PBSCT) from a haploidentically mismatched related donor. The procedure was well tolerated and early 3-lineage engraftment was documented. Four additional patients similarly treated underwent successful haploidentically mismatched related allogeneic PBSCT using the same protocol. Our successful pilot trial suggests that positive selection of AC133+ stem cells may be a useful method for safe transplantation with haploidentically mismatched stem cell allografts while avoiding acute and chronic GVHD. Future studies will be required to assess the clinical benefits of stem cell purification with AC133+ in comparison with CD34+ stem cells. Hydroxyethhil-starch (HES) is a macromolecule that augments the sedimentation rate of red blood cells and consequently it has been used in the harvest of granulocytes. The objective of this study was to evaluate the efficiency of adding 4 ml/ kg of 6% HES to the system in each collection of peripheral mononuclear blood cell (PMBC). Material and Methods: the Fenwall CS 3000 Plus® machine was used for cell separation. The settings for separation were 120-140 related to PMBC and 40-60 related to HTC. From 10/2001 to 06/ 2003, a total of 117 harvests were made in 62 patients. A mean of 4.7 (3.8-5.2) total blood volume equivalent to a mean of 27.1 l (16.5-35) was processed. An average of the initial/interim/final total cells and PMBC count in patients was made. Results: A mean of 3.6 x 11 (1.7-15.2) total cells and 0.71 x10 11 (0.19-1.5) PMBC, circulated in the cell separator. A mean of 2.9 x 10 11 (1.8-14.2) total cells and 0.59 x 10 11 PMBC were in the harvest. The efficiency of yield was 80.5% ( 48.6-93.4) for total cells and 83.1% (56.8-91) for PMBC. Final concentration of CD34+ cells was 6.2 x 10 6 (2.5-48) and in 50 out of 62 patients the final count was over 5 x 10 6 /kg as the ISHAGE method. Conclusions: the heterogeneity of the efficacy of the harvest of PMBC is well known. The data shown here are over the average in the referral literature. The adding of HES to the harvest allowed an increment of 23% of PMBC compared to a 115 pts. that previously ha been harvested without it. This fact probably contributed to a reduction of 31% of the number of collections from an historical mean 2.5 to 1.9. We did not observe graft delay or any other toxicity related to HES. As a major conclusion, HES contributes to a better efficacy in the collections of total and mononuclear cells. This fact may derive in a faster and most economic harvest of peripheral stem cells. This is the first report to our knowledge related to the use of HES in the context of peripheral stem cells collection.

C. S. Kim, M. H. Lee, C. H. Nahm, I. Kim, S. K. Kim, S. H. Pai, Inha University Hospital (Incheon, KOR) Chemotherapy followed by G-CSF administration is an effective way for mobilization of hematopoietic progenitor cell (HPC). At our hospital, we used G-CSF preceded by chemotherapy for HPC mobilization, including high dose cytarabine (3g/m sq over 2 hours BID every other day for 6 doses) for acute myeloid leukemia, cyclophosphamide (4g/m sq for 1 dose) for multiple myeloma, lymphoma, and breast cancer, and DHAP (dexamethasone 40mg for 4 consecutive days, cytarabin 2g/m sq over 2 hours BID for 1 day, cisplatin 100mg/m sq over 24 hours) for lymphoid malignancy. We started G-CSF at a fixed dose of 300ug SQ every day as soon as the leukocyte count began to rise after chemotherapy induced myelosuppression. Leukapheresis was commenced at the time when leukocyte count rose up to 1000/uL, and repeated for 2-4 consecutive days until the target number of CD34+ cell, at least 2X10 6 /kg, and another 2X10 6 /kg for backup whenever possible, was collected. Thirty five patient (male to female, 12:23; ages raging 4-60 with a median of 37; acute leukemia in 4, myeloma in 4, lymphoma in 14, breast cancer in 13) underwent a total of 174 courses (range per capita 2-16, median 4) of leukapheresis for HPC collection prior to autologous bood stem cell transplantation (ASCT) from January 1988 through December 2001. The CD34+ cell count collected in each series of leukapheresis ranged 0.23-79.3X10 6 /kg with a median of 5.6X10 6 /kg. The target amount of CD34+ cell could not be collected in one of 35, and her bone marrow was harvested before ASCT. All the patients achieved good engraftment after ASCT. The mean days +/-S.D. required for WBC count to be over 1,000/uL were 23.0+/-1.4 in cytarabin arm, 12.54+/-0.66 in DHAP arm, and 11.75+/-2.1 days in cyclophosphamide arm. Patient's age, sex, underlying malignancy, mobilization protocol, and WBC count or the degree of exposure to chemotherapy before mobilization did not show any statistically significant correlation with the rapidity or the amount of mobilized HSC collection on Kruskal-Wallis test. Ages cut off below or above 35 showed marginally significant correlation with the amount of HPC collection. Although, there seemed no useful clinical parameters predictable of mobilization efficacy, chemotherapy followed by G-CSF administration caused few problem and verified itself a good mobilization method.

Background: With Imatinib complete cytogenetic response can be attained in 40 to 70% of patients with CML in CP. The duration of this response is still unknown, thus is logical to try harvesting these patients for a future autograft procedure in case of loss of response or progression. We and others have reported difficulties in stem cell mobilisation from these patients. Parameters that might predict a successful collection in these patients will therefore be clinically useful. Patients and Methods: A total of 62 patients who had achieved major complete cytogenetic responses on Imatinib were studied. The median dose of Imatinib was 400mg daily (range 300-600mg). All patients received G-CSF (median 10mcg/kg/day, range 10-16) for at least 5 consecutive days prior to the harvest and until the day of the last stem cell collection. Imatinib was continued during the procedure. We aimed to collect >= 2 x 10 6 CD34+/kg. If this yield was not achieved with the first harvest a second attempt was performed within 24h. Peripheral blood samples were analyzed prior to the harvest for CD34 percentage of total WBC and CD34 absolute count (CD34 cells x. 10 3 /mL). Results: After G-CSF administration 45 patients had detectable CD34+ counts and underwent leukapheresis, 21 in a single procedure and 24 in two consecutive ones. Twenty of them achieved successful harvest (48%). The median CD34 count collected was 1.04 x 10 6 /kg (range 1-56), the median CD34pb was 4.49 x 10 3 /mL (range 0-146), the median CD34% was 0.02 (range 0-2.00) and the median WCC 29.5 x 10 9 /L (4-108).

In the univariate analysis a CD34+>10 x 10 3 /mL and a CD34>0.05% were significant variants to predict a successful harvest (p<0.0001 in both cases), the pb WCC showed significance as a continuous variable. Although in the multivariate analysis the only variable that remained significant was the absolute CD34+ count (relative risk 62, p>0.0001). Conclusions: the CD34 absolute count remains the best predictor of successful mobilisation in patients in cytogenetic response under Imatinib.

CD133 assessment in the peripheral blood before apheresis and in the autologous PBSC transplant is a valuable tool for prediction of the haematopoietic content of the graft F. Nicolini, G. Clapisson, T. Prebet, Q.H. Lé, A.S. Michallet, I. Philip, M. Michallet, Edouard Herriot Hospital, Centre Léon Bérard (Lyon, F) CD133 has emerged recently as a novel interesting surface marker expressed by different categories of primitive human progenitors from bone marrow, mobilized peripheral blood and cord blood. Although it has been demonstrated that CD133+ cells co-express immature markers such as CD34 and c-kit, it remains unclear whether this marker provides any distinct advantage over CD34 determination into the peripheral blood (PB) and in the autologous transplants per se to predict the hematopoietic content and the adequate hematopoietic recovery after autologous peripheral blood stem cell (PBSC) transplantation. To address this question, we have analyzed the PB from 23 patients [9 solid tumours (SD), 14 hematolopoietic malignancies (HM)], the PBSC transplants of 27 patients (10 SD, 17 HM) after various chemotherapy + G-CSF or steady state G-CSF ± SCF mobilization schedules. Assessment of CD133+ cells in the total PB before apheresis showed that 91 (± 10) % of CD133+ cells were CD34+ as well, and that the yield of CD133+ cells was closely positively related to the yield of CD133+ and CD34+ progenitors present in the autologous transplant S126 harvested (p = 0.003 and 0.01 respectively) and to the total CFU-C/Kg (p=0.01), but neither to total Nucleated Cells/Kg (p=0.09), CFU-GM/Kg (p=0.31), nor to CFU-GEMM/Kg (p=0.1). CD133+ progenitors present within the transplant were positively correlated to all progenitors/Kg present (BFU-Es (p=0.0005), CFU-GMs (p=0.0027), CFU-GEMM (p=0.012), total CFU-Cs (p<0.0001), total CD34+ (p<0.0001)). These results followed strictly the same pattern for CD34+ cells assessment in the whole PB before apheresis and in the apheresis product, except for CFU-GEMMs/Kg which were positively correlated with CD133+ cells present in the transplant (p=0.012), whereas it was not the case for CD34+ cells (p=0.54). In conclusion, the CD133 assessment in the PB before apheresis and in the transplant represents a valuable tool for the prediction of the hematopoietic content of the autologous PBSC graft, but does not give clear advantage over original CD34 determination.

Peripheral blood of adults and cord blood differ sharply in their content of progenitor and stem cells. Adult peripheral blood particularly after mobilization procedure contains a very large number of progenitors cells which allow early engraftment. Cord blood has low number of progenitors cells but it has quite high content of stem cells. For effectiveness therapy with stem cells rescue has been considered the number of infused hematopoietic cells and rather rarely theirs immunological quality. We evaluated profile of antigens expressed on the CD34+ cells obtained from mobilized peripheral blood and cord blood. Material and Methods: Samples of cord blood (CB, n=21) and products of leucapheresis (PL, n=22) were determined by flow cytometric analysis. The healthy donors were treated with G-CSF until the day leucapheresis with the help of COBE Spectra cell sorter. The immunophenotype of cell suspension was detected using panel of FITC and PE conjugated MoAbs reactive with antigens including: CD90, CD117, CD123, CD133, CD135 (for primitive hematopoietic stem cells), CD19, CD10, CD2 (for lymphopoietic lineage), CD33 (for myeloid lineage), CD71 (for erythroid and proliferating cells), CD38 (for progenitors committed to specific lineage). Results: The phenotyping analysis of CD34 cells derived from CB and PL showed similar in the both groups percentage of these cells with antigens coexpression as follows: Besides its cord blood included characteristic CD34+ subpopulation with phenotype CD38(-)CD133(+) which have been observed in higher percentage (14.5±4.2%) in CB unit than PL 8.8±5.2%). Conclusions: In examined groups were identified differences of immunophenotype among CD34+ cells in cord blood and peripheral blood. These results could explain helpfulness of cord blood for transplantation although one unit CB may have nonsufficient number of stem cells for adult engraftment.

Between 5% and 30% of patients with malignancies have "insufficient" collection of peripheral blood stem cells (PBSC) to proceed to transplantation, depending on the target cell dose used. The optimal strategy to obtain an adequate graft in such patients has not been established. To provide more information on the subject, we analyzed the hematologic recovery and transplant outcome of patients who had previously failed PBSC mobilization and followed different approaches of second-line stem cell harvest. From 1993 to 2003, 107 out of 745 patients (14%) with malignancies who were consecutively mobilized in our institution obtained an insufficient harvest (CD34+cells < 2 x 10 6 /kg). Such patients were categorized into three groups: group A included patients who did not proceed to a second stem cell harvest (n= 32); group B included those who were remobilized to collect PBSC (n= 41); group C included those who underwent bone marrow harvesting (n = 34). Autologous transplantation was subsequently performed in 11, 23 and 30 patients from groups A, B and C, respectively. A higher proportion of female patients with breast cancer was seen in group B. Median (range) CD34+ cells (x10 6 /kg) infused were 1.4 (0.9-1.7), 2.9 (0.9-5) and 0.6 (0.1-1.8) for patients transplanted in groups A, B and C, respectively. Group C patients additionally received a median bone marrow mononuclear cell content (x10 8 /kg) of 2.2 (range, 1.0-5.3). Following high-dose chemotherapy, median times (range) to achieve a granulocyte count higher than 0.5 x 10 9 /L and an unsupported platelet count higher than 20 and 50 x 10 9 /L were: 19 (7-25), 27 (12-47) and 29.5 days (18-90), respectively, for group A; 12 (10-35), 16 (7-360) and 30 days (15-360), respectively, for group B; 17 (10-48), 28 (11-180+) and 45 days (13-360+), respectively, for Group C. Thus, the hematopoietic reconstitution was significantly faster in group B than in the remaining ones. Median times (range) to hospital discharge after transplant were 32 (22-63), 24 (17-58) and 30 days (21-180) for patients in groups A, B and C, respectively (p=0.006). Transplant-related mortality was 9%, 4% and 10% in groups A, B and C, respectively. In conclusion, a complete hematologic recovery after transplantation can even be observed in patients with poor PBSC harvests. However, the use of additional PBCS from a second harvest may be a reasonable option to shorten the period of aplasia postransplant.

Umbilical cord blood (UCB) is an attractive stem cell source for transplantation in high-risk haematological malignancies as it can be obtained rapidly and is associated with a low risk of graftversus-host disease (GVHD). Preliminary concerns over reduced graft-versus-leukaemia (GVL) effect due to the naivety of UCB T cells have not been realised. We report single institution experience of unrelated donor UCB transplantation (UCBT) which offers further reassurance about its GVL potential. 15 patients (14 male) with a median age of 5.1 years (range 0.7-15.3) and weight of 18.3kg (range 5.0-41.0) were transplanted between June 1998 and February 2003 for AML (n=8), ALL (n=2), JMML (n=2), infantile myelofibrosis (n=1), anaplastic NHL (n=1) and Fanconi anaemia (FA)(n=1).

The conditioning regimen was myeloablative in 11 patients, reduced intensity in 3 and non-myeloablative in 1. All received GVHD prophylaxis with cyclosporin A (CyA) 5mg/kg from day -1 and methylprednisolone 2mg/kg from day+5 to day+14, then tapering to stop at day+25. G-CSF 5 microg/kg was given to 14 patients from day+7 until neutrophil recovery.

All patients received CMV negative, unrelated donor UCB grafts, which were 6/6 HLA matched with the recipient in 3, 5/6 matched in 7 and 4/6 matched in 5. The median infused cell dose was 5.0x107 TNC/kg (range 2.0-8.0), 1.2x10 5 CD34+/kg (range 0.5-4.9) and 4.3x10 4 CFU-GM/kg (range 1.5-52.3). Myeloid engraftment was achieved in 13 of 14 evaluable patients (93%) after a median of 26 days (range 13-50) with platelet recovery observed at a median of 38 days (range16-64) in 11 of 13 (85%) evaluable patients. Grade II-IV GVHD was documented in 2 patients (13%) and chronic GVHD was not observed. 5 patients (33%) have relapsed at a median of 74 days post transplant (range 19-94); 2 of these attained a further complete remission (CR) on withdrawal of CyA. Despite 14 of the 15 patients being at high risk of transplant-related mortality or relapse or both, 9 (60%) remain alive and in CR at a median of 21.4 months (range 8.6-60.9). 6 patients (40%) have died at a median of 2.3 months (range 0.3-5.8) of progressive disease in 3, VOD in 1, disseminated HSV and GVHD in 1 and adenovirus hepatitis in 1. These data represent the largest single UK institution experience of UCBT to date and confirm the therapeutic value of this approach in high-risk paediatric patients with haematological malignancies.

Objectives: The feasibility of autologous bone-marrow transplantation (ABMT) has recently been demonstrated in heavily pretreated patients non-mobilizing peripheral-blood progenitor-cells (PBPCs), suggesting that BM progenitor-cells are not as sensitive to chemotherapy as PBPCs. However, information regarding the impact of previous chemotherapy on the performance of BM grafts is scanty, and it constitutes the main objective of our study. Methods: We have retrospectively analyzed 40 consecutive lymphoma patients treated with the BEAM regimen and ABMT at our institution. The impact of the chemotherapeutic drugs (individual cumulative doses) received before transplant on stemcell yield, hematological recovery and transplant-related toxicity was assessed by univariate and multivariate analysis. Results: Univariate analysis failed to identify any variable that significantly affected progenitor-cell content (CFU-GM and MNC). Regarding the impact of pre-transplant chemotherapy on early engraftment, only cumulative doses of cytarabine (r = 0.28, p = 0.04) and cisplatin (r = 0.32, p = 0.02) had a negative influence on neutrophil recovery (to >0.5 x 10 9 /L), but this was not mantained in multivariate analysis. We did not find any chemotherapeutic drug that negatively affected platelet recovery (to >20 x 10 9 /L). By contrast, administration of several drugs significantly delayed the complete trilineage reconstitution, including doxorubicin, procarbazine, nitrogen mustard, cytarabine and cisplatin. In multivariate analysis, only previous doxorubicin retained statistical significance (190 vs 97 days to reach normal counts in PB between patients receiving and not receiving doxorubicin, p = 0.014). The early transplant-related mortality (TRM) observed in our series was 5%. We did not find any chemotherapeutic drug that increase the incidence of TRM or grade 3/4 OMS toxicities. Conclusions: Our results show that pre-transplant chemotherapy has little or no influence on progenitor-cell yield and short-term engraftment after ABMT, but, in contrast, has a much pronounced impact on long-term engraftment. In heavily pretreated lymphoma patients in whom a poor PBPC mobilization is expected, BMT may represent a reasonable option.

Is there any impact of stem cell source on long-term survival after allogeneic haematopoietic stem cell transplantation? Single-centre experience J. Pretnar, I. Preloznik Zupan, S. Zver, University Medical Centre Ljubljana (Ljubljana, SI) Objectives: This study was designed to evaluate the impact of stem cell source on long-term survival and event free survival after allogeneic haematopoietic stem cell transplantation (HSCT). We compared bone marrow (BM) and peripheral blood (PB) HSCT. Methods and patients: At the Department of Haematology, University Medical Centre Ljubljana, stem cell transplantation program is running since 1989. Between June 1989 and December 2002 sixty-nine adult patients with leukaemia were transplanted. There were 44 patients with acute leukaemia (30 with AML and 14 with ALL) and 25 patients with chronic granulocytic leukaemia (CGL). Median age was 36 years. Most of the donors were HLA-identical siblings. In 51 patients stem cell source was BM, in other 17 patients PB HSCT was performed. Most of peripheral HSCT-s were performed after 1999. Results: Survival curves were calculated using the Kaplan-Meier method. Overall survival (OS) was 60% at 4 years (65% vs. 55% for BM vs. PB HSCT) and did not show statistically significant differences between two sources of stem cells. Identical results were obtained for event free survival (EFS), which was 58 % (63% vs. 48% for BM vs. PB) at 4 years after HSCT and again without statistical differences between the two groups. However, there was a trend of better survival in CGL group of patients, where BM stem cells were used. Conclusion: In summary, our results show that source of HSC is not important for the long-term survival after HSCT. One of the most important clinical variables determining the success of hematopoietic stem cell transplantation (HSCT) is the number of viable CD34+ stem cells transfused to the patient. The amount of apoptosis in the hematopoietic stem cell products, attributable to the apheresis procedure, as well as the clinical consequences of transfusing high proportion of apoptotic cells is unknown. In the early stages of apoptosis which can also be described as programmed cell death, there is loss of cell membrane phospholipid asymmetry and expression of phosphatidylserine which can be determined by fluorescein stained AnnexinV. Thus there is a possibility that in the HSCT with high CD34+ cell yields, cells expressing phosphatidylserine can be recognized and destroyed by the phagocytic system. Seventeen healthy allogeneic stem cell donors were administered rhG-CSF (Neupogen, Amgen-Roche) 10µg/kg/day s.c. for 5 days. Two hours after the rhG-CSF treatment on the fifth day morning, peripheral stem cell apheresis was performed by continuous flow cell separation device and a mean of 5.9x10 6 (3-12.7)/kg (recipient) CD34+ PBSC was collected. Flow cytometric analysis (Beckman Coulter, Altra) of apoptosis was performed by using anti-CD34, anti-CD45, 7AAD (BC), Annexin V (Immunotech). The proportions of pre-apheresis viable and early apoptotic cells in the peripheral blood were 97.9% (88.6-100) and 0.3% (0-1.3) in CD45dimCD34+ cells, 98.3% (94.5-100) and 0.5% (0-2.7) in neutrophils, 98.8% (96.7-100) and 0.6% (0-2.8) in monocytes, 99.4% (97-100) and 0.6% (0-3) in lymphocytes, respectively. After the apheresis procedure the proportions of viable and early apoptotic cells in the products were 98.4% (94.1-99.8) and 1.3% (0-5.9) in CD45dimCD34+ cells, 96.9% (83.3-99.6) and 2.2% (0.1-10.2) in neutrophils, 97.1% (88.9-99.9) and 2.3% (0.1-10.7) in monocytes, 98.9% (94.4-100) and 1.1% (0-5.5) in lymphocytes, respectively. Compared to the pre-apheresis peripheral blood, both the CD34+ cells and the mature cell populations in the products tend to have higher proportions of apoptotic cells. However majority of the cells retain their viability. Recombinant human G-CSF, which is an anti-apoptotic cytokine, may oppose the apoptotic stimuli such as the extracorporeal circulation during the apheresis procedure and permit the transplantation of highly viable stem cell products with very low proportion of apoptotic cells.

Uncontrolled-rate freezing of peripheral blood progenitor cells allows successful engraftment by sparing primitive and committed haematopoietic progenitors

Uncontrolled-rate freezing (URF) techniques, which are fast and easy, could represent an attractive alternative to controlled-rate cryopreservation procedures which are time consuming and require high-level technical abilities. It was the aim of the present study to evaluate, on a routine basis, whether URF might spare primitive hematopoietic progenitors and maintain engrafting capacity. One-hundred ninenteen peripheral blood progenitor cells (PBPC) collections from 104 patients with hematological malignancies were cryopreserved in bags, with an URF procedure, in a cryoprotectant solution consisting of PBS, HSA and 10% DMSO and stored in liquid nitrogen. PBPC bags were tested before cryopreservation and at thawing for primitive (LTC-IC) and committed hematopoietic progenitors (CFU-Mix, BFU-E, CFU-GM) by means of long-and short-term culture assays, respectively. In addition, PBPC bags were evaluated for CD34+ cell numbers. Although thawing was associated with a statistically significant reduction of the absolute number of nucleated cells, recovery of LTC-IC, CFU-Mix, BFU-E, CFU-GM and CD34+ cells was not affected by the freezing/thawing procedures. No adverse effects were reported at thawing and only mild transient reactions were recorded in 22 patients during reinfusion of cryopreserved PBPC. All the patients underwent myeloablative therapy followed by reinfusion of PBPC, and prompt and rapid hematopoietic recovery was obtained in all patients. Our freezing procedure is as fast as easy, allows rapid hematopoietic recovery after myeloablative therapy by sparing primitive and committed hematopoietic progenitors. Our study strongly supports technical improvements aimed at cost reduction and feasibility of routine freezing procedures.

Intensive chemotherapy with autologous peripheral progenitor stem cell support: a single-centre experience A. Carneiro, F. Principe, C. Granato, F. Trigo, T. Costa, A. E.Santo, P. Guimaraes, M.J. Parreira, J.E. Guimaraes, Hospital Sao Joao (Porto, P) Aim: To analyse retrospectively the experience of autologous peripheral blood progenitor cell (PBPC) transplant from July 1995 to July 2003. Patients and Methods: We transplanted 87 patients, 41 females and 47 males, with a median age of 43 years (16-69), divided into: 19 acute myeloid leukaemia (AML) other than promyelocitic leukaemia, 7 acute lymphoblastic leukaemia (ALL), 28 multiple myeloma (MM), 28 non-Hodgkin's lymphoma (NHL) and 5 Hodgkin's disease (HD). General criteria for inclusion were age < 70 years, performance status 0-2, absence of systemic organ damage counterindicating intensive chemotherapy. Specific criteria for the different pathologies were: AML and ALL in 1st complete remission (CR) following consolidation chemotherapy and ineligible for allogeneic transplant; MM responsive to anthracycline-based treatment; NHL/HD in chemosensitive relapse or 2nd CR. Mobilization was performed using different chemotherapy regimens (consolidation course, high dose cytoxan or ESHAP) according to the condition, plus G-CSF. The median number of CD34/patient obtained was 8.7x10 9 /L. The cells infused were not previously submitted to selection procedure. The transplant was done initially in reverse isolation and from 2002 in HEPA-positive pressure rooms. The conditioning regimens were as follows: BuCy for acute leukaemia; high dose Melphalan for MM; BEAM for NHL/HD. All patients received oral antibiotics prophylaxis until 2001 and only acyclovir and fluconazol from then on. Results: Engraftment was observed in all cases. Only one patient, with NHL, died in the first 100 days after transplant of disease progression. Only 16/87 patients had a documented bacteraemia (18%). The estimated 5-year median overall survival posttransplant was 25% for AML/ALL, 55% for NHL, 100% for HD and 40% for MM. Conclusion: in our experience, autologous PBPC transplant is a relatively safe procedure, with a very low transplant-related mortality. Overall survival results are similar to those reported by most centres.

Anti-CD20 effect of haematopoietic stem cell mobilisation in patients with follicular non-Hodgkin lymphoma, after combined treatment with fludarabine and cyclophosphamide S. Garcia, R. Arrieta, M. Canales, M. Sanjurjo, F. Hernandez-Navarro, Hospital La Paz (Madrid, E) Introduction. The combination of anti-CD20 antibody and fludarabine is very active in patients with low-grade non-Hodgkin's lymphoma (NHL), probably due to an additive effect. However, prolonged neutropenia (also seen with the antibody alone) and mobilization delay have been described as adverse effects. Patients and methods. We studied differences in mobilization in patients with follicular NHL after chemotherapy with either FND (fludarabine 25 mg/m 2 x 3 d, mitoxantrone 10 mg/m 2 x 1 d, dexamethasone 20 mg/d x 5) or FluCy (fludarabina 25 mg/m 2 , cyclophosphamide 1000 mg/m 2 ) plus AntiCD20 (375 mg/m 2 ). Mobilization was performed with Ifo-Vp16 (ifosfamide 3,3 g/m 2 /d x 3 d, etoposide 150 mg/m 2 /12 h x 3 d) plus G-CSF 5 mcg/kg; or G-CSF 10 mcg/kg alone. Ten patients received FND and five FluCy, all of them with anti CD20. Mobilization was performed with Ifo-VP16 in the first nine patients and with G-CSF in six remaining. Results. Mobilization was very difficult in the last six patients with G-CSF alone: three doesn`t achieve it ten months after the end of therapy. One patient had neutropenia lasting for seven months, also associated with lymphopenia and hypogammaglobulinemia.

Conclusion. Therapy with anti-CD20 and Flu-Cy is effective in patients with follicular NHL. However, this treatment makes very difficult mobilization, along possible immune effect on neutrophils and commited hematopoietic progenitors.

Safety and prompt availability of UCB units can be improved by NAT

Nowadays umbilical cord blood (UCB) represents a largely employed source of haematopoietic stem cells for unrelated transplants thanks to worldwide spread of UCB Banks. In UCB banking, major goals are safety of stored units and prompt availability. For this purpose UCB donations are routinely screened for viral infections at collection and six months after, to close preseroconversion window. Once quarantine has concluded successfully, UCB units become available for transplant centers; on the contrary, UCB is discarded if any test produced abnormal results. For viral screening, the most advanced test is NAT, which can detect the genetic material of viruses, such as HIV or HCV, before antibodies or viral proteins are produced. Recent FDA license and European governments recommendations concerning blood safety raised interest about NAT and its extended use to UCB Banks. From June 2003, our institution has started testing UCB units by NAT regarding HCV, according to Italian transfusion laws. Also HIV-NAT has been performed. We preliminarily evaluated the impact of NAT on UCB units validation process. At delivery, for each cryopreserved UCB unit, a sample of maternal blood was collected for NAT assay. Once a pool of at least 18 UCB donations plus 2 internal controls has been set up, NAT was performed by HCV and HIV reagents (CobasAmpliscreen, Roche Diagnostics). In parallel, also traditional serologic screening was carried out. No positive results were detected by NAT. Antibodies assays were negative for all 150 tested donations. The maximum time between UCB collection and NAT assay was 15 days (8 days on average), depending on time needed to complete the pool; so the quarantine proved to be dramatically shortened (15 vs 180 days). Moreover, the second serologic control could be bypassed by NAT, leading to UCB units availability largely in advance. On the contrary, positive units might be eliminated before, avoiding costs of storing and maintenance, therefore balancing higher costs of NAT technology.In our experience, NAT revealed to have a positive impact on UCB units validation by ensuring safety and early availability. Moreover, the development of additional NAT assays, such as for HBV or HCMV, is also desirable for further improvement. In conclusion, besides the debate regarding NAT cost effectiveness is still open, we believe that the more advantages of new technologies are evident, the more implementation of reimbursement mechanisms becomes necessary.

A. Picardi, G. Ballatore, A. Tamburini, G. Suppo, L. Cupelli, T. Caravita, M. Mirabile, C. Malerba, M.I. Irno Consalvo, A.R. Camilli, A. Calugi, S. Amadori, University Tor Vergata (Rome, I) Standards for cord blood collection and banking indicate that acute fetal distress may be considered an exclusion criteria for cord blood banking. Diagnosis of fetal distress is based on several parameters such as fetal heart rate (FHR) pattern or meconium liquid staining, however, they are not always associated with postpartum asphyxia or acidemia. We evaluated the relationship between acute fetal distress and both abnormal placental blood gas values and abnormal newborn Apgar score. With the final aim to increase umbilical cord blood (UCB) banking, we studied whether the biological characteristics of UCB units collected from fetal distress delivery were comparable to units from healthy newborns. Obstetrical inclusion criteria were full term delivery, rupture of membranes < 12 h, normal ecographic fetal development, presence of non reassuring FHR and/or presence of meconium in amniotic liquid. Twenty-four units were studied and evaluated for nucleated and CD34+ cell count, viability, clonogenic assays, gas values and microbiological contamination. A random group of 24 units previously banked was used as control. UCB units of the study group were collected either from spontaneous (10) or cesarean delivery (14); the median values of volume, nucleated cell count, % of CD34+ cells and CFU-GM were fully comparable to the control group. Furthermore, all newborns with acute fetal distress showed normal Apgar score at 1 and 5 minutes post-partum and absence of metabolic acidemia. Only 1 unit collected from spontaneous delivery (4%) resulted contaminated by E. Coli at microbiological test. Overall, the results of this study have shown no significance differences between the two groups both in terms of units biological characteristics and newborns clinical conditions. These findings suggest that acute fetal distress does not preclude UCB collection and banking. The eligibility of these units for collection allowed in our experience an increase of 15 % UCB banking.

P. Solves, L. Larrea, V. Mirabet, G. Palomo, F. Carbonell-Uberos, M.A Soler, R. Roig, Valencia Transfusion Center (Valencia, E) Background: To achieve a viable bank, volume reduction of cord blood (CB) is essential for storage of a large number of units. For this purpose, several different methods have been employed, although the majority of them are not performed in a closed system and require the addition of exogenous material. Objective: To validate a method for volume reduction of CB units using a triple bag (Maco Pharma, MQT 2205PU) specially designed for cord blood, and a semi-automated closed system. Material and methods: CB was collected, into a collection bag containing 25-ml CPD-A solution, by trained obstetrical staff. After mixing, the CB collections that were not accepted for banking, were transferred to the central collection bag of Maco Pharma triple bag system. The cord blood was centrifuged in oval buckets at 3000 g for 12 min at 22°C, ensuring that the bags were well supported to prevent disruption of the buffy coat layer. A standard protocol programmed into the Optipress II (Baxter), together with the standard backplate for buffy coat preparation was used to process the CB units. The programme was set with the following parameters: buffy coat volume of 40 ml, a buffy coat level of 5.5 and a force of 25. Samples for nucleated cells and CD34+ cells counts, sterility control and progenitor cell assays (CFU) were removed from the CB pre and post-separation. Statistical analysis was performed with SSPP 8.0.

Results: A total of 30 cord blood units were processed. Mean time of volume reduction processing was 30 minutes, red blood cells depletion was 55.45% and lymphocyte recovery was 83%. All CB units were negative for bacterial and fungal contamination. Results are shown in the following table:

Conclusion: When comparing to another methodologies, volume reduction using top and bottom bags is a closed system that reduces time of processing but also preserves the quantity and quality of the progenitor cells. A less depletion of red blood cells is a minor disadvantage. 

Allogeneic peripheral blood stem cell transplantation (PBSCT) is used more increasingly as an alternative to allogeneic bone marrow transplantation (BMT) in patients (pts) with hematological malignancies. Definite efficacy of this treatment modality in the respect of all the aspects was not established yet. Aims: We have compared the use of allogeneic marrow with use of the peripheral blood cells from HLA identical sibling with respect to the engraftment, transfusions, transplant related mortality (TRM), acute and chronic GvHD, relapse and overall survival. Methods: We have analized 71 patients (pts) with different hematological malignancies (14 with sAA, 23-CML, 17-AML, 16-ALL and 1 pt-MM) treated in our Center with allogeneic SCT. Median age was 29 (9-52 years), M/F-48/23. Pts were divided into two groups depending on the stem cell source-bone marrow transplantation (BMT)-49 pts and peripheral blood SCT (PBSCT)-22 pts. All pts have received unmanipulated suspension of mononuclear cells (MNC) from HLA identical sibling. Conditioning regimens were adjusted to the primary disease and GvHD prophylaxis were the combination of CsA+ Mtx+ MPDN. Results: Considering the number of MNC, pts with PBSCT have received much bigger sample (2,46 vs 8,7x 10 8 /kg BW, p<0,001). Recovery of neutrophils >0,5x 10 9 /l and platelets >20x 10 9 /l were faster (p<0,01) in the group with PBSCT in compare with the group with BMT for 3, ie 4 days respectively. Requirements for transfusions of either RBC units or Plt units were much higher in the group with BMT (p<0,01). Those pts also had mucositis gr 2-3 more often (33,5% vs 10,7%, p<0,05). There was no significant difference between the groups considering the frequency of aGvHD (70,6% vs 81,2%, ns), but cGvHD was more frequent in pts with the allogeneic PBSCT (41,2% vs 79,6%, p<0,05). Stem cell source did not have significant influence on TRM (15,21% vs 35,02%, ns). Also, groups did not differ significantly in the relapses (20,8% vs 10%, ns). Pts with BMT had better overall survival comparing with the other group (log-rank test 2,1), because lot of pts with PBSCT had fatal form of atypical cGvHD. Conclusion: PB as the source of stem cells provides fast engraftment and consecutively the minority of early complications related to the SCT. Our future work sould estimate the frequency of relapses and total survival depending on the stem cell source at the larger, homologous groups considering the primary disease.

E.M. Villarón, J. Almeida, N. López, L.I. Sanchez-Abarca, F.M. Sanchez-Guijo, M. Alberca, B. Durán, C. Pata, A. Orfao, M.C. Del Cañizo, J.F. San Miguel, University Hospital of Salamanca, University of Salamanca (Salamanca, E) Introduction: Currently, hematopoietic stem cells from mobilized peripheral blood (HSC-PB) are commonly used in the stem cell transplantation setting. However, whether HSC-PB have the same immunophenotypic and functional characteristics as bone marrow hematopoietic progenitor cells (HSC-BM) remains unknown. Aim: to comparatively analyze the distribution of different hematopoietic progenitor cell (HPC) subpopulations between mobilized peripheral blood (PB) and bone marrow (BM) in healthy donors. Material: A total of 39 BM and 31 PB samples have been studied. In 9 cases, both BM pre-mobilization and PB postmobilization from the same healthy donor have been analyzed. Methods: Immunophenotypic analysis of CD34+ cell subpopulations was performed using the following four-color combinations of monoclonal antibodies (FITC/PE/PC5/APC): CD90/CD133/CD38/CD34 and CD71/CD13/CD45/CD34. In order to study committed progenitor cells we used "in vitro" standard colony-forming assays stimulated with IL-3+GM-CSF+SCF+Epo and to investigate the behaviour of the uncommitted cells from both cell sources, Delta Assays of plastic adherent progenitor cells (Pdelta;) were performed. Results: There were no significant differences between either cell sources regarding the percentage of total CD34+ cells. The relative distribution of the different CD34+cell subsets assessed by phenotype, clonogenic assays and Pdelta is shown in Table  1 .

Conclusions: Immunophenotypic studies showed that the percentage of lymphoid progenitors was higher in BM; by contrast, the percentage of myelo-monocytic progenitors was lower. Clonogenic and Pdelta; assays showed that the number of immature progenitor cells could be higher in PB. Background and Aim: The efficacy of mobilization with G-CSF is related to the reserve of hemopoietic progenitor (HP) cells. Several parameters, clinical and biological, have been used to predict the mobilization. We studied the steady-state counting of circulating HP CD34+ cells in association with the response to the administration of G-CSF at high doses by means of a multivariate analysis. Methods: The data of 56 consecutive donors were analyzed (healthy 16, MM 10, NHL 9, AL 7, CML 11, amyloidosis 2, solid tumor 1), aged 54 (19-69) years, who received G-CSF (16-24 µg/kg).Twenty-five of them were treated with intense chemotherapy (Ara-C, alkylating drugs, fludarabin). In these, the time elapsed since the last treatment received and the administration of G-CSF was 10 (4-36) weeks, and the number of cycles and chemotherapy schedules received were 4 (1-15) and 2 (1-6) respectively. Cell counts were obtained with a SE-9000 analyzer (Sysmex) and the quantification of CD34+ cells with flow cytometry (FACScan) using the MoAb CD34-PE (HPCA-II, BD) and dual platform technique. All samples were obtained prior to the administration of G-CSF and on day +4. Mobilization failure was defined when counting was < 5 CD34+ cells /µl on day +4. Statistical methods included a descriptive analysis, chi-squared test, Pearson's correlation and a ROC curve to investigate specificity of the significant analytical tests. Results: Failure was detected in 15/56 donors (27%). Statistical significance was only observed in healthy donors (p=.003), in the group without intensive chemotherapy including healthy donors (p=.000) or excluding them (p=.001), age (p=.001), hemoglobin, WBC, platelets and basal CD34+ counts (p=.000). Significant inverse correlations were observed between age and hemoglobin, WBC, platelets, and CD34+ cell counts (basally and on day +4). The significant correlation was direct between these analytical variables and the HP cell count response. The The major aim of this study was to report the preliminary results of unrelated donor umbilical cord blood transplantation (UD-UCBT) in patients with high-risk hematologic malignancies treated with a common conditioning regimen and graft-versushost disease (GVHD) prophylaxis. From April 2002 to October 2003, 37 patients underwent UD-UCBT. Conditioning consisted of thiotepa (5 mg/kg per day on days -9 and -8), busulfan (4 mg/kg per day on days -7, -6, and -5), cyclophosphamide (60 mg/kg per day on days -4 and -3), and antithymocyte globulin (Thymoglobuline, 2 mg/kg per day on days -5, -4, -3, and -2 in 8) and all received cyclosporine and prednisone for graft-versushost disease (GVHD) prophylaxis. Diagnosis were acute lymphoblastic leukemia in 17, acute myelogenous leukemia in 8, myelodysplastic syndrome in 6, chronic myelogenous leukemia in 3 and lymphoid malignancies in 3. The status of the disease at transplant was advanced in 17 cases. Median age was 24 years (range, 0.7-46). HLA match (HLA-A and -B by serology and -DRB1 by high-resolution DNA typing) was 6/6 in 3 (8%), 5/6 in 16 (43%), and 4/6 in 18 cases (49%). The median number of nucleated cells and CD34+ cells infused was 2.8 × 10 7 /kg (range, 1.4-21.7) and 1.25 × 10 5 /kg (range, 0.2-5.1) respectively. The median time to neutrophils > 0.5 × 10 9 /L was 22 days (range, 13-50) and the actuarial probability of myeloid engraftment was 80% at 60 days. Median time to > 20 × 10 9 platelets/L was 74 days (range, 27-88) and the actuarial probability of platelet engraftment was 71% at 90 days. The cell dose (CD34+ cells cryopreserved and infused, and CFU-GM cryopreserved) had a clear relationship with time to myeloid and platelet engraftment (P < 0.05 in all cases). Eleven patients (30%) developed grade II-IV acute GVHD (grade III-IV in 6 [16%]), and 7 of 16 (44%) patients at risk developed chronic GVHD, this becoming extensive in 4 (25%). With a median follow-up of 10 months, the probability of disease-free survival (DFS) at 1 year was 32% (95% CI, 16-48%). Stage of disease at transplant (P = 0.03) and CFU-GM at cryopreservation (P = 0.002) strongly influenced DFS. For patients transplanted in early disease stage DFS at 1 year was 42%. These results confirm: 1) that UD-UCBT should be considered a reasonable alternative for patients with hematologic malignancies and no appropriate bone marrow donor, and 2) the impact of cell dose and stage of disease in the outcome of UD-UCBT.

Mobilization of peripheral blood stem cells (PBSC) in healthy donors is induced by short term administration of recombinant human G-CSF. Although several factors were identified influencing the number of PBSC released from the bone marrow, the biological basis of the very different mobilization efficacy remains unclear. The aim of the present study was to evaluate if poor and good mobilizers show differences in the flow cytometrically investigated expression of adhesion molecules, known to play a role in the release of stem cells to peripheral blood. Furthermore, we analysed the expression of G-CSF receptor on CD34-positive cells. We investigated 74 aphereses from 70 healthy PBSC donors with a 4-color staining: CD34/CD45 combined with two different adhesion molecules in each sample (VLA-4, L-selectin, LFA-1, PECAM-1, CD44, and G-CSF receptor). Due to the peripheral CD34-positive cell count at the day of apheresis the donors were divided into three groups: < = 30/µl (poor mobilizer; n=22), 31-100/µl (standard mobilizer; n=31), > 100/µl (good mobilizer; n=21). The expression of VLA-4, LFA-1, and PECAM-1 (mean fluorescence intensity) was higher in poor mobilizers compared to good mobilizers reaching borderline significance for VLA-4 and LFA-1 (p=0.04 and p=0.04). L-selectin and CD44 showed only a trend towards a lower expression in poor mobilizers. Considering the percentage of antigen positivity LFA-1 was expressed in a significantly higher proportion of CD34-positive cells in poor mobilizers (p=0.01) whereas the percentages of all other tested antigens showed no differences. The expression of G-CSF receptors was similarly in all tested groups. Summarizing our results, we might assume that the extent of expression for VLA-4 and LFA-1 is correlated with the mobilization efficacy in healthy donors. The aim of this study was to identify significant prognostic factors using unrelated genomically HLA-A, -B and -DRB1 identical donors. Such data could help to choose the best donor. We studied 136 consecutive patients with hematological malignancies who received hematopoietic stem cell transplantation (HSCT) with a median age of 32 (range 0-55) years. Bone marrow grafts were given to 83 and peripheral blood stem cells (PBSC) to 53 of the patients. The cumulative incidence of grades II-IV acute graft-versus-host disease (GVHD) was 30% and chronic GVHD was 54%. At five years, overall transplant-related mortality (TRM) was 34% and patient survival was 50%. In Cox multivariate analysis, 32 potential risk-factors were analysed. Monoclonal antibody OKT-3 during conditioning was correlated with grades II-IV acute GVHD, chronic GVHD and TRM. HLA-DP mismatch was associated with poor TRM and poor survival. Cytomegalovirus seropositive patients with a seronegative donor had a decreased leukemia-free survival.

Five-year TRM was 14% with no risk-factor, 38% with one and 87% with two risk-factors. The five-year survival was 72%, 48% and 30% with no, 1 and 2 risk-factors, respectively. It is concluded that unrelated HSCT may be improved if an optimal donor and immunosuppression is chosen.

The probability of molecular HLA-C matching between patients and unrelated donors for promiscuous HLA-B allele B*5101

There is increasing evidence suggesting significant role of HLA-C matching in stem cell transplantations. The linkeage disequilibrium (LD) between HLA-B and -C is well documented even at the allelic level, however marked variation in the LD between various HLA-B and-C alleles was also noted (Prasad, Transplantation 1999). Although many HLA-B alleles (e.g. B*0702,*0801,*35) are strongly associated with single HLA-C alleles, others occur with many HLA-C alleles. Especially promiscuous HLA-C association demonstrates B* 5101 allele. We have tried to asses the probability of B*5101-Cw* matching in the recipients (R)-unrelated donors (URD) pairs and to determine the frequency of different B*5101-Cw associations. From the pool of 358 pts (Western Slavonic Caucasians) molecularly typed (4-digits by PCR-SSP) since 1999 for HLA-ABCDRDQ, 21 (5,9%) pts beared phenotype containing B*5101 where HLA-B*-Cw* association could be determined either by family studies or through haplotype frequency of the 2nd (nonB*5101) haplotype. For 17 of them altogether 28 potential URDs were typed, representing 49 probable "B*5101-Cw*" haplotypes on the whole. 11 different B*5101-Cw* associations were observed in total. The most frequent ones were Cw*1402 (22%,11 cases), 1502 (22%), 0102 (20%,10 cases) and 0401(10%,5 cases).The frequency of each of the remaining 7 HLA-C alleles (*0702,1602,0202,0303, 0404,1203 and *05) was below 10% and they formed together only 25% of all possible B*5101-Cw associations. From the 17 R-URD clusters there were 27 possible R-URD pairs with donors being otherwise completely molecularly matched for HLA-A,B,DR,DQ and also for Cw* allele of the 2nd haplotype, i.e. presumably with the same "B*5101" haplotype. Within this group, 19 (70%) R-URD pairs were mismatched for the (B*5101)-Cw*. The overall probability of molecular Cw* matching between R-URD pair bearing the same supposed HLA-A-B*5101-DR-DQ haplotype (i.e. otherwise A,B,DR,DQ molecularly matched) was thus only 30%. Even 2-3 URDs for the same patient mismatched only for different HLA-C allele associated with B*5101 were routinely observed. The limited number of samples obviated the attempt to define extended HLA-A-B*5101-C-DR-DQ haplotypes. Conclusion: If the R-URD pair shares HLA-B*5101 allele, the chance of complete matching at the HLA-C is extremely low despite the genotypic matching at the remaining HLA alleles. We should keep this in mind when arranging the URD search for such patients.

D. Stamatovic, L. Tukic, B. Balint, O. Tarabar, M. Elez, L. Simic, L. Ristic, M. Malesevic, Military Medical Academy (Belgrade, CS) Instruction: Allogeneic stem cell transplantation (SCT) are performed across the ABO blood group barrier in approximately one-third of all cases. There are three groups of ABO incompatibility:

minor, major and bidirectional ABO incompatibility. It is well known that ABO-incompatible SCT increases the risk of hemolytic reactions. However, only a few studies have systematically addressed the effect of ABO incompatibility on the incidence of GvHD, time to engraftment, relapse rate and overall survival following SCT. Aims: Retrospectively we have compared the ABO-compatible SCT with ABO-incompatible SCT with respect to the engraftment, acute and chronic GvHD, relapse and overall survival. Methods: We have analyzed 71 consecutive patients (pts) receiving allogeneic SCT, including 51 ABO identical, 10 minor, 7 major and 3 bidirectional ABO-incompatible SCT (20 pts). Median age was 29 years (range 9-52), 48 males, 23 females. The diagnoses were AML-17, ALL-16, CML-23, AA-14 and MM-1 pt. All pts have received unmanipulated suspension of mononuclear cells from HLA-identical sibling. Conditioning regimens were adjusted to the primary diseases and GvHD prophylaxis was the combination of CsA+ MTX+ MPDN. In minor and bidirectional ABO-incompatible SCT plasma was removed from the donor stem cells by centrifugation. In major ABOincompatible SCT isoagglutinins of the recipient were removed by plasma exchange. Results: Engraftment of neutrophils and platelets was not altered by ABO incompatibility (15,07± 4,3 vs 15,27± 6,32, ns; 19,70± 5,28 vs 18,76± 4,91, ns-respectively). Pts with ABO incompatibility had a higher incidence of grade 3-4 acute GvHD (22,6% vs 8,7%, p< 0,05), compared to ABO identity. There was no significant difference between the groups considering the frequency of chronic GvHD (52,5% vs 50%, ns). The relapse rate was not influenced by ABO compatibility. Pts with ABO compatible SCT had better overall survival comparing with ABOincompatible SCT (p< 0,05, log rank test 2,1). Conclusions: It is evident that ABO incompatibility have an impact not only on post-transplant hemolysis, but also on survival and the rate of severe acute GvHD after allogeneic SCT. Evaluation of the frequency of relepses and overall survival depending on ABO incompatibility should be taken at the larger, homologuos groups considering the primary disease and stem cell source and that would be the subject of our future work.

Outcomes of core and extended family donor searches -a single-centre analysis S. Vokurka, V. Koza, P. Jindra, K. Cerna, T. Karvunidis, D. Lysak, E. Bystricka, A. Volfova, L. Novak, Charles University Hospital (Pilsen, CZ) Introduction: Core family donor search (CFS) is a basic procedure in pts indicated for allo-SCT, in case they have at least one sibling. Extended family donor search (EFS)is initiated only in case of unsuccesfull CFS and only if individually recommended by HLA-experts. Duration of any search is a very important phenomenon, as prolonged searching increases risks of agressive malignancy relaps prior SCT. To check our capability of managing CFS and EFS, we have set this analysis. Objectives: To analyse duration, outcomes and complications of CFS and EFS in our pts. Methods: Retrospective single center analysis covering the period of 2000-02. The data files of the 226 searches coordinated in our department were analysed for number of HLAtyped siblings (CFS) and other relatives (EFS), search duration, outcomes and complications. Search duration was defined as a period starting when a Search request was given to a Coordinator, and finishing when Serach result was handed back to a Physician. Results CFS (n=184): 303 siblings were HLA-typed (44 in 2000 HLA-typed (44 in , 115 in 2001 HLA-typed (44 in , 144 in 2002 . Median duration was 13 (2-96) days (13 in 2000, 14 in 2001, 12 in 2002) . 27/184 (15%) of CFS were prolonged over 28 days and in 9/184 (5%) cases some siblings refused or hesitated to be HLA-typed. Donor was found for 67/184 (36%) of our pts, 97% were HLA-genoidentical. 2 donors (3%) refused donation. Results EFS (n=42): 148 members were HLA-typed ( 51 in 2000 HLA-typed ( 51 in , 48 in 2001 HLA-typed ( 51 in , 49 in 2002 . Median duration was 14 (2-160) days (16 in 2000, 10 in 2001, 14 in 2002) . 5/42 (12%) of EFS were prolonged over 28 days. Donor was found for 3/42 (7%) of our patients. Conclusions: We conclude, that our capability of managing CFS and EFS is satisfactory. Groving number of searches does not have negative impact on its duration and chance of finding a donor corresponds with data presented by others. Complicated, negative relations within a pts family can prolong search procedure, even refusal of a donation can occure. Discussion: Every effort shloud be further made to reduce duration and number of prolonged searches. Search duration up to 14 days could be optimal in case of well cooperating patient, family, Coordinator and HLA laboratory. Unrelated donor search (UDS) should not be neglected when EFS is on process. EFS with its limited outcomes could take precious time useful for UDS, especially in pts with a highly aggressive disease.

Autologous blood is commonly collected from bone marrow donors before the harvest and returned at the time of marrow donation to reduce the fall of Hb concentration. We reviewed the hospital charts of 231 female and 326 male bone marrow donors to determine how useful this practice is. 386 donors were siblings and 171 unrelated. The policy was to store one unit of blood from sibling donors except when the time to the donation was short, and from unrelated donors except when the recipient was a child. The blood was collected 2-5 weeks before the donation. All donors except seven were older than 16 years (range 12-68 years). Marrow was harvested with small aspirations of approximately 3 ml, the median total harvested volume was 900 (range 250-1350) ml. The median numbers of nucleated cells harvested from sibling and unrelated donors were 3.2 (range 0.9-7.4) and 4.8 (1.2-17.0) x 10 8 /kg patient weight, respectively. An autologous blood unit was stored from 85% of the female and from 78% of the male donors. An autologous transfusion was given to 80% of the female and 72% of the male donors. 14 donors did not receive autologous transfusion because the transplantation was postponed and the blood unit became outdated. Just before the marrow harvest the median Hb concentration was 127 g/l (range 104-152 g/l) in the female and 143 g/l (116-168 g/l) in the male donors autotransfused, and 136 g/l (117-154 g/l) in the female and 150 g/l (125-172 g/l) in the male donors not autotransfused. On the morning following the harvest the median Hb concentration was 105 g/l (79-135 g/l) in the female and 122 g/l (89-151 g/l) in the male donors autotransfused, and 102 g/l (75-133 g/l) in the female and 119 g/l (88-146 g/l) in the male donors not autotransfused. The postharvest Hb was less than 85 g/l in 4 female and less than 90 g/l in 23 female and 2 male donors. 14 of the donors with postharvest Hb less than 90 g/l had received an autologous transfusion. The post-donation Hb concentration did not decrease in any donor to levels detrimental to healthy persons regardless of whether autologous blood was transfused or not. If marrow is harvested in a manner to maximize the cell concentration and to minimize the volume, it is as a rule not necessary or useful to collect autologous blood for bone marrow donation, with rare exceptions.

Arterial access for peripheral blood stem cell collection J. Stein, J. Katz, O. Gelber, A. Grunspan, Y. Sverdlov, I. Yaniv, Schneider Children´s Medical Center of Israel (Petach-Tikva, IL) Peripheral blood pheresis is an efficient means for hematopoietic stem cell procurement. A major limitation of this procedure is the need for vascular access, which leads to the placement of central venous catheters in up to 20% of volunteer donors. Severe complications of central venous line-placement have been reported in adult stem cell donors, and the procedure is technically difficult to perform in obese adults and in young children. By contrast, percutaneous placement of arterial catheters (AC) is a simple and rapid procedure with a very low complication rate (<0.2%). We performed 47 peripheral blood cell collections using the Cobe Spectra® from 20 patients and donors using AC's placed by anesthesiologists. Patients arrived at the collection center after a 4 hour fast, and had platelet counts >50,000/ul. Conscious sedation (propofol 1 -2 mg/kg) was used in 45 procedures, while 2 were performed using local anesthetic alone. Nearly all procedures lasted less than 5 minutes, and were often completed within 2 minutes. 30 collections were performed for autologous use, 6 for unrelated patients, 3 for family members, 7 for extracorporeal photopheresis, and 1 for a donor lymphocyte collection. 20 donors were minors, ranging in weight from 9.7 to 57 kg. An existing indwelling central venous catheter was used as the return line for 15 autologous collections, while standard percutaneous intravenous lines were used in the remainder of cases. An average of 3.87 blood volumes were collected in each procedure, and the average time to collect a single blood volume was 47 minutes. The mean mononuclear cell count per collection was 7.93 x 10 8 /kg donor weight. Arterial access permitted maximal inlet flow rates (adjusted for size of the donor) to be used, expediting the collection process. Donor satisfaction with this procedure was excellent, and no complications occurred. We suggest that percutaneous AC's are a safe and efficient solution for peripheral blood stem cell donors collections in donors with venous access problems.

Phenotypic analysis of CD34+ and CD133+ subsets in successive collections of mobilised peripheral blood progenitors L. Gopcsa, A. Barta, A. Banyai, A. Poros, K. Paloczi, National Medical Center, Semmelweis University (Budapest, HUN) CD34+ and CD133+ cells obtained from peripheral blood represent the major source for stem cells used in autologous hematopoietic transplants. The aim of the study was to analyse the different stem cell subsets (CD133+CD34+, CD133+CD34and CD34+CD133-with or without CD7 co-expression, CD34+CD38+/-, CD34+CD117+/-, CD34+HLA-DR+/-and CD34+CD13+/-) in mobilized peripheral blood stem cell (PBSC) samples after salvage chemotherapy with recombinant human granulocyte colony stimulating factor (G-CSF) in patients with lymphoid malignancies. A total of 28 patients (17 female, 11 male) were entered in this study, including 14 with Non-Hodgkin's lymphoma, 7 with Hodgkin's disease and 7 with multiple myeloma. A total of 68 leukaphereses were analysed. The median count of nucleated cells, CD34+ cells and CD133+ cells were 7.3 x10 expressed the CD38, HLA-DR and CD13 antigens. Among the CD34+ cells, slightly less than one-thirds expressed CD117. Within the CD133+ cell compartment, the great majority of cells co-expressed CD34 antigen. The CD133+CD34-subpopulation was minor compartment and their percentage remained relative constant over leukaphereses. A relationship between the numbers of reinfused CD133+, CD34+ cells and the early engraftment were studied in 22 patients. The total CD34+ and CD133+ cells showed some correlation with early absolute neutrophil cell engraftment, but the CD34+CD13-subset was the best predictors. However, the CD34+CD13-and CD34+HLA-DRcells correlated with the early platelet engraftment. We also found some connections between the kinetics and the compositions of the CD34+ and CD133+ cells in consecutive PBSC collections. Comparative analysis of the subsets of CD34+ and CD133+ cells in the different patient's subgroups according to the stem cell yield and mobilization schemes were performed. Based on the results both CD34 and CD133 positivity are able to determine stem cells and early progenitors and equally important for clinical transplants. This work was supported by grants of NKFP 1/024/2001, Budapest, Hungary.

Introduction: In the last decade most centres have incorporated the use of peripheral blood cells in allogenic transplant programs. One of the advantages of this method is that general anaesthesia is avoided on donors, and that hematopoiesis recovery is more rapid than with bone marrow. Even though this method has many advantages, there are no guidelines to improve PBPC collection with wide registries showing a great heterogenity in the efficiency of yield products. We now present our experience in Large Volume Leucapheresis (defined as more than 3 times the total blood volume) processed on healthy donors as a simple and efficient procedure. Methods and Donors: 23 healthy donors (14 males and 9 females; median age 48, 11-73) were included in an allogenic peripheral blood transplantation program. They all received 12 mg/kg/day s.c of rhG-CSF (filgrastrim®, Amgen, Thousand Oaks, CA, USA) in two doses during 4 days. Our main objective was to yield at least 3x10 6 /kg CD 34+ cells. Leucapheresis was started on the fifth day after the administration of rhG-CSF. Large Volume Leucapheresis (LVL) was programmed (processing 15 to 25 L) and progenitor cells were collected through peripheral vein access in all cases, using a Cobe Spectra separator (COBE, Denver, CO, USA). Results: PBPC collection yield a median 6.22x10 8 /kg mononuclear cells (range 1.61-27.09) and 8x10 6 /kg CD 34+ cells (range 2.85-13.57). The median number of patient's blood volumes processed was 4L (range 3.2-4.45). All products were transplanted with rapid and sustained engraftment in all cases. No mayor adverse effects were observed and minor morbidity related to the PBPC collection was very low. Asymptomatic thrombocytopenia observed was in all cases transitory that turned to normal within a week. Results are shown in the table. Comments: As it has been observed in autograft programs for onco-haematologic patients, Large Volume Leucapheresis is a simple and safe method to collect progenitor cells from healthy donors. This method allows an adequate PBPC collection for transplantation with the simplification of a single harvesting procedure which is enough for prompt hematological engraftment. A HLA matched family donor (MFD) is a donor of choice in allogeneic bone marrow transplantation (BMT). Conventionally clinically eligible siblings and members of the extended family, for example in cases of consanguineous marriages, are tested to identify MFD. We have prospectively added two more tiers to this strategy assessing patients' children and parents as MFD. If no matched sibling (MSD) was identified the partner of patients with at least one child were HLA typed. For 99 patients 100 partners were typed and 14 were identified as sharing 1A, 1B, 1Cw, 1DR and 1DQ antigen. Nine of the 14 pairs shared the A, B, Cw, DR and DQ antigens seen in 1 of 3 common 'ancestral' haplotypes observed in a North European population; A1/B8/DR17, A3/B7/DR15 and A1/B57/DR7. Of this group (n=14) 18 children were typed and 4 were identified as HLA matched with the patient, with 3 out of 4 due to patient and partner sharing a common 'ancestral' haplotype. This 4% MFD identification rate is a conservative estimate since 4/14 of the patients either did not agree to have their child typed or the children are in the process of being typed. Two of these patients have been transplanted, are well and fully engrafted at 24 and 48 months. When available the parents of patients were also HLA typed. A total of 287 parents of 160 predominantly adult patients were typed and 7 patients (4.4%)were identified as having HLA matched parent. Of the 7 donors identified, 3 were MFD due to the parents sharing one of the common 'ancestral' haplotypes given above. These results show that assessing patients' children and parents can increase the identification of MFDs by at least 8.4%. In our programme we identify a MSD for 35.9% of patients but this additional source of MFDs is particularly important for those not eligible for an unrelated BMT and provides a transplant option for those patients, which would otherwise not be available. The observation that these MFD are often due to the sharing of a common 'ancestral' haplotype between patient/ partner or patients parents allows for strategic targeting of patients with these haplotypes. However, this strategy would have led to loss of one of our four child donors, where a 'ancestral' haplotype was not shared. The identification of common 'ancestral' haplotype in the patient would also allow strategies to identify MFDs in the patients' nieces and nephews. (16,000). Although the rate of addition will decrease as the panel size increases. An analysis of 1314 donor searches with 6/6 matches showed that those donors that were HLA-Cw typed were preferentially selected for confirmatory and extended typing. These results indicate that in addition to the resolution of the typing, the number of loci typed also has a significant impact in the selection of donors requested for further testing. In this study we retrospectively analyzed the outcome of the 115 patients admitted to our center between January 2000 and May 2003 who were found to be suitable for AHSCT. Also, we evaluated the effect of time from official transplantation decision to transplantation on early transplant related mortality and relapse incidence in 58 patients who underwent AHSCT. The diagnosis of the patients was: 39 AML, 30 CML, 16 ALL, 5 MM, 3 MDS, 8 solid tumors, 9 malign lymphoma, and 5 other diseases. AHSCT could be only performed in 58 (50%) of the patients. Twenty out of remaining 57 (35%) patients did not apply for the transplant procedures, 6 patients relapsed (10%), 6 patients (10%) are still waiting in the transplant list, 14 (24%) patients were excluded because of remission of the primary disease and co-morbid conditions. In 11 patients (19%) transplantation was not performed due to denial of the donor or recipient and social security coverage problems. In the AHSCT group median time from diagnosis (Dx) to official transplant (Tx) decision was median 6.53 (0.57-54.43) months, time from decision to transplant was median 4.90 (0.97-24.67) months and time from Dx to Tx was median 11.05 (3.33-62.47) months. Time from Dx to Tx decision and time from Dx to Tx had no impact on early transplant related mortality and relapse incidence in our patients. In conclusion, half of the patients can complete all transplant procedures in our center but the duration of the procedures on waiting list did not effect the outcome of transplanted patients in early post-transplant period.

U. Platzbecker, M. Bornhäuser, K. Zimmer, C. Rutt, G. Ehninger, K. Hölig, Universitätsklinikum Carl Gustav Carus, DKMS (Dresden, Tuebingen, D) Objectives: G-CSF is increasingly used to mobilize peripheral blood stem cells (PBSC) in healthy donors for allogeneic transplantation. Nevertheless, there are still limited data available on the efficacy and safety of repeated donations of PBSC. Methods: We investigated 67 healthy donors with a median age of 37 years (20-70) who underwent two separate mobilizations of PBSC. The median interval between first and second donation was 4.96 months (0.07 -46.91). For both first (FM) and second mobilization (SM) 7.5 µg/kg/day Lenograstim was administered and PBSC were collected from day 5 on. Results: G-CSF administration caused an elevation of the median WBC on day 5 of SM to 38.6 x10 9 /l (16-76), the median ANC to 29.7 x10 9 /l (13-66) and the median CD34+ concentration in the peripheral blood raised to 36.6/µl (6-135 Interestingly, a significant lower yield of CD34+ cells x 10 6 /kg donor weight was obtained on day 5 only in female (n=31, FM: 5.0 v. SM: 3.2, p=0.008) but not in male (n=36, FM: 5.9 v. SM: 5.4, p=0.24) donors. Donor age, weight, interval between mobilizations as well as WBC, ANC and concentration of CD34+ cells/µl in the blood on day 5 of SM were not statistically different between both groups. Side effects of G-CSF application and donation were comparable between both mobilizations in all donors. Univariate analysis showed that in donors with lower CD34+ blood concentrations on day 5 of FM (CD34-FM) and in females there was a higher risk for a yield of < 2x10 6 CD34+ cells/kg donor weight on day 5 of SM. A multivariate analysis revealed an interval of less than one month between donations (p=0.02), higher age (p=0.016) as well as a low body mass index (p=0.045) and CD34-FM (p=0.014) as independent factors associated with a higher probability to achieve < 2x10 6 CD34+ cells/kg on day 5 at the SM. Conclusion: A SM of PBSC is safe and feasible resulting in a sufficient number of progenitor cells in the majority of donors. Additionally, our study may help to identify donors, which are at increased risk for a low stem cell yield at a SM. This could lead to alternative strategies i.e. search for a different donor in order to reduce the distress of the donor and to avoid the clinical sequelae of transplanting a low number of hematopoietic cells in recipients requiring a second allograft.

A. Sperotto, F. Patriarca, M. Cerno, F. Silvestri, F. Zaja, C. Filì, A. Geromin, E. Calistri, R. Stocchi, D. Damiani, R. Fanin, Udine University Hospital (Udine, I) Background. The aim of our study was to retrospectively evaluate the outcome of unrelated bone marrow donor searches for 131 consecutive patients from February 1997 through April 2003. Design and Methods. At the time of search activation, 77 (58.5%) patients were affected by acute leukemia (AL), 24 (18.5%) by chronic myeloid leukemia (CML), 19 (14.5%) by lymphoma (L) and 11 (8.5%) by multiple myeloma (MM). In 82 (62.5%) patients a donor was identified: 69 (52.5%) patients were subsequently transplanted Characteristics of transplanted patients. Male/female ratio was 1.5. Median age 35.5 years (15 -59). 43 (62.5%) patients were affected by AL, 16 (23.5%) by CML, 5 (7.0%) by L and 5 (7.0%) by MM. 22 (32.0%) patients were transplanted early in the course of the disease (AL in first complete remission; L in at least partial remission, CML in chronic phase), while late transplant was performed in 47 (68.0%) patients. Results OS of the 131 patients were 42.5%. OS of the transplanted patients vs not transplanted ones were 52.5% and 27.5% respectively (p value =.05). OS of AL transplanted vs not transplanted were 59.5% and 22.0% respectively (p value=.027); while no difference in OS was observed for CML patients (transplanted vs not transplanted: 88.0% and 77.5% -p value = .41) Thirty -nine (56.5%) of the transplanted patients developed an aGVHD. 32 (46.5%) of the transplanted patients are alive and disease free, 3 (4.5%) are alive with disease, 9 (13.0%) died for disease and 25 (36.0%) died for transplant-related complications. OS of early vs late transplants was 75.0% and 17.5% respectively (p value= .017). Conclusions. In our experience, the probability of identifying a donor was higher than 60%: unrelated transplant has benefited a substantial number of patients lacking a matched sibling. Transplant seems also to improve the survival of patients, especially that of acute leukemia ones. Moreover transplants performed early in the course of the disease gives an advance in term of OS. Considering that the long time required for the search is often the major obstacle to the success of this program an early activation (at diagnosis for high-risk disease) should be strongly pursued.

I. Dolgopolov, R. Protsenko, L. Andreeva, V. Boyarshinov, R.

From 2001 to 2003 4 transplantations from family mismatched donors without T-depletion were performed in 2 pts with high-risk AML, 1 pt with CML in accelerating phase (relapsing after two syngenic transplants) and 1 pt with metastatic relapse of Ewing's sarcoma. In 1 cases donors were 4/6 matched mother, and in 2 -5/6 HLA matched brothers and in 1-5/6 matched mother. Conditioning regimen included fludarabine 180 mg/m2, busulfan 8 mg/kg and ATG 40 mg/kg. The median number of CD34+ and CD3+ were 5.8 (2.2-7.8)x10 6 /kg and 5.3 (2.8-8.6) x10 8 /kg, respectively. PBSC were transplanted after 30' incubation with vincristine and methylprednisolone without washing. GvHD prophylaxis consisted of short methotrexate and cyclosporine A. The WBC level >1.0x10 9 /l was reached on day+13 (11-17), and PLT transfusion independence was achieved on day+13 and +17 in 2 cases, in 2 cases pts required no PLT transfusion. All patients developed a full donor chimerism by d+100 or earlier. GvHD of grade I was seen in 2 pts, II in 1 and III in 1 pt, respectively. Treatment with steroids was effective. On November, 2003 2 pts (1 with AML and 1 with Ewing's sarcoma) are disease free, well and with full donor chimerism at 18 and 4 mo., respectively, with no evidence of GvHD. One pt with secondary AML died of disease progression on d+92. One pt with CML died of virus infection on d+38. Transplantation from a partially mismatched family is feasible and may be potentially curable in pts who need an allogeneic transplant but have no conventional donor. The lack of Tdepletion might lead to early recovery of antitumor immunity. GvHD is not fatal with an adequate prophylaxis. Low toxic regimen seems to be sufficient for stable donor hematopoiesis recovery and antitumor effect of transplant. Supported by Mr. J.Watkins donation.

Objectives: Usually mobilisation of CD34 positive cells with lenograstim in unrelated donors requires application of medication for 5 to 6 days. In our institution lenograstim was administered for 4 to 5 days, the progenitor cell apheresis took place on 4th and -if necessary-on 5th day. Retrospectively we analysed factors of influence for mobilisation success as gender, BMI (body mass index) or effective dosis of lenogastrim. Methods: 55 unrelated PBSC-donors (peripheral blood stem cell donors) received 11,8 (9,1-15,8) mikrogramm lenograstim sc. per kg BW (bodyweight) per day divided in two equal doses in 12 hours interval for 3 and a half day. 37 of the donors were male (67%), 18 female (33%). 33 donors had a BMI < 25 (60%) and 22 had a BMI > 25 (40%). Target transplant dosis was 4x106 CD34+ cells per kg BW of the recipient. Results: On day 4 mean peripheral CD 34+ cell concentration in all donors was 68 (14 -184) cells per mikroliter. 40 of 55 donors (73%) had CD34+ cell concentration of higher than 40 cells per mikroliter, they were classified as good mobilisers. In 39 of them (71%) we performed a single apheresis, in 16 (29%) two procedures. 15 donors (27%) were classified as poor mobilisers because of their CD 34+ cell concentration of less than 40 cells per mikroliter. In two of them (13%) a single apheresis was performed to reach requested CD34+ cell dosis. In 13 (87%) a second apheresis became necessary. 9 of poor mobilisers were male (60%), 6 were female (40%). The majority of them (80% N=12) had a BMI < 25. Conclusions: 39 of 55 donors (71%) completed apheresis cycle with only one procedure. This result is comparable with the standard mobilisation regimen. So the mobilisation regimen is feasible. In 13 donors, who did not reach a CD34+ cell concentration higher than 40 per mikroliter on day 4 and had to donate for a transplant recipient heavier than 50 kg, a second apheresis became always necessary. Among poor mobilisers were more donors with normal BMI (80%) than in the whole cohorte (60%). We explain the better mobilising effect of the regimen observed in donors with BMI > 25 with a higher effective dose, because adipose tissue cells does not express G-CSF-receptor.

Induced acute graft-versus-host reaction after syngeneic transplantation by short-course cyclosporine A administration possibly provides acute graft-versusmalignancy effect resulting in long lasting complete remissions H. Bertz, B. Hackanson, O. Schmah, A. Spyridonidis, M. Egger, K. Mikesch, F. Dörfel, J. Finke, University of Freiburg (Freiburg, D) Syngeneic transplantation is like autologous transplantation associated with a high incidence of relapse, because of the lack of HLA disparity and GvH-Reaction. Inducing GvHD with a Gv-Tumour effect is possible after autologous and syngeneic transplantation by cyclosporine A (CSA) administration. To induce a GvHR after confirmed twin transplantation pts. were treated at our institution with CSA starting day -3 with a though level of > 200ng/ml. On day +30 CSA was immediately stopped without tapering. Up to now 8 patients [5m/3f; median age 40 years (20-59)] were treated with this protocol. Diagnosis are CML (2), MM (2), highgrade NHL (2), sAML (1) and ALL (1) . Remissions at transplantation were CR1/CP1 (3), SD (2), CP2 (1), persistent induction failure (1) and relapse 1 (1;after autologous TX). BuCY (5), FBM (2) and TBI/CY (1) were the condition-ing regimens and PBSC (7) served mainly as graft source. All pts. received CSA 5mg/kg /d divided into two doses day -3 till day + 30; only the first patient received methylprednisolon, additionally. Results: All pts. engrafted and achieved CR. Leucocytes >1x10 9 /l were reached in median day +10 (9-15) and platelets >20x10 9 /l day +11 (7-15). Acute GvHD, requiring steroid treatment, occurred in 2/8 patients (25%), overall maximum grade 2, and limited cGvHD in 1/8 pts.. In median of 1228 days (677-3911) after transplantation six patients (75%) are alive with 4 (50%) in CR [AML (1), CML (1), hg. NHL (2)]. Both pts. with MM have signs of increasing paraprotein and receive DLI. Two pts. [ALL and CML; CP 2] relapsed and received a MUD transplantation, but died due to the underlying disease or MOF. We conclude, that it is possible to achieve long lasting CR after syngeneic transplantation. Cessation of short course CSA induces higher grade acute GvHD and may provide a Graft vs Malignancy effect. Concerning chimerism, 121 patients presented FDC, 21 regressive MC, 20 stable MC and 22 progressive MC. At last follow up, 116 patients are alive and 71 died (55% from disease progression and 45% from TRM). At 60 months post-transplant the probability of EFS was 43% (95% CI 35-52.4). The 3-year probability of EFS was 60% for patients in FDC, 25%, 29% and 10% for patients in RMC, SMC and PMC respectively. The multivariate analysis showed a significant impact on EFS of MC status, acute and chronic GVHD. This retrospective analysis clearly showed that MC is as well informative as other well known factors to influence EFS and that FDC profile remains a very good prognostic marker. Introduction: Chimaerism quantification is of great utility after allogeneic stem cell transplantation (SCT) because it allows prediction/early diagnosis of engraftment, graft rejection or disease relapse. However, from a methodological point of view, a gold standard for chimaerism quantification has not been defined yet. Objective: To compare FISH for the sex chromosomes (XY-FISH) with fluorescent microsatellite-PCR (STR-PCR) for chimaerism quantification after SCT. Material and methods: A total of 106 samples were analyzed with both techniques. Two independent groups of artificial mixtures of male and female cells (% male: 100, 75, 50, 25, 10, 5, 3, 1, 0.1, 0), as well as PB and BM samples from 10 patients showing different clinical evolution post-SCT were included in the study. XY-FISH (CEP XY; Vysis) was performed on routine smears scoring 500 nuclei in a blind fashion. Fluorescent STR-PCR was performed using AmpFlSTR SGM Plus kit (Applied Biosystems) and the PCR products were run on an ABI Prism 3100 (Applied Biosystems) DNA sequencer. Results: Results obtained with both techniques from the quantification of male ("recipient") cells in artificial mixtures showed a high correlation (r=0.998, p=0.01), as well as with the expected values (FISH r=0.999, PCR r=1.000, p=0.01). Sensitivity of the FISH assay has been previously established in our laboratory to be 1%. All informative STR markers identified on artificial mixtures were able to detect "recipient" (male) DNA in all samples with 3% or higher content of male DNA. Moreover, up to 0.1% male DNA could be detected in the first experiment with 2 STR markers (D3S1358 and TH01). Results obtained from the patients samples also showed similar results with both techniques and allowed the concomitant detection of mixed chimaerism in patients previously in complete chimaerism, as well as the restauration of complete chimaerism after DLI, in the same sample (data-point). Conclusions: Both techniques rendered similar results all along the quantification range (0-100%). Additionally, sensitivity is at least similar (FISH 1%/STR-PCR 3%; while consistency and clinical utility of the detection of small percentages (>1%) of recipient cells, observed with particular STR markers is established) and higher to that obtained with conventional STR-PCR (5-7%). Therefore, fluorescent STR-PCR can be currently considered as the "gold standard" for chimaerism quantification after SCT.

R.E. Ploemacher, K.W. Johnson, E.W.C. Rombouts, J. Baumgart, M. White-Scharf, J.D. Down, Erasmus MC, Biotransplant Incorporated, Medac GmbH (Rotterdam, NL; Charlestown, USA; Wedel, D) The alkylating prodrug Treosulfan (L-threitol 1,4 bismethanesulfonate) is currently undergoing clinical trials in hematopoietic stem cell (HSC) transplant conditioning protocols at myeloablative doses and as an alternative to Busulfan or total body irradiation. Previous studies comparing a number of different cytotoxic agents and fractionated radiation regimes have established that depletion of the long-term repopulating HSC in the recipient bone marrow (BM) predicts the extent of long-term hematopoietic engraftment after syngeneic HSC transplantation. In the present murine study we show that this drug is similar to Busulfan in its ability to deplete primitive HSC as determined by the cobblestone area forming cell (CAFC) assay. The level of HSC depletion in the recipient bone marrow was found to be both dose-and schedule-dependent and was reflected in the ability of Treosulfan to allow for long-term engraftment of syngeneic or allogeneic donor bone marrow. Under the cover of an immune suppressive regimen consisting of T-cell-depleting antibodies, Fludarabine and thymic irradiation, it is important to note that a low, non-myelosuppressive dose of Treosulfan (3 x 500 mg/kg) supported the establishment of stable, multilineage, mixed chimerism in C57BL/6 recipients of MHC-mismatched B10.A HSC transplantation without evidence for graft versus host disease (GVHD). In this case, the survival level of about 10 % competing HSCs remaining in the recipient bone marrow after low dose Treosulfan treatment corresponds to about 8 % stable donor HSC engraftment. Donor lymphocyte infusion performed at 10 weeks post transplant had the effect of transforming the state of mixed chimerism to full donor-type cells, again without evidence for GVHD. Donor-specific immunologic tolerance in the mixed chimeric animals was indicated by the acceptance of donor-type and rejection of third-party skin grafts. As Treosulfan has a low toxicity profile in clinical HSC transplantation, application of this drug at low doses may be considered as a useful component of a truly non-myeloablative conditioning protocol in providing for mixed hematopoietic chimerism and, consequently, in establishing a platform for adoptive immunotherapy or organ transplantation. We evaluated outcome of 18 patients with CML Ph+ and ALL Ph+ treated with Glivec that underwent allogeneic stem cell transplantation in our center. 15 pts were CML Ph+ (6 pts with biomolecular remission achieved with Glivec, 8 pts with hematologic remission in chronic phase 1 to 3, 1 pts in acceleration phase) and 3 patients were ALL Ph+ (2 pts that had relapsed, 1 pts in 2nd hematologic remission). Median age was 29 years (16 -48), median disease duration time --2,1 years (0,9-9,5), median Glivec therapy time -6 months (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) . In all cases therapy with Glivec was interrupted immediately before myeloablative regimen conditioning (1 pts: TBI+CTX, 2 pts:

TBI+Ctx+ATG, 12 pts: BuCy+ATG, 2pts:

Treosulfan+Fludarabine+ATG, 1pts BuCy). Two related donor and 16-unrelated donor HLA matched (in A, B, DR antigens) SCT were performed. The source of stem cells was peripherial blood in 7 and bone marrow in 11 cases. Median time to achieve ANC > 500/mm3 was 23 days (15-36), platelets > 50x10e9/l -27 days . 16 pts achieved full hematopoietic reconstitution. Transplant related mortality (TRM) was 16,6% (3 pts). Incidence of acute GvHD >2 grade -11,1% (2pts). 11 pts are in ongoing cytogenetic and biomolecular remission with a median follow -up observation of 6 months .

Glivec was administered posttransplant only to 1 pts (graft failure). 1 pts with ALL has relapsed, 1 pts with CML developed extramedullar relapse in CNS, testes and spina (with continous cytogenetic and biomolecular marrow remission), 1 pts developed secondary graft failure.

In conclusion, we found that Imatinib (Glivec) is not rising probability of acute GvHD and does not affect hematopoietic recovery. The plasticity of bone marrow derived stem cells has generated some enthusiasm regarding the potential clinical use in tissue regeneration. To investigate this possibility in the setting of myocardial infarction, we started a clinical phase I/II study. We included patients at least 3 weeks after a myocardial infarction needing aortocoronary bypass surgery. Bone marrow was obtained by sternal puncture during general anesthesia just prior to surgery. Bone marrow cells were density separated and resuspended in 2 ml volume. After bypass surgery 10 injections of 0.2 ml each were applied to the infarcted area. Before and 3, 6 and 12 months after the intervention, the myocardial function was assessed by ECG, echocardiogramm and scintigraphy. 14 patients (age 40 -71) have been treated so far with no significant adverse events attributable to the cell injections. The median total injected mononuclear cell number was 5.8 x 10 7 (range 0.8 -18.6). The median percentage of CD34+ progenitor cells was 1.58% (range 1.26 -3.28) and CD34+CD133+ 0.57% (range 0.1 -2.64), while endothelial progenitor cells were found with the following frequencies: CD34+KDR+ 0.02% (range 0 -0.05), CD34+vWF+ 0.73% (range 0.09 -2.47), CD34+CD144+ 0.44% (range 0.08 -0.64). So far 9 patients were assessed 3 months after the intervention. In 4 of 5 patients with a transmural infarction FDG-PET showed a 20-50% reduction in scar size. In one of these patients the infarcted area diminshed by 25% despite the fact that it was not revascularised. No significant change in ejection fraction was found. In conclusion our data show the feasibility of intramyocardial bone marrow cell injection during CABG surgery and provide preliminary evidence for myocardial regeneration after such a procedure. However more data are needed and only prospective placebo-controlled clinical trials will be able to answer the question regarding the clinical effectivness of this novel form of cellular therapy.

Enhancing bone defects repair using autologous bone marrow buffy-coat: report of 70 cases Bone marrow as a source of mesenchymal progenitors (MP) for the treatment of osteopenic defects has been introduced both in experimental models and, recently, in clinical practice. Under normal conditions, MP concentration in bone marrow is usually lower than 1% but they can be easily purified and expanded ex vivo.

Aim of this study is to evaluate the capability of mimally manipulated bone marrow cells to fasten bone regeneration following surgery for osteopenic defects.

Since June 2000 to November 2003 a total of 70 procedures have been performed for 61 patients diagnosed with various osteopenic disorders (mainly bone cysts and non-unions) who underwent bone marrow harvest from posterior iliac crests; a mean of 190 (75-273) mL were collected. Buffy coat was obtained in 38 cases by traditional procedure (centrifugation at 2000 rpm for 10 min). The following 32 cases were processed by SEPAX (BIOSAFE, Switzerland), a centrifugation device for automated processing of blood components in a closed and sterile environment.

Final results were a mean buffy coat volume of 27 (7-60) mL, containing 3 (0,23-7,40)x10 9 nucleated cells (TNCs). MP were evaluated by both cytofluorimetric assay of CD45/14 negative cells and the presence of cells generating fibroblast-like colonies (CFU-F). The mean number of CFU-F obtained was 40 (5-227)/1x10 6 TNC. Buffy coat cells were implanted either directly in the bone defect site or after resuspension with bone matrix and autologous platelets followed by Ca++ in order to induce clotting and platelet degranulation. The procedure was well tolerated and no side effects were recorded. Bone regeneration was shown faster as compared to historical controls. Both prospective trials and animal models are currently under development.

Correlation between the number of homed bone marrow mononuclear cells and the extension of the infarcted area in a rat model R. Pacchiana, R. Giordano, M. Ciulla, S. Ferrero, L. Lazzari, U. Gianelli, G. Busca, S. Bosari, F. Magrini, P. Rebulla, Cell Factory Ospedale Maggiore Policlinico, Istituto di Anatomia Patologica Ospedale San Paolo (Milan, I) Several clinical trials have recently exploited the ability of bone marrow (BM) derived cells to migrate to the infarcted myocardium and promote tissue repair. Nevertheless, the mechanism underlying this phenomenon is still unclear. Acute myocardial infarction (AMI) is invariably associated with inflammation and remarkable activation of the cytokine cascade. Since the production of inflammatory mediators is proportional to the extension of the damaged tissue, it seems reasonable that the infarct size may play a major role in chemoattracting BM progenitors. In this study we evaluated the hypothesis that the homing of bone marrow mononuclear cells (BMMNCs) is related to the extension of the necrotic area, in a rat model of myocardial cryodamage. Twelve donor and 12 recipient inbred isogenic adult (4 weeks old) Fisher rats were used to mimic autologous transplantation. Myocardial damage was obtained in recipient rats by cryoinjury. BMMNCs were purified labeled with PKH26, a red fluorescent cell dye, and infused 7 days after the injury through the femoral vein of recipient rats. One week after the infusion, the number of homed BMMNCs was assessed and the infarct size was correlated with the number of cells present in the damaged tissue. Labeled cells were found only in the injured myocardium of the treated animals (n=6), where a mean of 12 ± 3 PKH26+ cells per section examined were found; a significant correlation was found between the infarct size and the estimated number of cells (p=0.008). These data show that the homing of BMMNCs is related to the extent of the myocardial injury and suggest that some components of the inflammatory response after myocardial infarction may have a beficial effect on cardiac repair. In this regard, investigations are currently ongoing, aimed to the identification of molecules involved in mobilization and chemoattraction of blood derived progenitor cells after AMI.

Myocardial regenerative therapy from autologous bone marrow stem cells Sanchez, A. San Román, F. Fernandez-Avilés, J. García Frade, Hospital Rio Hortega (Valladolid, E) Experimental and clinical evidence suggests that bone marrow stem cells may differentiate into functioning myocytes and blood vessels after myocardial infarction. In september 2002 we started a phase I clinical trial on such regenerative therapy. Patients and methods:10 patients (age: mean/range=60/33-74 years) with non-viable (eco-dobutamine) anterior acute myocardial infarction (AMI) in the subacute phase were enrolled until September 2003. 50 ml of bone marrow were aspirated from the posterior iliac crest.The mononuclear fraction (MNC) was extracted by Phycoll density gradient, and cells were kept in culture medium overnight. The next day (13 + 4 days postinfarction) cells were infused in the anterior descending coronary artery by means of a balloon-catheter. Myocardial function was assessed at baseline and at 6-months follow-up by ECOdobutamine, talium scintiscan, magnetic resonance and PET scan. Results: A median of 57.5 X 10 6 MNC (range:11-125) were infused, with percents of 1.17 + 0.62 CD34+, 1.70 +1.00 CD117+, and 0.73+0.37 CD133+. After a median follow-up of 240 days (range:57-431) the only complications noted were femoral ischemia in one patient during cardiac catheterisation, and a transient ischemic attack 10-days post-infusion in another one. No arrythmias were apparent on Holter testing. 8 patients have reached the 6-month follow-up, with a 15% cardiac volume reduction and a 16% improvement in overall ventricular function and segmental dynamics. Comments: These preliminary results suggest that autologous bone marrow stem cell transplantation as a regenerative therapy may safely be carried out in AMI patients in the subacute phase. The efficacy data show some promise.

(CCl4)-induced liver injury E. Yannaki, E. Athanasiou, A. Xagorari, V. Constantinou, I.

On the basis of the recently recognized potential of bone marrow cells to give rise to hepatocytes, we investigated the possibility if only G-CSF could improve liver damage in an experimental injury model. To this goal, C57Bl6 mice were administered: Group A:G-CSF 200µ/kgX7days+CCl4 4ml/kg on day 8+G-CSFX4 days, Group B:G-CSFX7 days+CCl4 on day 8, Group C:CCl4 only. Liver histology, 5 days after CCl4, demonstrated in group A only mild centrolobular necrosis; in group B moderate necrosis, and in group C significant necrosis with severely disrupted architecture (injury grade I-II,I-III,III-IV, respectively). This was accompanied by a survival benefit in the G-CSF-treated groups. 15 days after CCl4 necrosis could still be detected only in group C. We also tested the G-CSF effect in a liver fibrosis model generated by CCl4 2ml/kgX2d/wkX2months. Group I and III received CCl4 for 2 months and group II CCl4 for 2 months+G-CSFX8d. Group I and II were sacrificed 9 days after last CCl4 dose and group III was sacrificed one month after. Histology after Gomori and Masson staining demonstrated typical cirrhosis in group I, while group II livers appeared relatively normal with fewer nodules. Group III livers had normal architecture with only occasional short fibril fragments. Since spontaneous recovery occurs also in the CCl4-treated group, in both injury models, we further explored to which degree this regeneration is endogenous or bmderived and whether the acceleration in recovery observed in G-CSF groups is due to mobilized HSCs. Female, lethally irradiated C57Bl6 mice, were transplanted with whole bm from male donors. 40 days post BMT, one group received G-CSFX7d+CCl4 on d8+G-CSFX4d and one group received CCl4 only. An indirect immunohistochemical method for sry protein detection, demonstrated clusters of donor-derived hepatocytes in both groups. 1.7% of the total hepatocytes were of donor origin in the G-CSF group compared to 0.7% in the control group. Overall, these data suggest that liver recovery after necrosis is basically liver-endogenous and in a lesser degree bm-derived. G-CSF ameliorates CCl4-induced liver injury and accelerates the regeneration process by mobilizing HSCs that home to the liver, although pharmacological effects other than mobilization, cannot be excluded. However, regardless of the underlying process, the potential of treating acute and chronic liver diseases is important.

E. Yannaki, E. Athanasiou, A. Xagorari, V. Constantinou, I.

There is cumulative evidence that bone marrow cells can contribute to hepatocytes when transplanted into irradiated recipients with liver injury. We investigated the possibility that transplantation of hematopoietic stem cells (HSCs) in unconditioned hosts with liver injury will potentially result in better liver engraftment, as the bulk of infused cells may home to the liver instead of distributing to the ablated bm and damaged liver. Moreover, cytokine-primed blood SCs could result in higher CD34+ yield and consequently in higher liver engraftment rate. In order to explore this potential, we transplanted C57Bl6 female, CCl4-treated mice, with PBSCs from male donors. Donors received G-CSF 100µ/kg+flt-3l 50 µ/kg for 6 days and on day 7, PB was collected and infused in equal cell dose to lethally irradiated and non-irradiated recipients, treated 24h before with CCl4 4ml/kg. Mice were sacrificed and livers analyzed 1-3 months post transplantation. Sry protein detection in liver sections of female recipients using an indirect immunohistochemical method, demonstrated in both groups, clusters of regenerating donor-derived hepatocytes. Double staining for sry and CAM5.2 showed the presence of hematopoietic origin cells that can differentiate into mature hepatocytes. A higher percentage of donor-derived hepatocytes was calculated in the non irradiated compared to the irradiated group (2.19%vs0 .61%). When the experiment was repeated with BM, again, more donor origin hepatocytes were present in the non irradiated compared to the irradiated group (1.70%vs0.98%).

No contribution was seen in other tissues, except for very low frequency of donor origin cells in tissues of few irradiated animals. Sry expression was also tested by PCR in blood and liver DNA of the female recipients. As expected, PB DNA of all irradiated mice was sry+ whereas in all unconditioned recipients was sry-. However, sry positivity was clearly seen in liver DNA from both groups. These findings suggest that lethal irradiation is not necessary for hematopoietic cells to engraft to the injured liver and moreover nonablated recipients may achieve better liver engraftment. However, it seems that the percentage of donor-derived hepatocytes is still low to reverse an established liver pathology. Experiments to further enhance the recruitment of HSCs to the liver are in process.

J. Toporski, K. Reczuch, D. Turkiewicz, D. Kratochwil, M. Dzidowski, R. Ryczan, B. Rybka, W. Banasiak, A. Chybicka, P. Ponikowski, Wroclaw Medical University, Military Hospital (Wroclaw, PL) In acute myocardial infarction (MI) infusion of bone marrowderived progenitor cells may favorably affect postinfarction remodeling. We evaluated safety, feasibility and clinical outcome of intracoronary infusion of autologous, bone marrow-derived mononuclear cells (BM-MNC) in patients with acute MI. Materials and methods: Five patients (4 men, mean age: 52 yrs, range: 35-69) with first, acute ST-elevation, anterior MI, admitted 6-42 hrs (median:16 hrs) after an onset of pain were studied. In addition to standard therapy (primary percutaneous coronary intervention [PCI] with stenting) patients received BM-MNC on the 4th or 5th day after MI. Bone marrow (BM) was harvested in intravenous general anesthesia by multiple aspiration of 2-5 ml. MNC were isolated from the BM by the Ficoll gradient centrifugation. The number of nucleated cells (NC),MNC, CD34+ cells and viability before and after isolation was evaluated. MNCs were infused over 1 minute (several fractions, 3 to 5 ml per fraction) into the left anterior descending coronary artery with subsequent 3-minute artery occlusion. Results: BM harvesting was well tolerated. The median volume of collected BM was 238 ml. Median NC count after isolation of MNC was 23900/mm 3 (range: 8240-88700). Median volume of infused MNC suspension was 15ml (range:12-15). Median number of infused CD34+ cells was 2.63E+6 (range: 0.93E+6 to 8.9E+6). Infusion of BM-MNC was safely performed in all patients. After a 4-month follow-up patients treated with BM-MNC demonstrated an improvement in global systolic left ventricular function as evidenced by an increase in left ventricular ejection fraction (LVEF) from 40+/-6% to 49+/-9% (range: +2% to +12%, p=0.04) and a decrease in wall motion score index (WMSI) from 1.8+/-0.2 to 1.5+/-0.3 (range: 0 to -0.4, p=0.06). Favorable changes in LVEF and WMSI assessed during stress echocardiography were observed: mean LVEF: 42% vs 61%, mean WMSI: 1.7 vs 1.2 (baseline vs 4 months after BM-MNC infusion). Using dobutamine echocardiography we documented increased viability of myocardium: among 13 akinetic segments at baseline, 4 months after therapy 5 were normokinetic, 6 hypokinetic with only 2 remaining akinetic. Conclusion: Intracoronary infusion of autologous BM-MNC can be safely performed in the early phase after acute MI. In patients already optimally treated with primary PCI, it results in an improvement in cardiac function and increases myocardial viability within the infarcted segments.

Quest for donor type cells in non-haematopoietic tissue in patients after HSCT. Analysis of tissue with high need for repair: hair and sperm A. Rovo, C. De Geyter, S. Meyer-Monard, D. Heim, J. Passweg, W. Holzgreve, A. Gratwohl, A. Tichelli, University Hospitals Basel (Basel, CH) Introduction: Several reports suggest that hematopoietic stem cell (HSC) develop unexpected plasticity and can form non hematopoietic tissue. Prospective quantitative studies in man are lacking. To investigate this hypothesis, we assess in a prospective study prevalence and extent of chimerism in non hematopoietic tissue with need for high tissue repair, and devoid of blood contamination, hair and sperm, in long term survivors of allogeneic HSCT. Methods: This study will recruit hundred patients. All consecutive male and female patients above 18 years of age, a minimal followup of two years and full donor chimerism of hemopoiesis at last control are included. The visit includes: anamnesis, clinical examination and laboratory assessment, chimerism from peripheral blood and hair follicles. Seminal fluid is analyzed according to WHO protocol, if azoospermia is ruled out, spermatozoa chimerism is performed. Chimerism is analyzed by PCR-based amplification of 9 different short tandem repeat (STR) loci and the amelogenin locus, detecting a minor cell population of 3%. The protocol has been approved by the Ethics Committee and patients were included only after written informed consent. Results: From April to October 2003, 65 patients were requested to participate in the study, 55 patients (85%) accepted (53 hairs and 22 semen). There were 36 male, median age 41 years (range=20-66), mean time after transplantation 9.3 years. Transplants were performed in 50 patients (91%) for hematological malignancies and in 5 patients (9%) for severe aplastic anemia. Type of allograft was bone marrow in 42 patients (76%), PBSC in 13 patients (24%). All recipients underwent myeloablative conditioning. Fifty two out of fifty three hair samples were evaluable for PCR analysis (1 sample Background: Among more strategies for regeneration of necrotic myocardium, the most promising approach seems to be the bone marrow stem cell transplantation. We tried to investigate the safety and feasibility of intracoronary infusion of AC133+ cells from bone marrow in patients with acute myocardial infarction (AMI). Methods: We enrolled 4 patients with recent myocardial infarction (4 to 28 days), successful reperfusion with stent implantation in AMI related coronary artery and left ventricular ejection fraction < 40%. We harvested 600 mL bone marrow from iliac crest, we isolated the buffy coat by centrifugation, we performed AC133+ cell selection with the CliniMACS and we infused intracoronary the AC133+ cells. Baseline clinical and laboratory evaluations plus ECG Holter monitoring, transthoracic and stress echocardiography, coronary angiography and myocardial perfusion scintigraphy (SPECT) with Technetium were performed. Results: We injected a mean of 6,1x10 6 AC133+ cells (range: 4,5-8), with a purity of 93,5% (range:92-95) and a viability of 97,2 (range 97-98); the recovery was 55% (range: 41-70,3). There was no cell clumping related to the AC133+ cell selection from buffy coat. Immediate and after 1 month no major periprocedural complications occured (CK, CK-MB, Troponin T serum level were in normal range), no infection, nor malignant arrhythmias (ECG Holter monitoring) were observed. Coronary angiography and SPECT will be repeated at 3, 6 and 12 months. Conclusions: These preliminary results suggest that intracoronary infusion of autologous bone marrow AC133+ cells is safe. Results of global left ventricular function and infarct tissue perfusion assessment are awaited. Barcelona, E; Duesseldorf, D) Fanconi anemia (FA) is a rare autosomal recessive disease characterized by congenital abnormalities, progressive bone marrow failure and cancer predisposition. As a result of a DNA repair defect, FA cells are hypersensitive to DNA crosslinking agents, like mitomycin C (MMC) or diepoxybutane (DEB). Allogeneic stem cell transplantation (SCT) is nowadays the only efficient therapy to replace the defective hematopoiesis of these patients. Unfortunately, only about one third of FA patients has an HLA-compatible related donor. Therefore, a significant number of patients are transplanted with hematopoietic grafts from alternative donors, resulting in poorer outputs. In this context, gene therapy is considered a new promising approach for the treatment of FA patients lacking a suitable donor. FA is, however, a heterogeneous genetic disease characterised by the presence of at least 11 complementation groups (FA-A, B, C, D1/BRCA2, D2, E, F, G, I, J, L), with 8 genes already cloned. The Spanish Network on Fanconi Anemia is currently conducting the subtyping of FA patients by means of retroviral complementation and western blot analyses from fresh PB lymphocytes, EBV transformed lymphoblasts and skin fibroblasts. These studies allowed us to verify the diagnosis of the patients, to establish a national classification of subtyped FA patients, and to determine patients with evidences of hematopoyetic mosaicism. In addition, these analyses facilitated to conduct mutation screening studies, and allowed us to identify those patients that could be benefited from a future gene therapy. So far, samples from 97 patients and relatives have been analysed. In 59 patients, the FA diagnosis was confirmed on the basis of the hypersensitivity of PB T cells to MMC. 78% of FA patients belonged to the FA-A complementation group, 3.6% to FA-G, 5.4% to FA-D2 and 5,4% of the patients are currently identified as FA-non A,C,G. Other 7,2% of FA patients are currently ascribed to the FA-D1/BRCA2 group or to other rare (FA-I, J) or new FA groups. Further studies aiming the correlation between the subtype and the clinical status of these patients are in progress. In addition, pre-clinical gene therapy studies aiming to optimise the transfer of FANC genes into FA-HSCs are in progress to facilitate the access of FA patients to this new therapeutic option. Fanconi's Anemia (FA) patients are most susceptible to alkylating agents and radiation at dose ranges commonly used for allogeneic stem cell transplantation (SCT). Non-myeloablative stem cell transplantation (NST) is particularly attractive for reducing toxicity and improving immediate and long-term outcome following SCT. Sixteen FA patients were transplanted at our center between 1989-2002. Initially, patients were transplanted with low-dose cyclophosphamide and ionizing irradiation (total lymphoid irradiation) (CY-TLI). Eight patients (age range 3-31 [median 10] years) were treated with NST consisting of fludarabine 30mg/msq/day x 6; cyclophosphamide 5mg/kg/day x2 and rabbit anti-T lymphocyte globulin (ATG) 10mg/kgx4. Four patients were already transforming with myeloblasts ranging 15-20%. The regimen was well tolerated with minimal transplant related toxicity; S142 4 patients had acute GVHD grade II; and 3 mild chronic GVHD. Seven out of 8 patients are alive DFS range between 1.6-7 (median 4.5) years. However, 1 rejection was observed when the protocol was applied for a matched unrelated donor (MUD). A second transplant from another unrelated donor was successfully accomplished without any complications using a combination of fludarabine, busulfan 4mg/kg/day x2 and Campath-1H (humanized anti-CD52), and therefore, it was decided that this protocol will also be used for a subsequent case. Seven out of 8 patients treated with NST with stem cells obtained from a matched sibling or MUD are alive and well with 100% donor cell chimerism and 1 with mismatched donor died. The contrast, among the CY-TLI group, only 50% are currently alive and disease free post transplantation. Post-transplant complications, including fever, infections, need for total parental nutrition, veno-occlusive disease of the liver and graft-versus-host disease, were much less frequent among patients conditioned with NST. Based on our data, fludarabine based NST may represent the treatment of choice for patients with FA with a matched sibling or MUD available. graft-versus-host disease(GVHD). GF is a predictive factor for overall survival after BMT. It may be associated with the reappearance of recipient cells, giving rise to mixed chimerism(MC). We have studied chimerism in SAA long-term survivors after allogeneic BMT. Patients and Methods: Hematopoietic chimerism study was studied in 20 patients(pts) receiving BMT for SAA between 1987-2000. Molecular analysis (VNTR) was used for sex matched donor/recipient pairs and XY chromosome FISH for sex mismatched pairs. The last follow-up included separate analysis on CD3+ and CD3-populations. Results: Twenty patients (median age 20 years, range 3-38; 15 M and 5 F) received a total of 25 transplants for SAA (2 pts received 3 grafts and 1 pt received 2 grafts), with a median follow-up of 8 years (range 8-192 months). All of them received BM (18 pts) or PBPCs (2 pts) from an identical sibling donor. Conditioning regimen was cyclophosphamide ± antithymocyte globulin. GVHD prophylaxis was cyclosporine A and methotrexate in most cases. Acute GVHD>II was diagnosed in 10 transplants and evolved to chronic GVHD (cGVHD) in 3 cases. Two pts had de novo cGVHD and 13 had no evidence of GVHD. Three experienced GF and received a second allograft, 2 received a third transplant. Presently, none has evidence of rejection or cGVHD. One pt died with severe cGVHD and invasive fungal infection. The chimerism analysis showed 1 complete autologous recovery, 16 complete donor chimera status (CC) and 3 mixed chimera status. None of the MC evidenced GVHD or GF. Ten of the 16 CC had significant GVHD. The MC status was demonstrated in the unselected bone marrow as well as in the CD3+ and CD3-cell fractions. Conclusions: Our results suggest that most long-term survivors of BMT for SAA are CC. In our pts MC was not associated with an increased risk of relapse. MC cases were diagnosed 2 months after BMT and remained stable off immunosuppression at 2, 4 and 5 years post-BMT. The presence of MC in the CD3+ cell populations is intriguing since it implies a state of tolerance between the donor and recipient immune systems, both retaining their immunocompetences since our 3 cases have no increased incidence of infections. Whether the persistence of MC will increase the risk of myelodysplasia in the host cells will only be known by a longer follow-up of a larger number of pts.

Cyclosporin-A response/dependence in paediatric patients with aplastic anaemia P. Saracco, C. Dufour, D. Longoni, S. Varotto, G. DelVecchio, A. Iori, M. Zecca, M. Cellini, P. Paolucci, D. Sperli, G. Zanazzo, G. Menna, P. Quarello, R. Cerchio, U. Ramenghi, A. Locasciulli for the AIEOP Immunosuppressive treatment (IST) with anti-thymocyte globulin (ATG) and CSA is the first-line therapy in children with AA lacking HLA-identical sibling. 15-25% of responders (R) are CSA-dependent with high risk of relapse at CSA withdrawal. Clonal disease (CD) develops in about 25% of patients (pts) after 10 yrs of follow up, and a relation between clonal evolution and G-CSF cumulative dose has been suggested; the role of longterm CSA therapy is still unclear. Retrospective study on pts with AA diagnosed between 91-99 in 13 Italian Paediatric Hematology and Oncology Association Centres and treated with IST (ATG 15 mg/kg/die days 1-5, CSA 5 mg/kg/die from days 1-180 +/-G-GSF). Aims: to assess and compare dose, treatment duration and tapering schedule of CSA and G-CSF among the various Centres; to detect CSA-dependent R; to assess a correlation between treatment (dose/duration) and CD. Control group: 10 pts who underwent HLA identical sibling BMT. Eligible pts: 47; mean age: 9 years; M/F 1.1. IST group: (37 pts): 31/37 alive (84%) and 25 responsive (67%); CSA treatment: 11 pts still on CSA tapering (30%) and 6 (16%) CSA dependent (mean TD 58 months); significant variability among Centres both in therapeutic dosage (8-12 mg/kg/die versus 5-8) and in tapering schedule after remission: short term (<3 months) versus long term (over 12 months) tapering. G-CSF treatment: mean duration 10 months; significantly higher cumulative dose in unresponsive pts compared to R (over 4000 µg/kg versus 1800-2000 ); CD developed within 15-48 months from diagnosis in 11% of pts. BMT group: 9/10 alive pts, 1 pt developed AML after 2nd BMT and died of BMT related toxicity. Survival of pts treated with IST is excellent but actuarial risk of relapse at 5 yrs is 38%; only 50% of pts on long-term CSA treatment is CSA-dependent; a more uniform approach for both CSA and G-CSF dose and tapering schedule is desirable among Centres. Risk of clonal evolution in our study is not different from that reported in groups of pts treated with IST without G-CSF. Background: Usage of low intensity regimen for allogenic stem cell transplantation (SCT) has resulted in decrease of organ toxicity and GVHD leading to reduction of mortality and morbidity in Fanconi's anemia (FA) patients. Objective: Between September 1994 and May 2003, seven patients (5 females and 2 males) with a median age 10 years (range 7-13) received allogenic SCT for FA in our center. Median follow up was 34 months (range 5 -110). In all cases the source of hematopoietic stem cells was bone marrow from HLA-identical siblings. All patients were separated on two groups according to the time of transplantation. 3 children were included in I group (between 1994 and 2000) , BU (6 mg/kg) and CY (40 mg/kg) was used as the preparative conditioning regimen; GVHD prophylaxis consisted of CsA 3 mg/kg. The II group (2001 included 4 patients, they received BU (4 mg/kg), FLU (150 mg/m 2 ) and ATGAM (90 mg/kg) as the preparative regimen; CsA 3 mg/kg, short course of MTX 5 mg/kg (+1, +4, +7) and daclizumab (anti CD25) 1 mg/kg (+4, +8, +15, +28) were used as the prophylaxis against GVHD.

Group I: Patients are showed engraftment on +9, +14 and +17 days. Regimen related toxicity included with mucositis II-III gr and alopecia. One patient developed acute GVHD grade IV, affecting skin, liver, gut. He had no response to the treatment with CsA, steroids and ATGAM, and died on day +134. Two children developed acute GVHD, grade III. They were successfully treated with steroids. But subsequently, chronic GVHD developed in both cases (limited with skin lesions, n=1; extensive, with skin, muscles, lung and joints lesions, n=1). In II group engraftment was showed on +11, +12 and +14 (in two cases) days. Signs of regimen related toxicity was absent in all cases. Two children had indications of acute GVHD grade I, its disappeared without specific treatment. Two patients developed acute GVHD grade II (with skin lesions n=1, and with skin and liver damage n=1), they successful treated with steroids 2 mg/kg. Severe infection complications and chronic GVHD were absent in this group. Conclusion: New approach to treatment of patients with FA showed the low incidents of chemotherapy related organ toxicity. Usage of CsA, MTX and daclizumab (in combine) reduced the risk of acute and chronic GVHD development. This type of preparative regimen and GVHD prophylaxis may therefore be more appropriate for the treatment of FA patients. Objective: Secondary malignancies represent a significant problem after allogenic bone marrow transplantation (BMT). We report a rare and atypical case. Case history: In 1987 severe aplastic anaemia was diagnosed in a 2.5 years old girl. After 2 months of steroid therapy a successful male sibling-donor BMT was undertaken. During follow up a slight thrombocytopenia was detected but the patient was in complete clinical and haematological remission until 1999. Then anaemia, progredient thrombopenia and granulocytopenia developed. Bone marrow showed dysplasia and mixed chimerism. Donor lymphocyte infusion was performed. Later on chronic graft-versus-host disease developed (with skin and gastrointestinal involvement). She received combined immunosuppressive medication (mycophenolat mofetil, steroid, cyclosporin-A). Her condition did not improve and she needed frequent hospitalisation (dystrophia, sepsis, cytopenia). In June 2000 bone marrow biopsy showed elevated number of monocytes (20 %). The biopsy was repeated in August 2000. The histology proved JMMoL. Cytogenetic and FISH examinations were performed with the following results: haematopoesis of donor origin, t(3;21), trisomy of chromosome 12. Genetical examinations were repeated in the healthy donor and no alteration could be detected. The patient received cytostatic treatment according to the EWOG-MDS-98 protocol without any success and the patient died in septicaemia in October 2000. Conclusion: We report a girl, who underwent a successful sibling donor BMT and 12 years thereafter, JMMoL developed. Genetically the tumour cells were of donor origin, however, in the healthy donor no chromosome aberration could be detected.

Persisting mesenchymal stem cells is the basis of successful bone marrow transplantation V. Rugal, K. Abdulkadyrov, M. Bercos, Institute Hematology (St. Petersburg, RUS) Survive transplanted stem cells in recipient organism is depend from many different factors and usually linger from days to mothers. When it is possible to detect some donor markers in recipient per many years, it is really to propose that transplantate contained cells with self-renewal potential and hystogenic possibilities equal with the same in embryo tissues cells. In 09.1959 one of the authors (then 6 years old boy) was treated in hypoplastic anemia with hemolytic component and nearly total lipoid substitution in bone marrow. Treatment included hemotransfusions, hormones; extract of bovine lever (compolon) and vitamins was unsuccessful. From 11,1959 to 01,1960 it was made 9 intraosseal allogenic BMT (3 from mother, 6 from strange donor-men). Every operation it was transplanted donor's bone marrow aspirates, getting ''ex tempore'' from os sternalis to recipient os calcaneum. Full remission was obtained 1,5 months after treatment. In 1971 at first, accidentally, it was founded Xchimerism at 75% segment nuclear cells (SNC) of peripheral blood. In 1979 level of those cells decreased to 45%. In 1982 immunological typing mothers and recipients lymphocytes showed full identity by HLA-A, B, C DR -antigens. To day there are near 30% SNC in recipient's peripheral blood has drumsticks. In 12.2001 molecular-biological typing recipient's leukocyte DNA confirmed the absence of polymorphism of genes, which code DR-antigens. We believe, this practical example is confirm the assumption about possibility of many years persistence and adequate function clonogenic mother's cells in adult, and their ability to provide recipient organism with stem cells after allogenic transplantation. Possible it may be whole life persistent mesenchymal cells. Then it is very likely to found some chromosomes or gene-markers of chimerism not only in blood but also at another tissues, which has mesenchymal genesis. May be permanent decrease of the level SNC caused by the gradual activation in recipient own stem cells.

Long-term immune reconstitution in patients treated with autologous stem cell transplantation for refractory autoimmune diseases R. Arnold, T. Alexander, G. Massenkeil, R. Burmester, A. Radbruch, F. Hiepe, A. Thiel, University Hospital Charite, German Rheumatism Research Center (Berlin, D) Aims: We performed here a detailed analysis of the newly developing immune systems in patients treated with immune ablation and subsequent autologous stem cell transplantation (ASCT) for severe autoimmune diseases. Methods: Peripheral blood lymphocytes were analyzed by multiparameter flow cytometry, including monitoring of the TCR-Vbeta repertoire on CD4+ lymphocytes. Thymic activity was determined by measuring T-cell receptor excision circles (TRECs) in distinct peripheral blood Th-cell subsets after ASCT by quantitative real-time PCR analysis. Patients and results: 12 patients with a median follow-up of 40 months have been included in the trial so far: polychonditis (n=1), SLE (n=6), systemic sclerosis (SSc, n=3), panniculitis (n=1) and MS (n=1). Clinical remission has been achieved in seven patients: polychondritis (n=1), SLE (n=5) and MS (n=1). In case of two patients suffering from SSc and in the patient with panniculits no changes in disease activity were observed. One patient reactivated SLE after being free of any clinical and serological symptoms for 17 months. One patient with SLE died due to transplantation related infection (aspergillosis), a SSc patient died of an acute deterioration of a chronic cor pulmonale. In all responding patients no autoreactive Th cells could be detected accompanied by complete serological remission. Lymphocyte compartments reconstituted functionally in all patients as shown by the reappearance and persistence of naive T-cells with high levels of TRECs and restored diversity of the Tcell-receptor repertoire. Reconstituted B cells were found to be of naïve phenotype (IgD+ CD27-). In case of the relapsing SLE patient recurring autoantibodies interestingly showed new specificities. Although we observed similar kinetics of lymphocyte repopulation in the non-responding SSc patients, autoantibody titers (ANA and Scl70) were not affected from the conditioning. Conclusion: The newly generated immune system in responding patients is tolerant to rheumatic autoantigens and is able to react to pathogens, suggesting regeneration of self-tolerance or ignorance. Our data demonstrate that ASCT can induce stable long-term clinical and serological remission in patients with severe standard-therapy refractory autoimmune diseases. A major future goal will be to reduce therapy-related risks of this curative cell therapy to include patients with severe autoimmune diseases, yet known to be responsive to standard treatment. Thirty patients with poor risk multiple sclerosis (MS) were included to the phase I/II clinical trial involving the high dose chemotherapy with autologous peripheral blood progenitor cell (PBPC) rescue. Twenty five patients underwent high dose conditioning BEAM with at least 2 months of follow up. T cell depletion in vitro was performed in 16 grafts. In 9 patients were grafts not purged as stem cell number did not reach the limit for in vitro manipulation or there was lack of resources.These patients received in vivo ATG 4mg/kg i.v. D+1, D+2 after transplantation. Median follow-up is 30 months (2 -64) . Median EDSS (Expanded Disability Status Scale) of grafted patients at inclusion were 6.5 (5.0-8.5), median EDSS of grafted patients at the last follow up was also 6.5 (5.0-10.0). Two patients out of 25 (8 %) improved significantly (by 1.0 point on EDSS), 7 patients (28 %) improved not significantly (by 0.5 point). Ten patients (40 %) did not change their EDSS. Four patients (16 %) gained their disability significantly (by 1.0 point on EDSS) despite the treatment, one of them died 31 months after the transplantation from disease progression (EDSS 10.0). Two other patients (8 %) worsened not significantly (by 0.5 point) on their EDSS. Patients who stabilized their disability or improved represent 76 %. The difference between the EDSS before and after the transplantation in the group was not significant (Wicoxon´s, repeated measure ANOVA, t-test). The development of disability between the group that was grafted with in vitro purged graft and the group with ATG i.v. was also not significant (Wilcoxon´s, Mann-Whitney). Twenty patients stabilized their MRI finding, in 2 patients decreased number and size of lesions were detected, 3 patients worsened their MRI. However, not all MRI were performed with gadollinium. Toxicity of the procedure differed in each individual. Two serious early events with respiratory failure have been observed. Both patients recovered. Another patient developed an inhibitor to FVIII. Twelve months after the transplantation percentage of CD4+ cells and CD4+CD45+ as well as IRI are still significantly decreased. As the significant majority of patients at least stabilized in their disability, we consider the results to be promising. Next patients will be included in the new international randomized clinical trial (ASTIMS). Supported by the grant IGA No. NF/6560-3.

High-dose cyclophosphamide and autologous peripheral blood stem cell transplantation for refractory chronic inflammatory bowel diseases -a two step approach K. Potthoff, W. Kreisel, H. Bertz, A. Schmitt-Gräff, J. Finke, University of Freiburg (Freiburg, D) Introduction: In about 5% chronic inflammatory bowel diseases (IBD) are refractory to current medical or surgical treatment. IBD are characterized by an uncontrolled immune response to unspecific antigens based on a genetic background. Therefore, aPBSCT might be an option for refractory IBD. Methods: We established a two step protocol for aPBSCT in IBD. A patient is regarded as refractory to medical therapy if corticosteroids, 5-ASA, immunosuppression (Aza, MTX, CyA), antibiotics and infliximab failed to induce a stable remission.

Step 1: Stem cell mobilization with cyclophosphamide (2.0 g/m 2 /d on two successive days). Stem cells are T-cell depleted and enriched by CD34-selection. Immunosuppressive therapy will be continued. In case of relapse step 2: High dose immunoablative chemotherapy with cyclophosphamide (50 mg/kg bw/d on four successive days) followed by aPBSCT. IBD specific therapy will be stopped. Immunosuppressive chemotherapy with cyclophosphamide (step 1) followed by immunoablative therapy with high dose cyclophosphamide and autologous PBSCT (step 2) may be an effective therapy for refractory IBD. Since 1997, autologous SCT is performed for children with treatment resistant Juvenile Idiopathic Arthritis (JIA). As the result of a multicenter survey it was recently decided to change to conditioning regimen into a Fludarabin Cyclophosphamide based regimen. To date 4 patients have been transplanted in 2003 in 3 different centers with this new protocol. In 3 of these there were symptoms of fever, rash, high ferritin and triglycerids and hemophagocytosis in the marrow during or shortly after the conditioning regimen. The regimen consisted of Fludarabin (30mg/m 2 for 5 days, Antithymocyte globulin (rabbit, Sangstat) 5mg/kg for 4 days and Cyclophosphamide 60mg/kg for 2 days. All 3 patients had the systemic onset form of JIA. They did not have the signs of systemic disease shortly before transplant. In the past they were resistant to antirheumatic drugs including anti TNF treatment. Stem cells were obtained from marrow (n=2) or after mobilization with Cyclophosphamide and G-CSF, yielding a final product of CD34 between 1.7 -19x 10 6 /Kg and of CD3: 1 x 10 5 /Kg. Four to 12 days after start of the fludarabin symptoms occurred of spiking fever, erythematous rash (not typical for systemic JIA). There was no epatomegaly. The lab showed aplasia in 1; in the second case leucocytes were just increasing again to 0.2 x 10 9 with 35000 platelets. In the third case there was a leukocytosis (18.500) with extreme lymphocytopenia (<100) and platelets of 38000. In all there was a coagulopathy with increased fibrinogen D dimers but with normal fibrinogen. Triglycerids and ferritin were greatly elevated. In 1 case there was a CMV cultured from the lungs, in the others there was no evidence of an infection. The CMV reactivation was treated with gancyclovir, the 2 other children were on prophylactic aciclovir. In 1 case a bone marrow aspirate was performed that showed abundant activated macrophages with some hemophagocytosis. On these grounds a diagnosis was made of hemophagocytosis (or Macrophage Activation Syndrome), a well known complication of systemic onset JIA. ATG related srum sickness seemed less likely. Treatment was started with high dose methylprednisolon (15mg/kg/day) and ciclosporin. All patients responded well to this treatment. Based on these experiences we conclude that fludarabin is contra-indicated as part of the conditioning regimen for systemic JIA. For future cases we advocate a cyclophosphamide ATG based conditioning protocol. Mobilization was performed with Cyclophosphamide 2 g/m2 plus G-CSF, conditioning was BEAM + equine ATG for multiple sclerosis (MS) and CY + ATG for other diseases and the graft was not manipulated. There were 3 deaths among 12 MS patients (1 aspiration pneumonia in a patient with EDSS 8.5, 1 esophageal bleeding in patient selfmedicated with non-steroidal anti-inflammatory drugs and 1 acute toxicity to BEAM), 1 death from acute kidney failure among 4 patients with lupus nephritis, 1 death from reactivated vasculitis after mobilization in a patient with overlapping SLE/SSc and 1 pulmonary death in a patient with pemphigus vulgaris. Five out 6 patients who died did not fulfill strict protocol criteria. Two lupus nephritis patients had acute renal failure requiring hemodialysis and intensive care but survived. Two patients developed anaphylaxis, one to etoposide which was omited from conditioning and other to horse ATG which was replaced by rabbit ATG. The 16 surviving patients (9 MS, 3 SLE, 3 SSc, 1 Takayasu's arteritis) are alive and well with no or minimal immunosuppression. In the future, morbidity and mortality of autologous HSCT for AID in Brazil should be reduced by better patient selection and management of transplant complications.

T. Alexander, G. Massenkeil, E. Gromnica-Ihle, R. Burmester, A. Radbruch, A. Thiel, F. Hiepe, R. Arnold, University Hospital Charite, German Rheumatism Research Center (Berlin, D) Aims: High dose ablative chemotherapy followed by autologous transplantation of highly purified hematopoietic CD34+ stem cells (ASCT) is currently being evaluated as a new strategy for the treatment of severe autoimmune diseases. We investigated the efficacy of this therapy for patients with SLE with respect to the reconstitution of the newly developing immune system. Methods: Mobilization was achieved with cyclophosphamide (2g/m2) and G-CSF. Conditioning regimen included cyclophosphamide (200mg/kg) and ATG. Clinical outcome was assessed using the SLE activity index (SLEDAI). Peripheral blood lymphocytes were analyzed by multiparameter flow cytometry, including monitoring of the TCR-Vbeta repertoire on CD4+ lymphocytes. Thymic activity was determined by measuring T-cell receptor excision circles (TRECs) in distinct peripheral blood Th-cell subsets by quantitative real-time PCR analysis. Results: From 1998, we have treated six patients with severe SLE, refractory to standard immunosuppression, including i.v. cyclophosphamide. At a median follow-up of 40 months four patients are in complete clinical remission. One patient died due to transplantation related infection (aspergillosis) and one patient exacerbated after being free of clinical symptoms for 17 months before he died of a pulmonary embolism 38 months after transplantation. Remission was accompanied by the complete disappearance of autoantibodies and a normalisation of complement factors (C3, C4). In case of the relapsing SLE we detected a changed anti-ENA autoantibody pattern after ASCT (anti Ro/SSA, anti La/SSB before Anti-Sm and anti-U1RNP after transplantation). Of note, one patient developed hashimoto thyreoditis 36 months after ASCT, in another patient MGUS was diagnosed 24 months after ASCT. Lymphocyte compartments reconstituted functionally in all patients as shown by the reappearance and persistence of naive T-cells with high levels of TRECs and restored diversity of the Tcell-receptor repertoire. Reconstituted B cells were found to be of naïve phenotype (IgD+ CD27-). Conclusion: Our data demonstrate that ASCT can induce longterm clinical and complete serological remission in patients with SLE, refractory to conventional immunosuppression. The durability of clinical remission has to be proven in longer followup periods. A major future goal will be to reduce therapy-related risks of this curative cell therapy to include patients with less severe SLE.

A.M. Marmont, F. Gualandi, A.M. Raiola, A. Dominietto, S. Bregante, M.T. van Lint, A. Bacigalupo, Stem Cell Transplantation Center, S. Martino´s Hospital (Genoa, I) Pure white cell aplasia is a rare autoimmune myelopathy (1) in which autoimmune mechanisms, whether antibody or cellmediated, suppress not only circulating neutrophils but also their bone marrow precursors. The bone marrow shows the exclusive absence of neutrophils precursors. Inhibition of granulocytopoiesis by autologous serum and/or T lymphocytes can generally be demonstrated. Immunosuppressive/ immunomodulating procedures are generally successful, but severe cases tend to relapse constantly. UPN 1436 is a 53 year old male (at time of transplant) who first presented with severe neutropenia in 1996. In September, 1998 there were 200 neutrophil and no granulocytic precursors in the otherwise normal myeloaspirate. He received the standard combination for SAA of HALG, cyclosporine and G-CSF. WBC peaked to 55x10 9 /L, with 85% neutrophils. Following severe relapse he received RALG in August, 2001, had a response, and relapsed. In January, 2002 he received HALG, remitted, and relapsed again. He had a series of neutropenic infections. He was conditioned with a RIC regimen (TT 10mg/Kg, CY 100 mg/Kg) and received BMT from his HLA-identical sister on September 17, 2002 (ANC 6.9x10 8 /Kg, CD34+ 7.86x10 6 /Kg). Hematologic reconstitution and clinical course were uneventful and full chimerism was achieved with normal granulocytopoiesis, but after day +140 neutrophils gradually disappeared and cytogenetics were 100% recipient. The patient received three consecutive DLI (1x10 6 , 1x10 7 , 1x10 7 ) and on day +280 was totally chimeric (marrow cytogenetics 100%, marrow STR 93%, CD3 STR 100%) with a normal neutrophil count (4900x10 9 /L). This is a further demonstration that DLI for relapse following allogeneic BMT for an autoimmune disease of marrow and blood may be curative. This effect may be defined as Graft-vs-Autoimmunity, and differs from the recognized GVL/GVT effects inasmuch as it also consists in the substitution of autoimmune lymphoid clones by normal lymphopoiesis (2) . In hematologic autoimmune disease the elimination of culprit antigens driving the autoimmune response may also be contributive. 

Objective: To study the mechanism of remission in multiple sclerosis(MS) induced by autologous hematopoietic stem cell transplantation(AHSCT)by an analysis of the alternation in Th l and Th2 subsets in peripheral blood cells of patients with MS before and after AHSCT. Method: Thl/Th2 subsets in peripheral blood cells of 7 patients with MS and 10 healthy persons were examined by a newly developed technique: detection of intracellular cytokine by flow cytometry. Peripheral blood samples were obtained from patients with MS before AHSCT and one month after AHSCT. It is considered that CD3+CD8-cells with IFN-g secreting are Thl cells; CD3+CD8-cells with IL-4 secreting are Th2 cells . The percentage of each group of cells was examined. Results: The data indicate that MS patients have increased percentages of Thl cells compared with health controls (P<0.01). The percentages of Thl cells decreased significantly in patients with MS after AHSCT (P<0.01). There is no significant difference before AHSCT and after AHSCT in Th2 cells. A serial study of one patient before AHSCT and one month and six months after AHSCT indicated that the patient is still at a lower status of Thl cells six months after AHSCT than it before AHSCT. Conclusion: There is a high level of Thl subsets in MS patients relating to pathogenesis of MS. AHSCT can suppress this high level of Thl cells in patients with MS. This suppression is helpful to remission of MS and it can be theoretically inferred to be one of the mechanisms of remission induced by AHSCT.

Allogenic HLA identical bone marrow transplantation in patient with juvenile idiopathic /chronic/arthritis /JIA

Autologous stem cell transplantation has been proposed as a therapeutic option for patients with severe JIA. Two years ago we made the first autologous CD 34 selected stem cell transplantation in patients with JIA. Six month after the stem cell transplantation the patient had a new flare. Here we report a child with a severe form of systemic JIA treated with allogenic HLA identical bone marow transplantation. The patient is a 9 years old girl who developed a severe systemic JIA at the age 3 years. The patient was refractory to coventional antirheumatic treatment with NSAIDs, methotrexate, pulse and oral cyclophosphamid and etanercept. She received more than 8 mg methylprednisolon/day during the last four years. The donor was the HLA identical brother of the patient. The allogenic bone marrow transplantation was approved by the parents, the National Children Hemopoetic Stem Cell Transplantation Committee and Ethical Committee of University Hospital of Miskolc.

Conditioning regimen: Busulfan 16 mg/kg, Cyclophosphamid 120 mg/kg, ATG 40 mg/kg. The patient received bone marrow mononuclear cells 2,4 x10 -8 /kg bw recipient. She engrafted at the postransplant day +13. She received GVHD prophylaxis cyclosporin and MTX and she had mild skin GVHD /II. grade/, treated by methylprednisolon and mycophenolat mofetil. After +70 day she had no fever, arthritis and morning stifness. At +51 posttransplant day VNTR examination demonstrated only donor origin cell in peripheral blood. Before the transplantation serum TNF alfa level 146 pg/ml /normal: 0-42 pg/ml/, and IL6 level 47,79 /normal: 0-26 pg/ml/, ESR: 55 mm/h, after the transplantation + 45 day TNF alfa level 0 pg/ml, IL6 level lower than 10 pg/ml, ESR: 13 mm/h. In the last ten years in pediatric rheumatology departments the incidence of death of severe JIA was very low around 1 %. But the chance for a normal life was very few/dwarfing, osteoporosis, nephrocalcinosis, serious limitation in motion and early death/. We cannot conclude that the allogenic BMT is indicated or not in severe JIA. But with our opinion the autologous stem cell transplantation is not other than an immunosuppresive treatment and it doesn't give chance for total recovery from severe juvenile idiopathic /chronic/ arthritis. Natural killer (NK) cell mediated cytolysis is stimulated and downregulated through the interaction of distinct human leukocyte antigen (HLA) class I molecules on target cell with specific killer cell immunoglobulin-like receptors (KIRs). It is currently discussed, that NK cells may contribute to autoimmunity. We have therefore studied the genetic distribution of inhibitory (DL) and stimulatory (DS) KIRs among 30 patients with psoriatic arthritis. As controls 99 healthy volunteers were included in this study. Analysis of KIRs was performed as described previously (S. Becker et. al, Human Immunol 64, 2003) using PCR amplification with sequence-specific primers for KIR gene segments. In additon the cell surface expression of activating repector NKp30, NKp44 and NKp46 war analysed by flow-cytometry. Our results indicate that psoriatic arthritis patients are characterized by an altered KIR gene repertoire. The inhibitory receptor KIR2DL1 and KIR3DL2 are decreased among psoriatic arthritis patients in comparison to controls (p<0.01) and a shift in KIR haplotype distribution. Interestingly, individual psoriatic arthritis patients showed the presence of activated NK cells as indicated by the expression of NKp44. Variation in KIRs could considerable alter the NK and/or T cell mediated immune response and may contribute to the disease pathogenesis.

Introduction: Apoptosis (programmed cell death) involved in the development and homeostasis of the Immune system. It is probable that dysregulations of apoptotic pathways are associated with pathogenesis of autoimmune diseases. Multiple sclerosis (MS) is an autoimmune disease of central nervous system that is created by inflammatory cells and autoreactive lymphocytes. In this study, apoptosis and Fas ligand (FasL), which is involved in apoptosis stimulation, were evaluated in peripheral blood mononuclear cells (PBMCs). Methods: in this research, 36 patients of MS, 30 patients with GBS and 25 healthy control subjects were studied. Apoptosis with TUNEL assay and FasL with indirect methods have been measured by flow cytometry. For detection of FasL it was used to mouse IgG1 anti-human FasL monoclonal antibody (mAb) and FITC-Rat anti-mouse IgG1 mAb. Results: apoptosis in PBMCs of MS patients did not differ significantly proportion of healthy controls and patients of GBS (p>0.05). FasL expression in lymphocytes and monocytes in peripheral blood of MS patients especially in end of relapsing Ms or remitting MS stage, a like with GBS patients were increased comparison of healthy control subjects (p<0.001). Conclusion: firstly, it seems likely that before induction of apoptosis in autoreactive or effector cells, these cells participated in autoimmune reactions. Secondly, with due attention to increasing of FasL in PBMCs of MS patients, it is probable other factors has been involved in inhibition of apoptosis that they may cause the cell death resistance in them and apoptosis have been dysregulated or delayed. Certainly, in order to prove this hypothesizes are needed to more researches. Objective: To determine the safety and efficacy of immune ablation and autologous hematopoietic stem cell transplantation (HSCT) in severe systemic lupus erythematosus (SLE) and refractory juvenile idiopathic arthritis (JIA). Methods: Two patients with persistently active SLE after oral cyclophosphamide therapy and two patients with refractory juvenile idiopathic arthritis (JIA) were recruited for HSCT. T-cell depletion was performed by CD-34 positive selection (Isolex) on the patients' bone marrow. The conditioning regimen was cyclophosphamide (160-200mg/kg), antithymocyte globulin (90mg/kg), and fractionated total body irradiation (4.42Gy). Outcome was evaluated by serum complement levels, serologic markers, function of diseased organs, and requirement of immunosuppressive medication. Results: The two patients with SLE were 20 and 21 years old respectively. The patients with JIA were 11 and 19 years old respectively. The mean follow-up duration after HSCT was 15 months (range 3-30 months). There was no major transplant related complication. All patients are alive with gradual and significant improvement. Complement and anti-double stranded DNA levels normalized with marked improvements in end organ functions for the two SLE patients. Joint pain and morning stiffness resolved totally for the first JIA patient (except bilateral hip osteoarthritic pain due to avascular necrosis before HSCT). Immunosuppresive medications were stopped in 3 patients. They all have good performance status. Conclusion: In patients who experienced persistence of organthreatening lupus or refractory JIA following standard aggressive therapy, HSCT may be performed safely, with marked clinical improvement and sustained withdrawal of immunosuppressive drugs. To assess the benefit of intensification with ASCT in CLL, we planned a prospective controlled trial. Previously untreated stage B or C CLL receive 3 monthly courses of ChOP (d1, I.V: hydroxorubicine 25 mg/m 2 , vincristine 1 mg/m 2 ; d1-5: Cytoxan 300 mg/m², prednisone 40 mg/m², both orally), and 3 courses of fludarabine (d1-5: 25 mg/m² IV). Patients who then fulfill the NCI complete response (CR) criteria are randomly allocated to ASCT intensification or no further treatment. Patients not completing the CR response criteria, irrespective of the response level, receive a salvage treatment with cisplatin (d1: 100 mg/m² IV), aracytine (d2: 4 g/m² IV) and dexamethasone (d1-4, 40 mg/d), and are randomly allocated to ASCT intensification or 3 monthly courses of fludarabine (25 mg/m² IV), and Cytoxan (300 mg/m² IV), both d1-3. For CR patients allocated to ASCT, unpurged peripheral blood stem cell (PBSC) are mobilised and collected in steady state with lenograstim (10 µg/kg/d) two months after completion of the last course of fludarabine; an additional mobilisation is done when necessary. For those patients (non CR) receiving the salvage treatment, mobilisation and collection are performed with lenograstim (5 µg/kg/d) starting d5 after the first and if necessary the second course of salvage treatment. From june 2001 to october 2003, hundred patients have been enrolled in this trial, and 66 have completed the monthly courses of ChOP and fludarabine: the CR rate is 55%. Among CR and non CR patients, 33 have been allocated to ASCT. The aim of this abstract is to focus on the results of the PBSC collection: in the CR group, 71% of patients yielded > 2.10 6 CD 34/kg, but 29% couldn't provide this minimal amount. In the non CR group, completion and failure of collection are observed in 74% and 26% respectively. Interestingly, > 90% of the patients have obtained a blood normalization (NCI criteria) after the 3 courses of ChOP. Accordingly, and considering the hasards of PBSC collection after the 3 additional courses of fludarabine, we decided to perform the mobilisation before the three courses of fludarabine for the next patients enrolled in this trial, which completion is expected for the end of 2005. Objective: A high rate of complete cytogenetic remission (CCR) after therapy with the tyrosine kinase inhibitor imatinib mesylate has been observed in pts with Ph+ CML. However, some concern exists about the long-term management of these pts, because relapse may occur due to residual resistant clones or to clonal evolution. We investigated the feasibility of Ph-CD34+ cell collection following G-CSF mobilization in pts who achieved CCR. Patients and methods: 18 CML pts (12 M, 6 F), with a median age of 52 yrs (28-66) were stimulated with rHu-G-CSF (filgrastim) at 10 microg/Kg/d x 4 days (1 pt underwent a 2nd mobilization after failing the first attempt). All but three previously were given IFN-alpha and cytosine arabinoside or HU. PBSC collection was performed when CD34+ > 15/microL in the PB. Imatinib was suspended in the week preceeding mobilization in 4 pts. Cytogenetic analysis was performed on at least 20 metaphases.The presence of bcr-abl+ cells was investigated in the yield by FISH or nested-PCR. Results: PBSC collection was performed on d +5 in 11 pts (62%) with a median of 51 CD34+ cells /microL and a median yield of 3 x 10 6 /Kg CD34+ cells through a single collection procedure, while the mobilization failed in 8 pts, these latter showed a median of 7 CD34+ cell/microL. Good mobilizers showed a higher WBC count on d+5 compared to those who didn¡¦t mobilize: 38.5 "b 6.7 vs 23.7 "b 5.2 x 109/L ( P< 0.05); duration of imatinib therapy was lower in mobilizers: 18 (12-36) months vs 33 (16-45) months (P= 0.034, Mann-Whitney). No differences were observed as to the lenght of IFN therapy and PBSC mobilization between the two groups. Noticeably, a successful CD34+ collection was achieved in all the pts who suspended imatinib. Bcr-abl+ cells were detected in 9 out of ten yields, only one pt resulted negative even at nested-PCR. Conclusions: CD34+ cell collection is feasible in CML pts who achieved CCR following imatinib mesylate therapy. These cells could be utilized for future autologous PBSC transplantation. Moreover, short-term interruption of imatinib could enhance CD34+ cell collection.

Conventional HLA-identical sibling bone marrow transplantation is able to cure chronic lymphocytic leukaemia. A study from the EBMT and IBMT Registries M. Michallet, A.S. Michallet, Q. Le, G. Bandini, P.A. Rowlings, H.J. Deeg, G. Gahrton, E. Montserrat, F. Nicolini, C. Rozman, A. Gratwohl, D. Niederweiser, C. Bredeson, M. Horowitz, Hopital E. Herriot, Institute of Hematology, IBMTR, Fred Hutchinson Research Cancer Center, Karolinska Institutet, Hospital Clínic, Kantonsspital Basel, University of Leipzig, General Hospital (Lyon, F; Bologna, I; Milwaukee, Seattle, USA; Huddinge, S; Barcelona, E; Basel, CH; Leipzig, D; Ottawa, CAN) In a previous study (M.Michallet et al Ann.Intern.Med.,1996; 124 : 311-15), we analysed 54 CLL patients (39 males and 15 females with a median age of 41) who underwent allogeneic bone marrow transplantation from HLA identical sibling donors between 1984 and 1992 and were reported to the EBMT and/or IBMT Registries. At transplantation, after a median interval of 37 months (m) from diagnosis, 3 patients were in Rai stage 0, 10 were in stage 1, 10 in stage 2, 7 in stage 3 and 22 in stage 4. Only 15% were chemosensitive pretransplant. After transplantation, 38 (70%) achieved a complete haematological remission, 17 developed acute GVHD grade > II and 17 (11 limited and 6 extensive) cGVHD. The 3-year probability of survival was 46% (95%CI [32-60]) with a median follow-up of 27 m and a maximal follow-up of 80 m : 100% for patients transplanted in Rai stage 0, 68% in stage 1, 30% in stage 2, 57% in stage 3 and 34% in stage 4. At the time of publication (1996), 31 patients died after transplant: 5 from relapse and 26 from TRM and 23 patients were alive in remission of whom 8 developed chronic GVHD. We recently updated the follow-up for the 23 surviving patients (14 females/9males;median age 38 years ) to investigate the durability of the observed remissions and to ascertain the possibility of CLL cure by conventional allogeneic transplantation. Currently, the median follow up is 10 years (range, 5 to 16 years). Nineteen of the 23 (83%) patients are still alive, 17 (74%) in complete haematological and immunophenotypical remission. Four patients died, at 7, 10, 11 and 16 years after transplantation: 3 died from progressive CLL (2 had received a T-cell depleted transplant) and one from bacterial sepsis. For the entire group of 54 patients transplanted, the 10 year probabilities of survival and leukemia-free survival are 41.2% (95% CI 27.9-54.6) and 36.6% (95% CI 23.3-49.9) respectively. Among 24 patients alive and in remission at five years posttransplant, the subsequently probability of being alive and leukemia-free at 10 years posttransplant is 80.9% (95% CI 65.9-97.8). This long-term study clearly confirms and underlines that CLL can be cured by conventional allogeneic HLA identical sibling donor bone marrow transplantation, with long-term continuous complete remission observed after a maximal follow-up of 16 years after transplantation. The older age associated with high transplant-related mortality (TRM) poses the major obstacle of conventional (myeloablative) allogeneic stem cell transplantation (SCT) in CLL pts. Reducedintensity conditioning (RIC) SCT could improve survival by reducing TRM while preserving GVL effect of alloSCT. The outcome of older CLL pts after RIC-SCT should thus be comparable to younger ones transplanted conventionally (i.e. myeloablative). We thus retrospectively analysed and compared the early outcome of CLL pts (n=22, median age 54, range 36-65) allotransplanted either conventionally (MYELO group, BU-CY conditioning, n=9 pts), or with RIC (RIC group, FLU-CY conditioning, n=13 pts). All pts had advanced disease stage, heavily pretreatment including previous autoSCT (4 pts, 18%) and poor risk features. Higher age at SCT for RIC group was the only difference among the pretransplant characteristics: 60 years (range 51-65) for RIC vs. 47 (36-59) for MYELO (p=0,0001) whereas disease status, proportion of previously autotransplanted, follow-up and the proportion of unrelated donor (23% vs 33%, p=0,66) were similar between groups. For the whole CLL group (median follow-up of 10 mts, 3-65), the 1-year Kaplan-Meier OS and PFS probabilities were 44% and 18%, whereas 100 days and 1 year cumulative TRM were 23% and 50%. After splitting to RIC vs. MYELO the number of pts alive at 100 days and 1 year after SCT was similar: RIC vs MYELO: 10/13 (77%) vs 7/9 (78%) and 8/13 (62%) vs. 3/9 (34%), reflecting the same 100-day and 1year cumulative TRM (23% vs. 22% p=1,0 and 38% vs. 66%, p=0,39). The incidence of gr. II-IV aGVHD was also similar (38% vs 44%, p>0,1). Graft failures occured on the whole in 4 pts (18%), there was a statistically insignificant trend for higher rate of graft rejection for RIC: 4 pts (31%) vs. 0 pt for MYELO (p=0,11). Altogether 4 (18%) pts relapsed or progressed, all in RIC group (p=0,46). Due to the short follow-up no mortality owing to CLL progression was observed. The estimated probabilities of 1 year OS for RIC and MYELO were 58% and 30 % (p=0,4) whereas the corresponding figures for PFS were 21% and 30% (p=0,3). These preliminary results seem to indicate 1. comparable early outcome for allotransplanted CLL pts regardless of age when RIC SCT is applied for elderly 2. presumably higher toxicity but better disease control for conventional SCT. Conclusion: Obstacle of high TRM after alloSCT in elder CLL pts could be overcome by the use of RIC. Patients and method: We analysed consecutive USCT performed between November 1994 and January 2003 in ten centres participating in the study. In order to analyse the impact of HLA typing on survival, only donor-recipient pairs analyzed, as a minimum, at serologic level for A and B loci and at allelic level for DRB1 locus were included (n= 92). The median (range) age of the series was 31 years (15-51). Forty nine SCT (53%) were performed within the first two years after diagnosis. In 82 cases stem cells were obtained from bone marrow and in 10 from peripheral blood. Conditioning regime was Cy + TBI in 86% of cases and CyA + short MTX were used for GvHD prophylaxis in 89%. In addition to DRB1, 73 patients had DQB1 locus and 25 class I loci typed at allelic level. Eighteen pairs had one HLA mismatch and 4 had two. Results: Median (range) time to neutrophil and platelet recovery was 20 (10-100) and 24 (10-360) days, respectively. Ten patients presented a primary graft failure. Incidence of grade II-IV, III-IV acute GvHD, and extensive chronic GVHD was 69%, 37%, and 39%, respectively. Main complications were VOD (11 cases), haemorrhagic cystitis (7), thrombotic microangiopathy (5), interstitial pneumonia (5), CMV infection (30) and CMV disease (8). Three patients relapsed. Forty three died due to infection (26 cases), graft failure (4), VOD (3), GVHD (7) or relapse (1) . Actuarial probability of survival (SRV) was 51% at 6 years for the whole series. Variables associated with a better SRV in univariate analysis were: age under 25 years (63%; p=0.05), interval diagnosis-SCT <2 years (59%, p=0.06), SCT performed after 1997 (57%, p=0.06), use of "early" CyA (80%, p<0.001), A, B, C, DRB1 and DQB1 identity at allelic level (92%, p=0.01), absence of grade II-IV acute GvHD (81%, p=0.00002) and absence of extensive chronic GvHD (83%, p<0.0001). In multivariate analysis variables associated with a better prognosis were age lower than 25 years, "early" CsA and absence of grade II-IV acute GvHD. Conclusions: This experience shows that, at the present time, USCT in patients with CML in 1CP offer similar results than SCT from HLA-identical siblings, specially when using A, B, C DRB1 and DRQB1 identical donors at allelic level. Purpose: In a recently published study (Schetelig et al, JCO, 2003) we demonstrated a graft versus leukemia effect in patients with CLL. However, GVHD caused significant morbidity and mortality. In an attempt to lower the rate of severe GVHD we replaced ATG by Campath in a new protocol. Here, we report preliminary safety data of 18 patients treated within the new study. Patients and Methods: Eighteen patients with a median age of 53 years (range, 43 to 64 years) and advanced CLL were included. A median of 3 prior chemotherapy regimens had been given before transplantation. Sixteen pts had received Fludarabine among them 7 pts with fludarabine-resistant disease. High risk cytogenetic features were identified in 9 pts. After conditioning with fludarabine (150 mg/m 2 ), busulfan (8 mg/kg) and Campath (75 mg) peripheral blood stem cells from related (n=4) or unrelated donors (n=14) were transplanted. GVHD prophylaxis consisted of CSA. Results: After a median follow-up of 4 months (range, 0 -17 months), 15 pts are alive. Chemotherapy was tolerated without significant extramedullary toxicitiy. Regeneration of neutrophils (>0.5/nl) and platelets (>20/nl) required a median of 14 (range, 17-25) and 10 (range, 0-27) days, respectively. Acute GvHD grades 0-I/II/III-IV was observed in 13/5/0 pts, respectively. Limited chronic GVHD occurred in 4 and extensive disease in one out of 9 evaluable pts. Three pts experienced late secondary graft failure on days 134, 152 and 324 in the presence of persisting CLL and one pt had progressive disease despite stable graft function on day 136. Three out of 13 pts at risk of CMV infection experienced CMV-antigenemia. Severe non-fatal encephalitis (HHV6 and EBV as suspected agents) was observed in two pts and hemorrhagic cystitis (Polyoma-DNA detected) in one patient. Two pts died from pneumonia (infectious agent unknown) on days 19 and 35 after HSCT and one pt died subsequent to treatment of relapse. One-year overall survival was 72% (95% CI, 42% to 100%). One-year probability of non-relapse and relapse mortality was 13% (95% CI, 0% to 30%) and 17% (95% CI, 0% to 47%), respectively. In conclusion, conditioning with reduced-intensity busulfan and fludarabine combined with 75 mg Campath in vivo and GVHD prophylaxis with CSA has low regimen-related toxicity and prevents severe acute and chronic GVHD effectively. Viral infections and late secondary graft failure remain major problems. Introduction: Allogeneic stem cell tranplantation (ASCT) is curative for patients (pts) with Ph+ CML. Imatinib induces durable remissions especially in chronic phase (CP). Physicians are faced with new challenges as to when and whom to transplant. In this retrospective study, the impact of prior imatinib therapy on the outcome of ASCT is analysed. The effectiveness of imatinib in relapse after ASCT is also evaluated. Patients and Methods: 96 pts (55 m, 41 f) had ASCT at the university of Leipzig between 1997 and 2003. Median age was 38.5 y (range 16-64). 33/96 pts (34.4%) received imatinib prior to ASCT for CP refractory to IFN-alpha (n=12), AP (n=15), and BP (n=6). ASCT was done because of resistance to imatinib (n=15), haematological response only (n=14) or because of advanced disease at diagnosis despite cytogenetic remission (n=4). 63/96 (65.6%) pts in CP (n=41), AP/2.CP (n=18), BP/2.AP (n=4) had ASCT without imatinib. Results: Median interval between diagnosis and ASCT was 22 mos (range 2-258). For the entire cohort, disease phase at ASCT was an important predictive factor for OS and DFS at 1 y. The probability of OS, and DFS for pts in CP at ASCT, and for pts beyond CP were 77%, 66%, and 55%, 33% respectively (P=0.0005, 0.0006). Interval between diagnosis and ASCT as well as imatinib prior to ASCT had no negative effect on OS and DFS. TRM at 1 y for pts in CP and beyond CP was 22%, and 29% respectively (P=0.307). Relapse incidence (RI) was higher in pts beyond CP (53%) versus 13% in pts in CP (P 0.0004). Prior imatinib had no effect on RI. > 3 y interval between diagnosis and ASCT (P=0.007) and resistance to imatinib prior to ASCT (P=0.014) were associated with higher RI. Pts treated with imatinib for relapse after ASCT had a similar OS at 1 y to pts without relapse (67%, 64% respectively) which was significantly better than OS for relapsed pts treated with immunotherapy (reduction of immunosuppression and/or DLI) (P=0.0614). Conclusions: 1. CP at ASCT is the most relevant factor influencing outcome. 2. Imatinib does not appear to have a negative effect on outcome after ASCT. Results may be improved with imatinib prior to ASCT in pts with advanced disease by reducing neoplastic cell burden. 3. Imatinib is effective for relapse after ASCT 4. Mechanisms of resistance as point mutations need to be identified to predict response after ASCT in pts resistant to imatinib prior to ASCT. 5. Our results need to be confirmed in larger prospective studies. Background: Imatinib has been reported to be effective in treating post-transplant relapse of CML. Patients: In this study we analyze 22 CML patients, with a median age of 33 years (range 21-60), who relapsed after transplant, at a median interval of 488 days, and received Imatinib 200 mg/day, at a median interval of 218 days (range 0-1478) from relapse. Twelve patients received Imatinib as second line therapy after failing donor lymphocyte infusions (DLI) (group A), whereas 10 patients received Imatinib as first line treatment (group B). Thirteen out 22 patients are still taking Imatinib, with a median duration of therapy of 9 months/patient (range 3-18). Group A comprised more patients with advanced phase of disease (p=0.05), less patients with molecular relapse (p=0.1), less patients with unrelated donors (p=0.01), a shorter median interval transplant-relapse (p=0.04). Toxicity: None of 8 patients with GVHD worsened, and no patient experienced "de novo" GVHD. 48% of patients had hematological toxicity: severe leucopenia (grade 3-4 WHO)(19%), severe anemia (14%), severe thrombocytopenia (5%). Non hematological side effects were mild and included oedema (24%), diarrhea (9.5%); 43% of patients experienced an infection (0% grade 3-4) at 6 months after Imatinib (median, range 1-16). Responses: At 3 months a complete or major cytogenetic response was seen in 36% of group A and 89% of group B patients. In 5 patients of group A, who failed a first course of DLI and a course Imatinib, a course of DLI+Imatinib induced 3 complete cytogenetic responses (all with molecular residual disease-MRD-positive). Two patients of group B received DLI as consolidation of MRD: at the last follow-up are both in complete molecular remission. At last follow-up (median 369 days, range 120-698), 95% of the patients are alive (2 patients died of disease progression); 90% have a haematological response, 63% had a cytogenetic response (complete in 50%). Six out of eleven patients in complete cytogenetic response are in molecular remission. Conclusions: (a) we confirm that Imatinib is an additional therapeutic option for CML patients relapsing after an allogeneic transplant, (b) it is more effective given as first line therapy for molecular relapse and (c) the combination of DLI+Imatinib may rescue patients refractory to both DLI and Imatinib given as single treatments.

The ability to mobilise progenitor cells is impaired in patients with chronic lymphocytic leukaemia compared to other haematological malignancies and this is independent of marrow infiltration R. A. Lovell, K. Holder, M. Karanth, C.D. Fegan, G.E.D. Pratt, D.W. Milligan, Birmingham Heartlands Hospital (Birmingham, UK) Mobilisation of peripheral blood stem cells (PBSCs) is a key step in the ability to undertake stem cell autografts for a variety of haematological malignancies. A number of mobilisation schedules are available which balance the morbidity of the chemotherapy dose with mobilising efficiency. Ourselves and others have noted that progenitor cells may be harder to obtain from patients with CLL. We have therefore analyzed our data to examine mobilisation outcomes in patients with CLL compared to other patients with haematological malignancy undergoing mobilisation using the same regimens. Patients were enrolled into a trial comparing mobilisation with either intermediate dose cyclophosphamide + lenograstim 5 µg/kg or lenograstim alone at 10µg/kg. In those patients who had received fludarabine a period of at least three months was left prior to harvesting. All patients who received treatment and were in stable PR or CR. 81 patients were randomised (6 HD;16 NHL; 35 myeloma; 24 CLL). The median age was 54 years (range 20-68). A previous report of this trial has shown no difference in the two arms in the ability to mobilise stem cells. We have now analysed the impact of disease type on mobilising efficiency. Failure to mobilise was defined as the inability to harvest 2x10 6 CD34 cell/kg in three aphereses. The overall failure rate was low at 28 %. There was no difference in the mobilising success for patients with Hodgkin's disease, non Hodgkin's lymphoma and multiple myeloma. However there was a highly significant difference in patients with CLL compared to the rest of the cohort (P= 0.0001). This was also strongly significant when compared to the myeloma group (P= 0.0002). It appears the ability of CLL patients to mobilize sufficient stem cells is markedly reduced even when compared to myeloma patients who have bone marrow infiltration. This effect may in part be due to previous treatment, possibly fludarabine, but may also reflect an altered bone marrow microenvironment. Careful consideration of mobilization protocols for CLL patients is suggested and further investigation of these patients is warranted to guide future therapy. Unrelated transplantation is option for the patients without sibling donor, but these transplantations are supposed to be less successful due to higher TRM. The 3 year probability of survival of 613 patients, receiving unrelated transplants for CML in chronic phase within the first year of diagnosis was 54% (IBMTR 1994 (IBMTR -1999 . We analysed our own data from 105 transplantations performed for CML between 1988-2003. 46 patients received HLA-identical sibling and 30 patients unrelated transplants for CML-CP. 29 transplants were done for advanced stage of CML. Characterisation of the cohort of the patients is: HLA-identical sibling BMT group: med. age-36 years (16-55) (28 males, 18 females).

Conditioning: CyTBI-9, BuCy-34, BuFluATG-3. 32 patients survive (14-181 m), with 5 patients suffering from extensive cGVHD. 10 patients recevied DLI and 4 pts STI for relapse. 14 patiens died after transplantation ( 7 of them due to cGVHD, 4 due to toxicity of conditioning, 1 due to relapse, 1 due to aGVHD and 1 due to infection ). MUD-BMT group: med. age-40 years (19-57) (21 males, 9 females). Conditioning: CyTBIATG-14, BuCyATG-8, FluTBIATG-6, BuFluATG-2. 20 pts survive 5-70 m. 2 pts have extensive cGVHD. 6 patients received DLI for relapse. 10 patients died (7 of them due to infection, 2 due to aGVHD and one due to relapse ) Results: 3 year probabilities of survival is 70% in the HLA-id sib. BMT and 65% in the MUD-BMT. Further analysis showed, that the probability of 3 years survival is 80% in the 21 patients receiving unrelated transplants between 1999-2003. We did not find such improvement in the group of the patients receiving graft from HLA-identical sibling during the same time period. The outcome of the patients transplanted for advanced disease is worse with the 3-year probabilities of survival only 20%. Discussion: Our results of MUD-BMT for CML-CP are similar to the results of HLA-identical sibling transplantation and are even better in the group of patients transplanted since 1999. We postulate, that low risk of death due to GVHD in our group of MUD-BMT is influenced by ATG we have been using since 1995. Based on these results we beleive, that there is no reason to differentiate indication for BMT in dependence on the type of the donor available (HLA id. sibling vs. MUD).

A sequential chemoimmunotherapy protocol can obtain a high proportion of molecular remission in chronic lymphocytic leukaemia V. Pitini, C. Arrigo, C. Amata, G. La Gattuta, L. Siracusano, D. Teti, Policlinico Universitario Messina (Messina, I) The choice of therapy for chronic lymphocytic leukemia in stage B and C is difficult, but during the last decade there have been two major changes in the management of this disease in younger patients. The first was the realization that Fludarabine might be the best first-line therapy and the second was that younger patients might be suitable for an autograft with durable molecular remission after transplant. However, new studies should now be designed to elucidate the value of in vivo and/or in vitro purging. Here, we report the results of a pilot study using a sequential chemoimmunotherapy protocol which combines Fludarabine (25 mg/m 2 per day for three days), Cyclophosphamide (250 mg/m 2 per day for three days), and Rituximab (375 mg/m 2 on day 1), monthly x4, followed by Alemtuzumab, given at 10mg subcutaneously three times a week after a stepped up dosing during the first week (3mg as the first dose and increasing, as tolerated, to 10mg) x 12 weeks before mobilization with Cyclophosphamide at a dose of 5 g/m 2 and G-CSF (10 micrg/Kg/d). Prophylaxis with trimethoprimsulfamethoxazole and Acyclovir was required during and for 6-months after Alemtuzumab therapy. Minimal residual disease was detected by the cytofluorimetric analysis of peripheral blood and bone marrow samples using a triple combination strategy (CD20/CD5/CD19, CD22/CD23/CD19, and kappa/lambda/CD19), with a level of sensitivity of 5 X 10 -5 and performed in parallel with a non specific allele CDRIII polymerase chain reaction analysis. Evaluation of minimal residual disease was performed before the conditioning regimen (BEAM). Between November 2002 and February 2003, 5 patients (4 males,1 female, median age of 50 years), were enrolled. All patients, before BEAM, had complete clinical remission. Four of the five patients achieved negativity for CDIII rearrangement. All patients proceeded to mobilization with successful harvest with median yield of 5.9 x 106/Kg CD34+ cells. There were no serious infection complications observed at the time of autografting. To date all patients are alive and in complete remission. Whether these results will be translated into an improved survival remains to be seen.

Imatinib mesylate therapy in patients with chronic myeloid leukaemia previously treated with autologous stem cell transplantation M. Cortés, A. Sureda, R. Martino, A. Aventín, J. Nomdedeu, J. Briones, D. Valcárcel, S. Brunet, J. Sierra, Hospital Santa Creu i Sant Pau (Barcelona, E) Imatinib mesylate (STI 571, Glivec) is a highly effective and welltolerated treatment for patients with chronic myeloid leukemia (CML). Autologous stem cell transplantation (ASCT) is a noncurative therapeutic strategy with the objective to reduce tumor burden in CML patients not candidates for allogeneic stem cell transplantation. We have reviewed the long-term outcome of 27 patients [19 men and 8 women, median age of 48 (30-63) years] diagnosed of CML in first chronic phase (CP) who received an ASCT at a median time of 34 (7-99) months after diagnosis with the combination of cyclophosfamide (Cy) plus total body irradiation (n = 9, 35%), busulfan (BU) plus Cy (n = 7, 27%) or Bu alone (n = 10, 38%) as conditioning regimen. Twenty five patients (92%) received maintenance therapy with alphainterferon (IFN) after ASCT. Three patients are still under IFN therapy at 4, 7 and 8 years after ASCT and in continuous complete cytogenetic remission (CCR). Six patients discontinued IFN therapy due to disease progression and the remaining 15 patients (55%) were switched to STI571 therapy due to intolerance/resistance to IFN (n = 10, 67%), progression to accelerated phase (AP) (n = 3, 20%) or to blastic crisis (BC) (n=2, 13%). Daily doses of STI571 were 400 mg po and 600 mg for IFN-intolerant patients and AP/BC patients, respectively. All IFN-intolerant patients achieved a complete hematological remission (CHR) at a median time of 4.5 weeks (3 -8) after starting therapy; half of them (n = 5) achieved a CCR 6 months after starting STI571 and all of them remain in CCR with a median follow up of 18 months. Two patients discontinued STI therapy due to disease progression and one due to grade 4 hematological toxicity. Of the 3 patients treated in AP, 2 patients achieved a CHR at 12 and 14 weeks after beginning therapy and one of them a CCR with a follow up of 18 months. None of the patients treated with STI571 for BC responded. Of the patients in CCR, 60% of them presented a 10E4log reduction in the number of bcr/abl transcripts evaluated by quantitative PCR 6 months after starting therapy and the remaining 40% a 10E3 log reduction with the maximal reduction observed at 12 months after starting STI571 therapy. In our experience, STI571 is able to achieve a significant percentage of CCR in CML patients treated with ASCT and resistant to IFN with minimal hematological toxicity. Results in advanced phases are poor. CML carries high risk for graft versus host disease (GVHD) but there is no published study analyzing factors affecting transplant outcome exclusively for CML patients. With this aim, we combined the results of allogeneic transplants (ASCT) performed in three centers, two from Poland (PL) and one from Turkey for CML. Patients: All CML patients transplanted in PL (Dec 1991 -Nov 2002 and Ankara (March 1995 -March 2003 were entered into the same database and evaluated. A total of 200 patients (120 male, 80), chronic / advanced phase: 180/20; age: 35.8 (5-54) received either bone marrow (BM)(n=107) or peripheral blood stem cells (PBSC)(n= 93) following a conditioning regimen of busulfan-cyclophosphamide (BuCy)(91%) or fludarabine-based reduced intensity regimen (RIC). All the ASCTs performed before 1995 were BMT. After 1995, BMT (n=92) was equal to PBSCT's. Both univariate and multivariate analysis were performed using the logistic regression model for evaluating the predictive factors on the risks of acute (aGVHD) and chronic GVHD (cGVHD). Results: Cumulative analysis on all patients showed 96 % engraftment at day 17 for both an absolute neutrophil count of 0.5 x 109/l and 20 x 10 9 /l platelets . The incidence of aGVHD (grade II to IV) was 32.5 %. In patients surviving day 100 (n=162) the frequency of cGVHD was 53.5%. Early transplant-related mortality was observed 17%. By logistic regression analysis, ASCT type had a significant effect on the incidence of severe aGVHD (BuCy vs RIC, 35% vs 12.5%, p=0.03, RR: 5.97). A positive correlation between the frequency of cGVHD and recipient's age (p=0.004, RR: 1.08) and a negative correlation with donor's age (p=0.02, RR:0.94) has been found. At five years, cumulative overall survival (OS) was 54.4±4.1% and 46.64±4.4% for DFS. Both OS and DFS following ASCT performed before and after 1995 revealed similar results. Univariate analysis showed that recipient gender (p=0.0019), female donor and male recipient (p=0.01), stage, aGVHD (P<0.00001), extensive cGVHD (p=0.0004), Gratwohl score (p=0.00001) is influencing DFS. Stem cell source was not a risk factor.

Multivariate analysis confirmed recipient gender (p=0.01)(OS), extensive cGVHD (p<0.0001)(DFS) and aGVHD (p=0.016) (OS) be risk factors, respectively. Conclusion: This multicentric retrospective analysis emphasizes the importance of donor-recipient gender, age, Gratwohl score, ASCT type and GVHD but not stem cell source on OS and DFS in following SCT in CML.

L. Shvidel, M. Shtalrid, L. Bassous, N. Harpaz, A. Berrebi, Kaplan Hospital (Rehovot, IL) Contamination of peripheral blood stem cell harvests by tumor cells contributes to the high frequency of relapse following autografting in CLL. Thw anti-CD20 monoclonal antibody rituximab induces a rapid decrease of circulating B cells and has been useful for in vivo purging prior to peripheral stem cell collection. Since November 1999, we have performed stem cell harvests after pretreatment with rituximab in eight patients with advanced-stage CLL and one with de novo PLL. Seven patients received rituximab as consolidation for treatment of minimal residual disease following chemotherapy with fludarabine and cyclophosphamide. Two other extensively pretreated patients, who relapsed after autologous stem cell transplantation (APSCT), were treated concurrently with rituximab and chemotherapy (CHOP or fludarabine-based) for active disease. PSC mobilization was performed using cyclophosphamide 1.5 g/m 2 and G-CSF. The median CD34+ cell count was 4.4x10 6 /kg (range, 1.0-12.0), and the median percentage of CD5/CD19+ cells was 1.2 (range, 0.5-13.0). Four patients were autografted, hematological recovery was uncomplicated. Two patients transplanted while in relapse, achieved partial response. Two other patients who underwent transplantation following first-line chemotherapy achieved complete responses for 29+ and 9+ months post autografting. Currently, their blood counts are normal and immunophenotyping reveals 7.1% and 2.2% circulating CD5/CD19+ cells, subsequently. In conclusion, in vivo purging with rituximab prior to stem cell collection is able to reduce contamination of the harvest by CLL cells and does not affect the yield or function of stem cells.

Recently, clinical trials have examined rituximab with fludarabinebased chemotherapy as first-line treatment for CLL. The effect of this modality on in vivo purging of CLL cells will define the optimal schedule of rituximab in autografting. We present the data of a pilot experience of autologous transplantation with alemtuzumab (Campath-1H) in-vivo purged PBSC in B-CLL. Treatment protocol included (a) debulking with DHAP (2 courses), followed by (b) Campath-1H 30 mg up to 8 doses, (c) cyclophosphamide (CY) 4 to 7 g/sqm plus G-CSF and stem cell apheresis (following a single supplementary administration of Campath-1H 30 mg to deplete neoplastic B cells from circulation), and (d) autograft with a high-dose regimen of mitoxantrone 60 mg/sqm plus melphalan 180 mg/sqm. Only advanced stage (BII-CIV) patients <60 years who proved resistant to or had relapsed after fludarabine were eligible to treatment. Ten patients (median age 53 y, range 37-60; M=8, F=2) were enrolled. One was stage BI (LDT<12 mo), 6 were stage BII, and 3 stage C. Median time from onset of disease was 37 months (range 7-74). The patients had previously received 1 to 3 CT lines (median 2). Immunoglobulin heavy-chain variable genes (VH) mutational status was assessed on CLL cells prior to treatment start, showing a non-mutated status in 9 out of the 10 patients. Response to DHAP (CR =1; PR=6) and to Campath-1H (CR=4; PR=5) was excellent, with circulating clonal CD5+/19+ lymphocytes dropping from (median) 4.7 to 0.2 x 10 9 /L (99.9% depletion). However, in terms of CD34+ cell recovery the mobilization step with intermediate/high-dose CY plus G-CSF was unsuccessful in 3 out of the 8 treated patients. Up to now, 4 patients have been autografted, 3 with PBSC and 1 with BM cells. Two of them are in clinical and molecular (PCR for IgH gene rearrangement) remission at 1 and 8 months from autograft, respectively, another attained PR, and a fourth relapsed after 6 months duration CR. Two patients are still waiting to be transplanted, while four quit the program due to consensus withdrawal despite persisting complete remission (n=1) or disease progression (n=3). During treatment the patients underwent a number of infectious complications (1 Aspergillus, 2 HZV, 1 ECHO, 1 Coxackie, 1 streptococcal and 3 CMV reactivations). Prolonged neutropenia was observed in 2 cases, and autoimmune hemolytic anemia in 1. In conclusion, the majority of B-CLL patients failing fludarabine will respond to DHAP and Campath-1H given sequentially, but CD34+ cell collection may be inadequate after such treatment despite an excellent in-vivo purging effect. The immune-suppression induced by Campath-1H has to be considered carefully.

New approaches to the second transplant for thalassaemia J. Gaziev, C. Giardini, P. Polchi, P. Sodani, G. Lucarelli, Pesaro Hospital (Pesaro, I) Unlike hematological malignancies patients with thalassemia have an increased risk of graft failure after BMT. Up to 10% of patients with thalassemia develop graft failure following transplant. Our historical experience of second BMT using different conditioning regimens showed a higher incidence of graft failure and transplant related mortality. In 1977 we started a prospective study with a new preparative regimen for second transplants in thalassemia. Sixteen patients (6 males and 10 females) with median age of 8 years (range 3-27 years) received hydroxiurea 30 mg/kg/day, azathioprine 3 mg/kg/day (day -45 to -12) and fludarabine 20 mg/m 2 /day (day -17 to -13) before conditioning with BU14 CY200 (day -9 to -2) to reduce the hyperplastic bone marrow and increase immunosuppression. Results of an interim analysis of data showed a higher incidence of graft failure and low disease-free survival. Ten out of 16 patients (63%) had engraftment and 5 of them subsequently lost their grafts. The incidence of primary graft failure was 37%. Three patients died. The probability of overall survival was 81%, DFS-25%, graft failure-69% and transplant related mortality-20%. Overall this regimen was well tolerated and transplant related toxicity was not significant. Because of a higher incidence of graft failure we closed this protocol for second transplants.

Recently we have modified this preparative regimen with increasing the dose of fludarabine to 40 mg/m 2 /day and adding thiotepa 10 mg/kg/day and antithymocyte globulin 2,5 mg/kg/day. Two patients were given a second and one patient a third transplant after this regimen. All 3 patients had a prompt full engraftment and no significant toxicity has been seen.

A. Ghavamzadeh, M. Jahani, A. Mousavi, B. Bahar, M. Iravani, K. Alimoghaddam, S. Gholibeikian, F. Safavifar, P. Heydari, Dr. Shariati Hospital (Tehran, IR) Objectives: Chronic Graft-Versus-Host-Disease (cGVHD) is an important complication of allogeneic blood and marrow transplantation. In this study we assess cGVHD outcome in bone marrow (BM) and peripheral blood stem cell transplantation (PBSCT) in thalassemia major patients in our center. Methods: We studied engraftment kinetics, outcome of cGVHD allogeneic PBSCT vs. BMT in 184 Beta Thalassemia Major Patients in our center between 1991 to November 2003. Patients' mean age was 6 years (2.5-17years). Classification of their disease was class 1=70, Class 2=71, Class 3=43. Conditioning regimens in class 1and 2 were Cyclophosphamide 50 mg/kg for 4 days + Busulfan 3.5 mg/kg for 4 days and in class 3, Cyclophosphamide 40 mg/kg for 4 days + Busulfan 4 mg/ kg for 4 days. We used Cyclosporin 3 mg/kg (IV) from day -2 to +5 and then 12.5 mg/kg (PO) plus Methotraxate 10 mg/m 2 in day +1 and then 6 mg/m 2 on days +3,+6 as GVHD prophylaxis regimen. Results: Myeloid and platelet engraftment (PMN >0.5x10 9 /L and PLT> 20 x 10 9 /L) occurred at median 11 and 20 days after PBSCT Vs. 21 and 27days after BMT respectively (p-value=0.02) . Incidence of Acute GVHD (grade II-IV) was near in both groups (58.1% in PBSCT Vs. 52% in BMT). In our first assessment cGVHD occurrence was 28.3% (Limited 16.3% and Extensive 12%) from which 18.4% was in PBSCT (Limited 9.2%, Extensive 9.2%) versus 9.7% in BMT (Limited 7%, Extensive 2.7%) p-value was 0.00. But gradually after treatment in a period of time (1 to 48 month) with median 17 months, some of them regressed.

Finally 14.6% of the patients remained with cGVHD (limited=9.2%, Extensive =5.4%) from which 2.7% was in BMT (Limited=1.1%, Extensive=1.6%) versus 11.9% was in PBSCT (Limited= 7.6%, Extensive=4.3%). Mortality rate of cGVHD was 6.6% occurring only in PBSCT. And Mortality rate of rejection was 13.3% occurring only in BMT group. Overall survival (5-10 years) was 80% and thalassemia disease free survival (5-10 years), 70% .There were no significant differences between the two groups. Conclusion: Engraftment in PBSCT was faster than BMT. Occurrence of cGVHD (Limited and Extensive) in PBSCT was more than BMT but occurrence of rejection and it's mortality in BMT was more than PBSCT. Although, cGVHD is an important complication of PBSCT, but it is treatable and needs good care and follow up. Early progenitors express ABO antigens and are targets for recipient derived iso-hemagglutinins in major ABO mismatch HSCT. We report a case of pure red cell aplasia (PRCA) in a boy undergoing BMT for thalassemia, mimicking loss of erythroid graft as characterized by beta/alpha synthetic ratios. A 10 yr old boy with beta-thalassemia major (Pesaro class 1) due to beta0 IVS II-1 (G-A) homozygosity with coexistent heterozygosity for alpha+ thalassemia (-a/aa), underwent HLA identical sibling non-thalassemic donor BMT. Conditioning: Fludarabine 30 mg/m²/day x 5; Cyclophosphamide 60 mg/kg/day x 2; Melphalan 140 mg/m²/day x 1; ATG 2.5 mg/kg/day x 4. GvHD prophylaxis was Cyclosporine 2 mg/kg/day. The graft was red cell depleted due to donor/recipient blood group mismatch (A Rh pos/O Rh pos). A total of 1.7 x 10 9 /kg nucleated cells was infused. Delayed engraftment was evident (ANC >0.5 x 10 9 /l at +40 days, platelets >50 x10 9 /l at +33 and >100 x10 9 /l at +54 days). Immune reconstitution was not delayed. Erythroid regeneration was profoundly delayed requiring transfusion support until +5 months. Pre-BMT recipient anti-A titres were:IgM 1:128 and IgG 1:8 increasing to 1:2000 and 1:500 respectively, and remaining detectable for 4 months without evidence of hemolysis. Full donor engrafment of peripheral blood leucocytes using VNTR analysis was documented on day +26. VNTR analysis of bone marrow and PBMC was also undertaken at +12 weeks, in the presence of normal white cell and platelet recovery but no detected erythroid regeneration. Bone marrow culture at this time documented reduced levels of BFU-E but mainly of donor origin when analyzed by VNTR. Beta/alpha synthetic ratios were those of the recipient (0.11). Erythroblasts were completely of recipient origin as measured using VNTR. This suggested loss of donor red cell engraftment. At +20 weeks the beta/alpha ratio improved (0.6 -0.7). The child remains well and transfusion independent, with normal blood counts and full donor engraftment at +1 year The beta/alpha ratio is 0.96, as expected from the gene expression of the non-thalassemic donor. PRCA post HSCT is rare. We report a case where simulataneous analysis of globin chain synthesis and VNTR chimerism gave seemingly conflicting results. This unique combination effects insights into the dynamic process of red cell engraftment following ABO major incompatibility transplantation. Analyses of BFU-E colonies enabled confirmation of donor red cell engraftment.

Bone marrow transplantation for thalassemia using different conditioning regimens including ''short'' anti-thymocyte globulin therapy M. Zakerinia, H. Nourani Khojaste, M. Ramzi, M. Haghshenash, Shiraz University of Medical Sciences (Shiraz, IR) Summary: Allogeneic bone marrow transplantation (BMT) was performed on 113 Iranian transfusion dependent thalassemia major patients from May 1993 through September 2003. To have a longer follow up, at least 1.5 years, we here report BMT of 90 patients who were transplanted up to December 2001. The donors were human leukocyte antigen (HLA)-identical, mixed lymphocyte culture (MLC) nonreactive siblings (n=74) and parents (n=6), HLA-identical MLC reactive siblings (n=5) and parents (n=1), and one HLA-antigen mismatch siblings (n=4). The induction regimen in eleven patients was oral busulfan (BU) 14mg/kg and IV cyclophasphamide (CY) 200 mg/kg, in fifteen patients was BU 15mg/kg and CY 200 mg/kg, in forty seven patients was BU 15 mg/kg, CY 200mg/kg, and short antithymocyte globulin (ATG) 40 mg/kg (10mg/kg on days -2, -1, +1, +2), and in fifteen patients was BU 15 mg/kg, CY 200 mg/kg, and ATG 60-100 mg/kg (10mg/kg3-5 days before and 3-5 days after BMT). Graft-versus-host disease (GVHD) prophylaxis was consisted of cyclosporin and prednisolone. The group who received BU 14 and CY 200, as compared to the group who received BU 15and CY 200, although in a lower age range and lower risk group; median age 7 yrs. vs 10 yrs. and 46% vs 7% were included in the Lucarelli's risk group class I (the best prognostic group) respectively, had a lower disease-free survival (DFS); 64% vs 80%, with a follow up of 7 -9.5 yrs. That is why from 9 yrs. ago our standard protocol for BU is 15 mg/kg. The group who received " short" ATG (40mg/kg) and BU 15, CY 200 had almost the same out come as did the group who received higher dose of ATG (60-100 mg/kg), DFS: 72% vs 73% respectively, despite the fact that half of both groups were included in the lucarelli's risk group class III (the worst prognostic group), 49% vs 53%. We had a better DFS for the patients who received BU 15, CY 200, and no ATG, as compared to the group with ATG (80% vs 72%), but 27% of them developed grade IV acute GVHD and 54% developed chronic GVHD. In the group with short ATG only 15% and 17% developed grade IV acute and chronic GVHD respectively. There was no significant difference for platelets and white blood cell (WBC) drops and engraftment days, and the no of packed red blood cell transfusions in the different groups. The median hospital stay was longer for the group with BU 15, Cy 200 (81 vs 61-65 days), second bone marrow infusion was needed in 6% and 20% of patients who received ATG 40 and 60-100 respectively (1-2 mo post BMT). Conclusion: BU 15 is more effective than BU 14 for it's myeloablative properties, and by adding "short" ATG to the conditioning regimen the incidence of grade IV acute, and chronic GVHD is much less in thalassemic patients, especially if HLA-disparity is present.

Intensified immunosuppression for treatment of impending graft rejection in thalassaemic patients U. Duffner, B. Strahm, U. Kontny, C. Niemeyer, University of Freiburg (Freiburg, D) Allogeneic BMT from an HLA-identical donor is the only proven curative therapy for homozygous beta-thalassemia. Graft rejection remains a major problem especially for high risk patients with iron overload. Presence of >25% residual host cells (RHC) at 2 months after BMT or >15% at 3 months has been reported to predict graft rejection with a probability of 96% and 72%, respectively. Here, we report on 3 patients with increasing RHC treated with intensified immunosuppression (IIS). Patient 1 is a 14 year old boy transfused since the age of 3 months. Chelation therapy was started at the age of 8 years; at BMT his serum ferritin was 2433 ng/ml reflecting severe iron overload. The conditioning regimen consisted of busulfan (Bu) 16 mg/kg and cyclophosphamid (Cy) 200 mg/kg. RHS was monitored by short-tandem-repeat markers. Following RHC < 5 % at day 33, RHC gradually increased to 20% on day 119. IIS was implemented by increasing ciclosporin (CsA) trough levels from 150-250 ng/ml to 350-450 ng/ml. With this regimen RHC decreased to 0% at day 658 when IIS was stopped. Patient 2 is a 2 year old boy with a serum ferritin of 1475 ng/ml at BMT. He was conditioned like patient 1. Chimerism was complete at day 42, but RHC slowly increased thereafter to 15 % at day 79. Despite increasing CsA trough level from 150-250 ng/ml to 350-450 ng/ml, RHC increased to 50 % at day 182. With the introduction of mycophenolatmofetil (MMF) at a dose of 1200 mg/m2/d RHC gradually decreased to the current level of 7.5 % at day 476. Patient 3 is a 9 year old girl with portal liver fibrosis and a serum ferritin of 1475 ng/ml at BMT. The conditioning consisted of hydroxyurea 30 mg/kg/d and azathioprin 3 mg/kg/d for 5 weeks followed by fludarabin 100 mg/m 2 , Bu 12 mg/kg and Cy 160 mg/kg. RHC increased from 15 % at day 32 to 50% at day 70 when IIS was started by increasing CsA trough levels from 150-250 ng/ml to 350-450 ng/ml and adding MMF at dose of 1200 mg/m². Initially RHC rose to 70% at day 77 but later decreased. Currently, at day 126, RHC is 20 %. We conclude that impending graft rejection can be prevented by IIS presumably by inhibiting immunocompetent host cells. Despite improvement in immunosuppressive regimens, acute and chronic graft versus host disease (GVHD) continue to be major causes of morbidity and mortality after allogeneic stem cell transplantation (SCT). Based on available data demonstrating that, in matched related donor transplants, moderate T-cell depletion (10(5) T cells/kg) with infusion of mega doses of CD34 cells results in engraftment without GvHD, it was proposed to use this approach for non-malignant allogeneic transplant in whom GvL is not a prerequisite. Patients: 12 patients, median age -1 year (5 mo -8 yrs), were treated.

Four patients had thalassemia major, 3 immunodeficiency, 3 metabolic disorders (Hurler syndrome (2), San Felippo (1), 1 each with adrenoleucodystrophy (ALD) and familial hemophagocytic syndrome. Peripheral blood stem cells were collected from matched related donors after G-CSF priming. Conditioning included busulphan 4mg/kg on days-9,-8,-7,-6, cyclophosphamide 50 mg/kg on days -5,-4,-3,-2, ATG (fresenius) 5mg/kg on days-9,-8,-7,-6,-5, fludarabine 40mg/m 2 on days -9,-8,-7,-6,-5. T cell depletion was performed by positive selection of CD34 cells by immunomagnetic beads (CliniMACS). On day 0 high doses of CD34 cells were given, median cell dose -10.7 x10 6 CD34/kg (7.4-50 x10 6 ). 10 5 T cells per kg were added. No prophylaxis for GVHD was given post transplant. Engraftment occurred between day +8 and +12 post transplant (median-10 days). There was no GVHD in any patient. No severe infections occurred during or post transplant. All patients are alive and well with no clinical evidence of disease. Mixed donor chimerism was documented in all patients. Immunological reconstitution with normal CD4 cells occurred at 6 months post transplant. Median follow up of all 12 patients is 15 months (2-58 months) .

Conclusions: A suggested new approach to SCT in nonmalignant diseases with matched donor is described. This is achieved by high dose of CD34 cells; moderate T cell depletion and no post transplant immunosuppression.

G. Kriván, Á. Bátai, B. Kapás, S. Lueff, M. Molnár, P. Reményi, M. Réti, A. Tremmel, T. Masszi, St. László Hospital, OPNI (Budapest, HUN) X-linked adrenoleukodystrophy (X-ALD) is the most common inherited peroxisomal disease characterized by myelopathy, peripheral neuropathy and cerebral demyelination which develop in association with the accumulation of very-long-chain fatty acids. Three boys (aged 12 y and 7 years) with X-ALD were transplanted since 1999 in St. László Hospital, Budapest. Patient 1 had neurological symptoms only, but all had brain MRI changes before transplant. The last two pts transplanted in August 2003 were monozygotic twins. Lacking of HLA identical donors all the three patients were transplanted with CD34+ selected paternal grafts. Conditioning regimens consisted of Bu (16 mg/kg)-Cy (200 mg/kg) (pt 1) and Bu (16 mg/kg)-Thiotepa (10 mg/kg)-Cy (200 mg/kg)-ATG (80 mg/kg) (pts 2,3). Graft size were 7.5x10 6 /kg (pt 1) and 6.8x10 6 /kg CD34+ cells (pts 2,3), and 4.2x10 5 /CD3+ cells (pts 2,3). All pts has engrafted, but pt1 later has rejected the graft and showed continuous neurological deterioriation. The monozygotic twins were transplanted simultaneously thus the disease and transplant process could have been observed and easily compared. Pts 2 and 3 has engrafted on day +14 and +13, respectively. The disease course was complicated by febrile neutropenia and mucositis (both pts), hemorrhagic cystitis caused by polyoma virus BK (pt 2). On day +39 pt 3 has developed grade III skin GvHD which responded to only 5 mg/kg methylprednisolon, while the other twin remained free of symptoms. Three months after transplant pts are full chimeras. While methylprednisolon dose was tapered by 1 mg/kg and cyclosporin A was discontinued owing to microangiopathic hemolysis in pt 3, the other twin has developed grade II skin GvHD as well, which was controlled properly by methylprednisolon. It might be remarkable that monozygotic twins under the same transplant conditions could show quite different transplant course. Control brain MRI on day +98 revealed significant improvement of previous changes in both children.

Cartilage hair hypoplasia: successful unrelated haematopoietic stem cell transplantation V. Bordon, E. Vandecruys, C. Dhooge, G. Laureys, Y. Benoit, Ghent University Hospital (Ghent, B) Cartilage Hair Hypoplasia (CHH) is a rare autosomal recessive disease, characterised by prenatal onset of growth failure with metaphyseal chrondrodysplasia and different grades of immunodeficiency. We report a girl with CHH, who presented with short stature and short limbs, on radiographs short metacarpals, short and curved femora and severe failure to thrive during early childhood. The haematological examination showed anemia due to red cell hypoplasia of the bone marrow, with progressive neutropenia, abnormal humoral immunity, low T-cells counts (CD3+ 232/µL), abnormal lymphocytes transformation test (LTT) and migration test. Mutation analysis revealed a heterozygous for two RMRP mutations: +40G>A and +63>A, the parents were not consanguineous and healthy carriers for these mutations. During the first 2 year of life, she had several severe pulmonary infections, intractable diarrhoea and intestinal pseudo-obstruction with requirement of total parenteral nutrition. Because of the bad clinical condition of the child and the permanent need of hospitalization, she underwent an allogeneic-MUD stem cell transplantation at the age of 23 months after myeloablative conditioning with Busulfan (16 mg/kg) Cyclophosphamide (200 mg/kg) and ATG (20 mg/kg) 2 months after transplant she developed a severe EBVlymphoproliferative disease, with good response after stop of the immunosupressive treatment, interferon-alfa and acyclovir. The T cell counts were reconstituted 10 months after transplant, with normalisation of LTT after 4 years post-HSCT. Now the patient is alive and well 5 years after transplant, but the longitudinal growth is markedly below the 3th percentile and the radiological osseous changes characteristics for CHH persist. We conclude that in this child with an unusual severe form of CHH, a HSCT has fully corrected the immune deficiency, but no benefit has been seen in the course of the condrodysplasia. Introduction: B cell lymphoproliferative disorders are often characterised by the production of monoclonal immunoglobulins that are detected in the laboratory by electrophoresis and immunofixation electrophoresis (IFE). Bence Jones myeloma or Light Chain Multiple Myeloma (LCMM) is defined by the absence of an intact immunoglobulin paraprotein and the presence of immunoglobulin light chains in urine and the light chains are important for disease monitoring. Nephelometric serum free light chain (FLC) assays have recently been shown to be useful in the diagnosis and monitoring of LCMM and more sensitive than urine assays for determining complete remission (CR). The EBMT definition of CR includes the absence of detectable paraprotein in both serum and urine (by IFE) and the aim of this study was to compare rates of CR in LCMM patients, judged by IFE in serum and urine, with the serum FLC assays. Patients and Methods: We examined 36 archived, matched urine and serum samples from the UK MRC Myeloma trials (V to VII trial) from patients judged to be in plateau by urine FLC measurement. Plateau had been defined as a three month period of stability following chemotherapy in which the paraprotein levels had not changed. Sera and urine were tested for kappa and lambda light chains by IFE (Sebia) and the results compared with serum FLC levels obtained by nephelometric analysis (The Binding Site). Results: 17 out of 36 patients had no detectable light chains in either serum or urine by IFE. The remaining 19 patients showed a monoclonal light chain band in either serum only (5 patients) or urine only (5 patients) or both (9 patients). However, when serum FLC were measured and the kappa/lambda ratio examined, 11 out 17 of the serological CR patients had abnormal kappa/lambda ratios (mean ratio of 9.3 compared to the normal mean of 0.63) indicating the presence of residual disease in contrast to the IFE results. The 19 patients with detectable paraprotein in serum and/or urine all had abnormal kappa/lambda ratios. Conclusions: These data indicate that serum FLC levels are a more sensitive measure of CR than IFE of serum or urine. Urine assessment of FLC is less sensitive because the kidneys can catabolize many grams of FLC per day, thereby preventing entry into the urine, while serum IFE has a sensitivity limit of approximately 150mg/L for monoclonal FLC detection compared to approximately 1 mg/L for the FLC assays. The treatment of relapse in MM after allogeneic SCT (allo-SCT) remains disappointing. Several studies demonstrated the efficacy of THAL in MM pts, but none of them have specifically addressed the impact of this drug in the treatment of disease progression after allo-SCT. To assess the efficacy and the safety of THAL in this setting, we conducted a retrospective study on 28 (16M/12F) consecutive pts from 9 french centers, who received THAL as salvage therapy after allo-SCT, between May 2002 and June 2003. The median age at time of relapse was 53 y (38-64). Pts had an IgG M-component (n=15), IgA (n=6) and IgD (n=2). The remaining 5 pts had light chain (n=4) and non-secretory MM (n=1). At diagnosis, cytogenetic was missed in 14 pts while 10 had a chr.13 deletion and 4 had a normal karyotype. Prior to allo-SCT, pts had received either standard chemotherapy alone (n=7) or at least one autologous SCT (n=21). Only 3 pts had received THAL before allo-SCT because of disease progression. At time of allo-SCT, 18 pts were in plateau phase, one in relapse and 9 had refractory progressive disease. Twenty-seven pts underwent SCT from an HLA-identical sibling and one from an HLA-identical unrelated donor. The conditioning regimen was myeloablative (n=4) and nonmyeloablative (n=24). Twenty-four pts experienced disease response after allo-SCT while 4 pts remained with progressive disease. Relapse occurred 51 to 574 days (median, 377) after allo-SCT in the 24 responders. Before the beginning of THAL, 15 pts had already received another course of salvage therapy including DLI (n=11), local radiotherapy (n=2), dexamethasone (n=1) and interferon (n=1), of whom only 4 pts could achieve a partial response. Thus, at time of onset of THAL therapy, 24 pts had a progressive disease. Twenty-four pts were evaluable for chronic GVHD, of whom 8 had an extensive form and 3 a limited form. THAL treatment was given at a median dose of 200 mg/d (50-600). At the reference date of July 01, 2003, the median follow-up from the start of THAL treatment was 301 d (7-1147). THAL was discontinued in 12 pts because of treatment failure (n=6), toxicity (n=4), exacerbation of chronic GVHD (n=1) and second allo-SCT (n=1). Overall, the median time of effective THAL therapy was 147+ d (7+ to 1147+). Responses were recorded in 14 of 25 evaluable pts (55%) as: nearly complete response (n=2) major response (n=4), partial response (n=3) and minor response (n=5). Nine pts progressed and 2 were still in stable disease. Three pts were excluded from response evaluation because of short follow-up less than 15 d. THAL had no benefical effect on chronic GVHD. The 2-year estimated survival from the onset of THAL was 46%. Ten pts died of disease progression (n=8) or transplant-related mortality (n=2). When looking for prognostic factors for survival or response, only an IgG MM subtype was found to adversely influence the response rate to THAL treatment (P=.04). Conclusion, although MM relapse after allo-SCT is of poor prognosis, THAL seems to be an effective and feasible option in this setting. Furthermore, our results compares favorably to what have been reported in refractory MM pts who did not receive allo-SCT in terms of response and survival rates. The small proportion of pts achieving a nearly complete response (8%) emphasizes the need for prospective trials using THAL in combination with other anti-MM drugs or DLI.

Allogeneic transplantation after non-myeloablative conditioning consolidation therapy following high-dose melphalan and autologous stem cell transplantation improve outcome in high-risk multiple myeloma M. Martino, G. Beltrami, M.T. Corsetti, G. Penna, G. Messina, F. Morabito, P. Iacopino, A. Carella, Bone Marrow Transplantation Unit (Reggio Calabria, San Giovanni Rotondo, I) To reduce the treatment-related mortality but retain the cytoreductive effect of high-dose chemotherapy as well as the graft-versus-myeloma effect in patients with MM, we designed a tandem transplantation program consisting of high-dose chemotherapy supported by autologous stem cell transplantation (auto-SCT) followed by a dose-reduced conditioning regimen with allogeneic stem cell transplantation (allo-SCT) to induce a GVM effect. We treated 26 pts, 12 males and 14 females, with a median age of 49 years (range 36-62). While 10 pts were enrolled after relapse from previously treatment (5/10 received previously single o double HDM with autoSCT), the tandem auto/allo program was used as upfront therapy in 16 pts. /Kg (range 1.2-10.0) and CSP/MTX was given to control graft rejection and GVHD. No myelosuppression and mucositis were observed and no pts received trasfusional support. Acute GVHD was diagnosed in 10 pts (5 grade I-II; 5 III-IV). Chronic GVHD was observed in 6 pts (1 extensive; 4 limited). Donor lymphocyte infusions were given to 9 pts either to attain full donor chimerism or to eradicate residual disease. Ten pts remain in continuous CR according to the EBMT criteria with negative immunofixation; 8 pts had a PR, most of those with still decreasing monoclonal band. With a median follow-up of 19 mo, 20 pts still alive while 6 pts have died because of aGvHD (1 pt.) or progressive disease (5 pts). The actuarial survival was 69.7+11% and the progression-free survival was 63.7+11%. Interestingly, the progression-free survival probability was 69.3+17 % in 16 pts who received more than 4.0 x106/Kg allogeneic CD34+ cells as compared to 48.0+16 % in 10 pts who received a minor allogeneic CD34+ cell dose. These data show that NST induces excellent disease control in MM, after reducing tumor burden with HDM and autologous stem cell support. Also, this combined autografting-allografting is well tolerated in older pts with a low TRM. GVHD is the single most serious complication.

Salvage of multiple myeloma patients relapse after an autologous stem cell transplantation, in the era of thalidomide and reduced-intensity allogeneic stem cell transplantation I. Hardan, A. Kneller, A. Shimoni, M. Berkowicz, A. Avigdor, M. Yeshurun, P. Raanani, Y. Davidowich, N. Shemtov, I. Ben Bassat, A. Nagler, Tel-Aviv University (Tel-Hashomer, IL) Autologous stem cell transplant (autoSCT) has become the gold standard therapy for young patients (pts) with multiple myeloma (MM). However this is not a curative approach and the prognosis for pts with post autoSCT progression is poor. Thalidomide (Thal), reduced intensity allogeneic SCT (RIC SCT), and a 2nd autoSCT are novel approaches in this setting. Methods: We applied the following treatment strategy for post autoSCT relapse : 1. Pts with aggressive relapse were given Thal ± Dexamethasone (Dexa), followed at response by RIC SCT with Mel/Flu/ATG or a 2nd autoSCT, depending on eligibility and donor availability. Pts not responding to Thal were given DTPACE or intermediate dose Mel (IDM) prior to SCT. 2. Pts with smoldering post autoSCT progression were followed with a watch and wait policy and Thal ± Dexa was administrated only upon a clinical indication. A second SCT procedure was offered in this group at escape from response (for pts with more than PR to Thal) or following DTPACE/ IDM for no responders to Thal. Results: Seventy-three pts (median age 56 y) have been treated according to this strategy. Twenty seven pts underwent RIC SCT (24 related, 3 unrelated donors), 10 pts had a 2nd autoSCT and 36 pts continued Thal therapy. The response rate (= or >PR) to Thal was 52%. Treatment related mortality was 8% [6 pts, all after RIC SCT, (22% in this group)]. With a median follow up of 34 months from progression: 1.The overall survival rate for the entire group is 60% (66%, 60% and 52% for continuing Thal, 2nd autoSCT, and RIC SCT groups, respectively). 2.

The progression free survival (PFS) rate is 52% for the entire group (53%, 52% and 50% for Thal, RIC SCT, and 2nd autoSCT groups). 3. In 19 patients the 2nd PFS period exceeded the first PFS period (from first autoSCT until progression). In a multivariant analysis, the duration of first PF period, response to Thal, and low risk score (b2MG, albumin level and performance status) at relapse were associated with improved prognosis. Availability of an HLA matched donor has a tendency to be a positive prognostic parameter but did not reach a statistical significance. Conclusions: The introduction of novel modalities of therapy in MM seems to significantly improve the ability to salvage pts relapsing after autoSCT. The exact timing and scheme of therapy that should be followed as well as the role of novel therapies such as proteosome inhibitors and new iMids in this setting should further be explored Introduction: In 1997 we started the new therapeutic strategy of the treatment of relapsing disease named "T2 model" in patients, who underwent retransplantation in the case of second/third relapse. Main principle of T 2 model is "the same treatment as during the first transplantation "+ "something more = experimental treatment". The aim is the stabilization of the disease for the period, which should be longer than before the first relapse. An engraftment, toxicity of myeloablative regimen and event free survival (EFS) were evaluated in patients in intraindividual analysis. Methods and patients (pts.): Total of 32 pts. mainly with first relapse during 24 months after first transplantation (18/32) underwent retransplantationn (median follow-up 67 months). High dose of dexamethasone reinduction therapy was followed by transplantation with identical myeloablative dose of melphalan (200 mg/m 2 ) as before. Toxicity of first and second transplantation was compared according SWOG criteria. The main experimental groups were: IL-2 (10 pts. received IL-2 activated graft followed by one month immunotherapy); thalidomide (15 pts. received maintenance thalidomide therapy 100mg daily if tolerated), 4 pts. received pamidronate instead of clodronate therapy and 3 patients received consolidation chemotherapy once in 3-4 months. EFS after first transplantation was compared with EFS after second one (EFS 1 & EFS 2). Results: Sensitivity to reinduction therapy was 90%. The engraftments of PBSC (100%) were similar after first and second transplantation (median of Leu>1x10 9 /L -12.0 vs. 11.5 days; median of platelets > 50x10 9 /L -13.0 vs.13.0 days). TRM was 3.7% for the second transplantation. All standard toxicity was very similar. Till 31.1.2003 total of 22 pts. already relapsed (69%) including 12 deaths (37%). Total of 8 pts. reached EFS 2 better than EFS 1 including 2 pts. in IL-2 group, 4 pts. in thalidomide group 1 pt. in pamidronate group and consolidation group. Median overall survival for all patients was not reached and 25 % percentil of OS is 50 months despite poor prognosis of MM patients with early relapse. Conclusion: The retransplantation procedure has similar engraftment and acceptable toxicity in patients with MM. Influence of retransplantation on the next EFS 2 and OS is encouraging especially in the group of pts. treated with thalidomide. We investigated in a retrospective multicenter study the impact of deletion 13q by fluorescence in situ hybridization (FISH) on outcome after dose-reduced allografting in patients with multiple myeloma. In 68 out of 140 patients data on deletion 13q detected by FISH were available: 37 patients were FISH negative and 31 FISH positive for deletion 13q14. Patients in both groups were well balanced regarding age, b2 microglobulin, relapse after a prior autograft, donor sex, related and unrelated donor, graft source, CD34+ cell dose, and chemosensitivity at time of transplantation. Patients with deletion 13 had a worse event-free survival and overall survival than patients without deletion 13 (18% vs 42%, p=0.05 and 22% vs 67% p=0.03, respectively). Patients with deletion 13 experienced a higher relapse rate (82% vs 58%, p=0.05), but similar incidence of treatment related mortality at one year (24 vs 18%). In the multivariate analysis, deletion 13q remained a significant risk factor for higher relapserate (HR 3.28; 95% CI: 1.31 8.24; p=0.01) and for event-free survival (HR 1.94; 95% CI: 1.03 3.67; p=0.04). For overall survival, two or more prior high-dose chemotherapies were associated with a significant higher probabilty of death (HR: 2.48 ; 95% CI: 1.19-5.17, p=0.02), while patients with deletion 13q had a nearly two times higher risk for death after treatment (HR: 1.94; 95% CI: 0.95-3.98, p=0.07) after dose-reduced allogeneic stem cell transplantation.

Multiple myeloma (MM) is the second most common hematological malignancies with an incidence of 4.1/100.000 per year. Disease control can be achieved by high-dose chemotherapy followed by autologous stem transplantation but relapse occurs in over 90% of all patients. Thus the development of novel concepts for eradication of minimal residual disease are highly warranted. Adoptive immunotherapy represent an attractive approach for augmenting tumor immunity and may be achieved by genetically modifying T-cells with an MM-specific chimeric immunoreceptor targeting the cell-surface antigen HM 1.24. In contrast to healthy plasma cells HM 1.24 is homogenously over expressed by primary MM cells and is not detected in other hematopoietic and nonhematopoietic tissues. An anti-HM 1.24 humanized single-chain immunoglobulin was de novo synthesized and fused with the CD3-z intracellular signaling domain. Primary CD4+ and CD8+ T-cells were genetically modified by retroviral mediated gene-transfer with the constructed receptor and Cr-release assays performed 10 days after transduction with unselected CD4+ and CD8+ T-cells. 5/5 MM cell lines were specifically lysed (up to 23%; E/T ratio 30:1) by transduced CD8+ T-cells. In comparison there was no specific lysis detected by the parental T-cells incubated with the MM cell lines, nor with the EBV transformed B-cells, nor K562 as targets that do not express HM 1.24. No specific lysis of MM cell lines was detected by transduced CD4+ T-cells. In contrast intracellular cytokine (ICC) staining with both transduced CD4+ and CD8+ T-cells showed robust IFN-g production upon stimulation with MM cell lines. In order to enrich for CD8+ T-cells able to lysis MM efficiently, transduced CD8+ T-cells were selected by the IFN-g secretion assay and expanded. Cr-release assays performed with selected CD8+ T-cells resulted in 54% specific lysis of MM cell lines at an E/T ratio of 5:1. To determine if primary MM cells can stimulated transduced T-cells, ICC staining was performed with T-cells incubated for 6 hours with primary MM cells. Both CD4+ and CD8+ T-cells showed IFN-g production which reached similar levels as ICC experiments with MM cell lines as stimulators. In summary, CD4+ and CD8+ Tcells can be render to be highly specific for MM cells by expression of a humanized anti-HM 1.24 chimeric immunoreceptors. Such T-ce ls can further be selected for functionality by the IFN-g secretion assay resulting in high specific lysis of MM cells. Despite the development of high dose chemotherapy, there has been little progress in improving the cure of patients with multiple myeloma (MM). This report describes the results of 41 MM patients who received an antithymocyte globulin, fludarabine and busulfan-based reduced intensity conditioning (RIC) for allogeneic stem cell transplantation (allo-SCT) from HLAidentical siblings. 29 patients (70%) were in PR, one patient in CR, and 11 (27%) in progressive disease at time of allo-SCT. 34 patients (83%) received at least one autologous transplantation in the course of their disease prior to allo-SCT, but this was not part of the allo-SCT strategy. Median time between allo-SCT and autologous transplantation was 4 (range, 2-57) months. Median time between MM initial diagnosis and allo-SCT was 24 (range, 6-146) months. The cumulative incidences of grade II-IV and grade III-IV acute graft-versus-host disease (GVHD) were 40% (95% CI, 24%-56%) and 8% (95% CI, 2%-22%) respectively. 10 patients developed limited chronic GVHD, whereas 7 developed an extensive form giving a cumulative incidence of chronic GVHD of 51% (95% CI, 34%-68%). With a median follow-up of 389 days, the overall cumulative incidence of transplant-related mortality (TRM) was 17% (95% CI, 6%-28%). In addition to the one patient who was in CR at time of allo-SCT, 21 other patients (53%; 95% CI, 38%-68%) could achieve an objective disease response during the follow-up period (complete remission, n=10; partial remission, n=11). 2 patients remained in stable disease, 3 patients were not assessable for disease response because of early death, and 14 patients (34%; 95% CI, 20%-48%) did not have any disease response and progressed after allo-SCT. Among the patients who survived beyond day 100 after allo-SCT and who had an objective disease response, 13 (65%) experienced limited or extensive chronic GVHD as compared to only 4 (31%) in the rest of the series (P=.05). The Kaplan-Meier estimate of overall survival (OS) at 2 years was 62% (95% CI, 47%-76%). Progression-free survival (PFS) and OS were respectively significantly higher in patients with chronic GVHD as compared to patients without chronic GVHD (P= .005 for PFS and P=.01 for OS). Collectively, these data demonstrate that RIC allo-SCT can mediate a potentially curative graft-versus myeloma effect with a low rate of toxicity and TRM. Background: Ultimately all patients relapse after high-dose therapy (HDT) supported by single autologous stem cell transplantation (ASCT). Double autografting may improve survival. This study is a nonrandomised comparison between single and double up-front ASCT in patients with newly diagnosed myeloma. Patients and methods: 101 consecutive patients received HDT and ASCT at our institution between years 1992 and 2003. Since 1992 patients <71 years underwent single autografting (n=74) and since 1996 patients <61 years double autografting (n=27). Initially patients received conventional chemotherapy, typically VAD, before mobilisation with 2 g/m² cyclophosphamide + G-CSF. HDT was MEL140 mg/m² + TBI (n=21) or MEL200 mg/m² once or twice (n=80). Blood graft was used in the majority of patients. Post-transplant G-CSF was used. The groups were comparable, but the double ASCT group tended to have more light chain and more stage III disease. The EBMT response criteria were used together with additional VGPR defined as otherwise CR but immunofixation positive. Kaplan Meier curves for OS and PFS were produced, and significance tested with log-rank test. Results: Responses to single and double ASCT were: CR 40.5 and 52 %, VGPR 31 and 18.5 %, and PR 23 and 30 %, resp. Median follow-up times of living patients are 62 and 46 months for the single (n=41) and double (n=21) ASCT, resp. Median OS for the single ASCT group is 60 months, and has not been reached in the double ASCT group (P=0.077). OS at 5 years for the two groups are 50 and 70 %, resp. OS was similar whether CR or VGPR was achieved after ASCT. Median PFS is 24 months for the single and has not been reached in the double ASCT group (P=0.098). PFS at 5 years are 40 and 62 %, resp. There were no differences in toxicity or engraftment after the first and second autografting. Transplant-related mortality was 1 % in the single and 0 in the double ASCT group. Conclusion: Both single and double ASCT results in CR+VGPR rate of 70 % but CR rate tends to be higher with double ASCT. Double ASCT suggests to improve both OS and PFS when compared to single ASCT but the difference is not significant, possibly due to the low number of double ASCTs. Double autografting is very well tolerated.

High-dose chemotherapy (HDT) improves the outcome of patients with multiple Myeloma (MM) in comparison to conventional chemotherapy. Dose-escalating strategies including tandem HDT are currently evaluated to further improve remission rates and survival of patients. Therefore we conducted a randomized multicenter trial to compare an intensified conditioning regimen with the current standard high-dose melphalan. Between 1997 and 1999 a total of 56 patients with stage II and III disease, who were matched for age (median 56 years), number of previous therapies (median time from diagnosis to transplant 7 months) and different risk factors (beta2-microglobulin, LDH, CRP, cytogentic abnormalities), were randomized. All patients received 2 courses of oral idarubicine (40mg/m 2 ) and dexamethasone and 2 courses of intravenous cyclophosphamide (2g/m 2 ) and adriamycine (50mg/m2) in combination with G-CSF followed by peripheral stem cell collection. Thirty patients were treated with melphalan 200mg/m 2 (HD-M) whereas 26 patients received idarubicine 42mg/m 2 , melphalan 200mg/m 2 and cyclophosphamide 120mg/kg (HD-IMC) followed by autologous blood stem cell transplantation. Acute toxicity was higher with HD-IMC, including 5 (20%) treatment-related deaths due to infections versus none (0%) in the HD-M group. This lead to early termination of the study. Severity of mucositis (grade IV 19 versus 8 pts., p=0.001), CRP (20 versus 7 mg/dl, p<0.001), days of fever (11 versus 3, p<0.001), days with iv-antibiotics (13 versus 4, p<0.001), number of erythrocyte-(6 versus 2, p=0.0001) and platelet-transfusions (16 versus 4, p=0.0003) were significantly higher after HD-IMC. Although response rate (CR: 31% versus 7% and PR 75% versus 52%) and time-to-progression (median 30 versus 15 months) were better for HD-IMC in comparison to HD-M, there was no significant difference in overall survival (median 50 months) after a median follow-up of 50 months. In conclusion intensified conditioning before autologous stem cell transplantation had an intolerable high treatment-related mortality and did not improve overall survival in patients with multiple myeloma. 

We have previously shown that in "planned" double transplantation the median survival is superior (60 v 51mo; H.R = 0.89, CI 0.79-1.00, p =0.05) to patients not planned to receive a second transplant despite < 60% of the planned patients receiving their second transplant. It should be noted this survival advantage is reversed after 70mo (H.R 3.01, CI 1.07-8.46, p = 0.04). We have now used a multi state approach to further analyse relapse and transplant related mortality (separately) in relation to second transplantation and also survival after relapse in order to determine the role of time to second transplantation. Thus patients who have had one transplant may be either alive and relapse free, or relapsed or dead and may or may not have had a second transplant before or after relapse. This approach utilises Cox models with time-dependent covariates for the occurrence of: second transplantation (and time to it), corrected for known risk factors. The results have been summarised into ''transition probabilities'' which can be represented graphically. This approach suggests the advantage for tandem transplantation is lost if the second transplant is performed more than two years from the first and that patients having a second transplant within six months of the first, have the best prognosis. However, it should be stressed that this is an observational study and thus provides a description of what was observed in the population but cannot be used to draw causal conclusions as the factors leading to early and late transplantation are unknown.

Aims: The median survival of patients (pts) with systemis AL amyloidosis (AL) without therapy is 13 months. Autologous stem cell transplantation (ASCT) after high dose melphalan (HD-M) chemotherapy has been reported to be effective in pts with AL. We treated n=28 pts with AL amyloidosis in order to evaluate efficacy and toxicity of the therapeutic regimen combining VAD chemotherapy (vincristine/adriamycin/dexamethason) with highdose melphalan followed by ASCT. Methods: From May 1998 to November 2002 n=28 pts (19 male, 9 female) with biopsy-proven systemic AL amyloidosis and main involvement of heart (n=5), kidney (n=13), liver or intestinum (n=6) and other organs (n=4) were initially treated with 1-5 cycles of VAD (median 3 cycles) in order to reduce the plasma cell load. Stem cells were mobilized in n=24 pts with G-CSF after 4 g/m² cyclophosphamide (n=4), or CAD (1 g/m² cyclophosphamide, 15 mg/m² adrimycin and dexamethasone; n=7) or 12 g/m² ifosfamide (n=13). High dose melphalan was applied to n=24 pts. Melphalan dose was reduced according to renal function: n=15 received 200 mg/m², n=3 -140 mg/m², n=6 -100 mg/m². Results: N=4 (14%) pts died prior to stem cell mobilization: n=2 due to disease progress, n=2 due to VAD toxicity. Stem cells could be mobilized successfully in all n=24 pts who received mobilizing chemotherapy and G-CSF. N=3 pts (12%) succumbed before day +100 after ASCT. Causes of death were: cerebral hemorrhagy on day +9, neutropenic sepsis on day +10 and intestinal hemorrhagy on day +48. A further patient died 10 months after ASCT due to progressive heart failure. The median follow up of n=20 surviving pts is 29 months (12 -66 months). Remission rates of clonal plasma cell disease are: n=7 CR, n=3 PR, n=5 SD, n=5 not available. Remission rates of organ function are: n=14 improved, n=4 stable, n=2 slow progress. Conclusions: High dose melphalan followed by ASCT after initial VAD chemotherapy is feasible in pts with systemic ALamyloidosis, but the toxicity is high: VAD toxicity -7% deaths, 100-days TRM after HD-M and ASCT -12%. Haematological response and remission of clonal plasma cell disease was achieved in 50% patients surviving after therapy and seems to be essential for clinical improvement of organ function.

Although the conventional allogeneic stem cell transplantation is capable of producing remission and long-term survival for patients with multiple myeloma (MM), this procedure has been limited by the substantial transplant-related mortality. Reduced intensity conditioning allogeneic stem cell transplantation (RIST) is a new procedure for its feasibility and lower mortality. However, the best conditioning regimen and its efficacy in MM still remains unclear. To investigate the status of RIST for MM in Japan, we conducted a retrospective survey of Japanese MM patients who underwent RIST at 11 hospitals participating in Japan Myeloma Study Group (JMSG). Total 45 patients were included in this study. They were 23 males and 22 females, with median age of 53 years (range 39 to 63). Eighteen patients previously received autologous stem cell transplantation. Conditioning regimen consisted of fludarabine-based regimen in 24 patients, and TBI (1-2 Gy)-based regimen in 18 patients. Thirty-five patients received peripheral blood stem cells (PBSC) and nine patients received bone marrow (BM) and one patient received both. GVHD prophylaxis consisted of cyclosporine in 28 patients and tacrolimus in 17 patients. Complete chimerism was obtained in 37 of 41 evaluable patients. Twenty-eight of 42 (67%) evaluable patients developed grade II -IV acute graft-vs-host disease (aGVHD), and eight patients (19%) developed grade III -IV aGVHD. Chronic GVHD developed in 26 of 35 (74%) evaluable patients (nine in limited and 15 in extensive type). Treatment-related toxicities of more than grade III were as follows: liver in six, gastrointestinal system in three, kidney in three, cardiovascular system in three, lung in three and skin in two patients. CMV infection including antigenemia was observed in 19 patients. Complete remission was obtained in 19 of 44 (43%) evaluable patients. Early death within 6 months was observed in 7 of 38 (18%) evaluable patients. To date, 31 patients are alive with 10 patients being in complete remission with a median follow-up of 18.2 months (range 2 -40). These observations suggest that RIST is feasible with reliable donor engraftment and relatively low TRM. However, GVHD and disease recurrence particularly in patients with relapsed or refractory disease are the major obstacles to overcome. Further studies are required to establish the role of RIST in the treatment strategy for MM.

High dose chemotherapy (HDT) with autologous stem cell transplantation (SCT) has proven superior to conventional therapy in patients with multiple myeloma under the age of 70 years. HDT improves both overall survival (OS) and event free survival (EFS). Since median EFS is no longer than 35 months even with tandem autotransplant, relapse is common after intensive pretreatment. Thus, novel therapeutic modalities are warranted. Myelosuppression has to be considered as the critical toxicity after prior SCT. Bendamustine hydrochloride is a bifunctional alkylating agent that has been used alone or in combination regimens for the treatment of a broad range of cancers. In patients with multiple myeloma significant more complete responses (CR) were obtained studying bedamustine/prednisone compared to melphalan/prednisone. Here we present a dose-finding phase I/II study of bendamustine in patients relapsing from single or tandem autotransplantation. Patients up to the age of 70 years with a white blood count > 3.0 x 10 9 /l and a platelet count of > 100 x 10 9 /l were eligible if serum creatinine was < 2 x upper limit of normal (ULN) and serum bilirubin and transaminase levels < 3 x ULN. Bendamustine was administered intravenously on 2 consecutive days over 30 min. and repeated in the same dosage every 4 weeks for a minimum of 2 and a maximum of 6 cycles. 28 patients (pts.) were included: 5 patients received 2 x 60 mg/m 2 (2 -6 cycles); 8 patients 2 x 70 mg/m 2 (1-5 cycles); 6 patients 2 x 80 mg/m 2 (2 -4 cycles); 6 patients 2 x 90 mg/m 2 (1 -4 cycles) and, up to now, 3 patients were started on 2 x 100 mg/m². Currently 26 patients are evaluable for toxicity and response. Neither patient developed dose-limiting hematotoxicity as defined by an absolute neutrophil count (ANC) < 0.5 x 10 9 /l for > 7 days or an ANC < 0.1 x 10 9 /l for > 3 days or a platelet count < 25 x 10 9 /l. One patient who stopped taking PCP prophylaxis developed pneumocystis carinii pneumonitis, no other serious infectious complications were observed. 12 (46%) patients responded (MR or PR), whereas 14 patients either showed stable or progressive disease, respectively. Thus, bendamustine hydrochloride in dosages up to 2 x 100 mg/m² every 4 weeks seems to be very well tolerated even in heavily pretreated patients after HDT. We will aim at an improvement of the efficacy of bendamustine (RR 46%) if applied in even higher dosages in relapse after autologous SCT. 

Thalidomide has proven to be effective in antimyeloma treatment.

It inhibits angiogenesis and has an immunomodulatory effect. The aim of this study was to assess the effectiveness of mobilization and collection of progenitor stem cells of haematopoiesis as well as haematological recovery following autologous transplantation of progenitor cells in patients with resistant multiple myeloma, effectively treated with thalidomide. Material and Methods: Autologous transplantation (PBSCT) was conducted in 10 patients treated in our Department for multiple S161 myeloma resistant to standard chemotherapy. 7 males and 3 females aged at average 55 (range 42-64) were treated. The patients had the following types of MM: 4 IgG kappa, 2 IgG lambda, 2 IgA kappa, 1 IgA lambda and 1 IgD lambda. All the patients had reached the IIIA clinical stage according to Durie Salmon. Positive response to treatment with Thalidomide (at least PR according to SWOG) was achieved in all patients. The initial dose of 200mg/d of thalidomide was increased to 400mg/d after 4 weeks of treatment. Administration of thalidomide was combined with Dexamethazone at a dose of 40mg/d for 4 days every 28 days. After a period of between 8-24 weeks of treatment with thalidomide (approximately 17 weeks), when best results were achieved, patients were qualified for stem cells mobilization in order that autologous transplantation could be conducted. Cyclophosphamide at a dose of 4g/m2 and G-CSF (Neupogen) at a dose of 5 mgm/kg/day were employed in the course of mobilization. Results: Mobilization was successful in all cases, 2-3 cytaphereses were conducted at average. The following results were obtained: 2,74x10 8 /kg MNC (1, 21) , 4,45x10 6 /kg CD34+ cells/kg (1, 3) , 30,85x10 4 /kg CFU-GM cells/kg. Regeneration of granulopoiesis and platelets was achieved in all patients; median time to ANC>0,5G/l was 18 days, PLT>50G/l was 17 days, respectively. The observation time following autoPBSCT is 10 months (4-18). Complete remission was achieved in 3 patients and partial remission in 7 patients. Relapse occurred in 2 cases, respectively 8 and 12 months after transplantation. Conclusions: Mobilization of progenitor stem cells, with thalidomide and Dexamethazone is possible in resistant types of multiple myeloma. The effects of mobilization and haematological recovery in patients pretreated with thalidomide and those treated with conventional chemotherapy are comparable.

A. Trojan, R. Giger, N. Rist, D. Jäger, C. Taverna, University Hospital (Zurich, CH) CD8+ T effector cells play a crucial role in the immune response to virally infected cells. Thalidomide can act as a T-cell costimulator resulting in enhanced cytokine production (IL-2 and IFN-y) of virus-specific CD8+ T cells and their cytotoxic activity. Both expansion of virus-specific CD8+ T cells and an increase of IFN-y in these cells demonstrated a CMV and Influenza specific T-cell response. Probably the antigen specifc TCR stimulation upregulates T cell activation signals thereby mounting also the production of IL-2 and IL-12. We have tested the effect of Thalidomide in blood of 5 healthy HLA-*A0201+ volunteers using quantitative PCR (qPCR). In vitro cultures of PBMCs were pulsed with Influenza peptide GILGFVFTL (6 microgramm/ml each for 2h, in the presence of Beta2-microglobulin) and taken in culture with therapeutic concentrations of Thalidomide (100 micro-gramm/ml). On day 7, cells were restimulated with Influenza peptide and tested for IFN-y ranscription. After 2h peptide pulsing, total RNA was extracted and transcribed into cDNA; IFN-a gene expression was measured by ABI Prism 7700 Sequence Detection System (Applied Biosystems, USA). Since stimulation with HLA class I-restricted epitope defines CD8+ T cells as the only relevant population, IFN-y mRNA copy numbers were corrected for CD8 mRNA copies from the same sample creating a ratio that reflects the status of CD8+ T cell activation. Our results demonstrate that T cell activation (indicated as IFN-ã /CD8+ ratios) upon Influenza peptide stimulation exhibited in average 72% of the activity that was obtained from concomitantly cultured PBMCs using the standard IL-7 and IL-2 expansion procedure. Multiple myeloma (MM), either in first line therapy or in conditioning regimes for trans-plantation, represents one condition in which there is evidence of clinical benefit from Thalidomide. We therefore tested the effect of this drug on T-cell reactivity in one patient with MM. After in vitro peptide stimulation of PBMCs cultured with Thalidomide (as outlined above) qPCR results revealed a significant activity of CD8+ T cells comparable with that observed in volunteers. However, in vivo reactivity after two weeks of peroral thalidomide intake (200 mg/day; approx. 40 microgramm/ml), according to INF-y/CD8 ratios, revealed an impared but measurable activity. Further studies will have to explore whether Thalidomide can mount an effective anti-antigen specific T cell response in therapeutical settings. The ratio was abnormal in 20 (18%) patients. These data suggest that even though the M protein or the paraprotein is undetectable by immunofixation, serum FLC remain abnormal in some patients. There was no correlation between abnormal ratio and high levels of light chains, 8/30 (26.7%) patients with either high kappa or lambda FLC had an abnormal ratio vs 12/77 (15.6%) with both normal light chains (P= 0.2). Most patients with raised FLC had normal kappa/lambda ratios with elevated levels of both kappa and lambda. This was not associated with increased risk of relapse and is probably a result of a disordered immune reconstitution. However, abnormal ratios significantly predicted relapse (see table) and may be a sensitive marker of minimal residual disease. Prospective longitudinal studies are required to confirm and further investigate this potentially important observation.

Introduction: The curability of multiple myeloma is only possible by means of allogeneic transplant, which is a procedure available just for a few patients and criticized because of its high mortality. After our group preliminary experience in follicular lymphoma (Haematologica 2002; 87:400-407), we have started a program of idiotypic vaccination after autologous peripheral blood stem cell transplantation (PBSCT) with dendritic cells pulsed with the purified paraprotein from patients with myeloma. Here we communicate our initial experience in the selection and vaccination of patients. Patients and methods: We choose patients who were diagnosed of myeloma and who were suitable for PBSCT. We isolate the paraprotein from a sample at diagnosis and after treatment with VAD courses, we perform the mobilization and collection of PBSC, obtaining a purified fraction of CD34+ in order to generate dendritic cells. After reevaluation at +3 months post-PBSCT, the patients in minimal residual disease state began the vaccination program: 3 subcutaneous doses of dendritic cells pulsed with the purified paraprotein every two weeks, 5 sc doses of paraprotein + KLH + GM-CSF monthly, 1 boost dose of pulsed dendritic cells six months after the beginning of vaccination and a final boost x 2 doses of paraprotein + KLH + GM-CSF. Results: We have evaluated 23 patients suitable for the vaccination program. PBSCT could not be performed in 4 cases due to previous complications, and 7 are still receiving chemotherapy treatment. PBSCT was performed in 12 patients: 1 died of refractory disease, 2 are awaiting for reevaluation, 3 have not reached enough response and 7 cases have begun the vaccination program. Four patients have completed the vaccination program without any local or systemic adverse effects. So far, we have generated enough dendritic cells in all of them: 21.4, 91.5, 49.79 and 4.94 x10 6 CD1a+ cells have been administered respectively. In the 4 cases, humoral and cellular anti-KLH responses without any specific anti-idyotipic response have been induced. Conclusion: The vaccination treatment seems to be feasible: the in vitro clinical scale dendritic cells generation has been optimal, is still too early to establish its clinical utility and it does not seems to cause significant toxicity. (Supported by grant FIS 01/0913).

Aim: In an ongoing randomized prospective study we are comparing the efficacy of neridronate and zoledronate in reducing myeloma-related skeletal morbidity in patients undergoing autologous stem cell transplantation. Patients and study design: The study group presently includes 14 patients (7 males and 7 females; age range 53-74 yrs) with multiple myeloma. Type of myeloma: IgG (n 5), IgA (n 5), light chain (n 4). Clinical staging: IA (n 2), IIA (n 5), IIIA (n 5), IIIB (n 2). When enrolled in the study, all patients had evidence of osteolytic lesions/pathologic fractures and/or secondary osteopenia (-2.5 S.D. < T-Score < -1 S.D.) or osteoporosis (T-Score < -2.5 S.D.), in the absence of metabolic bone disorders. Patients received a double (n 10) or a single (n 4) autologous peripheral cell transplantation. Patients were randomly allocated to receive monthly zoledronate (4 mg via 15-min infusion) or neridronate (100 mg via 150-min infusion). Bone Mineral Density (BMD) with Dual energy X-ray Absorptiometry (DEXA) has been assessed at baseline and after a 6-month follow-up. Serum calcium, phosphor and alkaline phosphatase levels have been assessed monthly. Further evaluation of BMD and osteolytic lesions/pathologic fractures will be performed after a 12-month follow-up. In the two groups, there are not major differences with respect to mean age, performance status, disease type and stage, median time from diagnosis, renal functioning, severity of bone involvement and extent of antineoplastic therapy. Results: Here we show our data regarding lumbar spine (L1-L3) and hip BMD % change after six months of therapy. Neridronate: Mean BMD % change, Spine (±SD): 2.8 (1.69); Mean BMD % change, Hip (±SD): 2.07 (2.86). Zoledronate: Mean BMD % change, Spine (±SD): -0.78 (7.86); Mean BMD % change, Hip (±SD): 0.48 (4.04). Conclusions: Our results suggest that, over a 6-month treatment period, spine and hip BMD % changes using intravenous neridronate are comparable to those observed using intravenous zoledronate.

In a retrospective analysis 27 patients with first relapse of multiple myeloma after high-dose chemotherapy (HDT) and autologous stem cell transplantation (SCT) were treated with single agent thalidomide. Patients had stage III disease, median age was 56 years, 74% had IgG subtype, median ß2microglobulin level was 2,43 mg/L (range: 1,5-32,6). HDT consisted of either single melphalan (n=18) or melphalan, cyclophosphamid and idarubicin (n=9). 20 Patients received inteferon and 7 patients no maintenance therapy. After relapse, thalidomide was given in a median daily dose of 200 mg (range: 100-400). The median follow up was 63 months (range: 23-137). During treatment with thalidomide WHO grade 1-2 toxicities were observed in 74% of patients whereas no grade 3-4 toxicities were seen. PNP (58%), constipation (39%) and fatigue (13%) were the most common adverse events. Response rates were as follows: 54% PR, 3% vgPR, 10% MR, 10% stable disease and 23% progressive disease. The overall response rate (57%) to single agent thalidomide in our homogenous group of patients with first relapse after HDT was higher than reported for relapsed and refractory patients who had more advanced disease. In our patients median EFS and OS from the beginning of thalidomide treatment was 12 and 88 months, respectively. Univariate analysis was performed to identify prognostic factors for a better outcome of thalidomide treatment. Factors analysed were age, ß2-microglobulin level, myeloma subtype, type of conditioning high-dose regimens, response to HDT, duration of remission after SCT, application of interferon maintenance therapy and thalidomide dosage. The only predictive factor for a better EFS and OS after thalidomide treatment was the duration of remission after HDT irrespectively of kind of remission achieved. Patients who had achieved any remission after HDT lasting for more than 12 months had a median EFS of 26 months whereas patients whose remission after HDT lasted less than 12 months had a median EFS of 5 months (p=0,03). In conclusion, treatment with single agent thalidomide is well tolerated and effective in patients with first relapse after HDT and autologous SCT. The duration of remission after prior HDT is predictive for EFS and OS after thalidomide therapy indicating that tumor clones with high chemosensitivity also display a high susceptibility to immunmodulatory effects.

Starting from 1996, first line therapy for our patients aged less than 70 affected by advanced stage multiple myeloma (Salmon II or III) includes three DAV courses (adriamycin 50 mg/sqm and vincristine 1 mg. total dose in single bolus infusion on day 1 and dexamethasone 40 mg. days 1 to 4) followed by high dose melphalan (140 or 200 mg/sqm) and one or two autologous stem cells transplantations (ASCT). Seventy-four such pts have been diagnosed in the period 1996-2002 ("cases"); fifty-three of them have received at least one ASCT; four did not receive this therapy due to comorbidity; nine were included in the program but they did not reach ASCT due to progression, toxicity of DAV courses or refractory disease; eight received MP as first line therapy because they were included in a randomized collaborative trial. A control group was created with 74 patients diagnosed in the years 1989 -1995 ("controls") matched for stage and age (+-3 years); in this group 5 pts were initially treated with VAD-like courses, 32 with melphalan and prednisone, 35 with polichemotherapy (with two or more alkylating agents, without anthracyclines) 2 pts died before beginning chemotherapy. No differences were present between the two groups as to bone marrow plasma cell (mean 48.9 % and 47.9%), classes (IgG: 49 and 46; IgA 16 and 15; IgD 0 and 2; BJ 7 and 9; NS 2 and 2), monoclonal component among IgG and IgA myeloma (mean 4.37 and 4.59 g/l). Results: survival has been analyzed on an "intention to treat" basis; overall survival is significantly better for "cases" than for "controls" (median survival not yet reached vs. 39 months) (p=0.03). Statistically significant difference among "cases" and "controls" is maintained in Salmon stage II (p=0.03); among pts in stage III, the two curves are similar up to twenty months; after this time the survival of "cases" becomes better. Conclusions: our case-control study in advanced stage multiple myeloma patients shows a survival benefit of ASCT when compared to conventional therapy.

Autologous Transplantation is recognised as the standard of care for younger patients with myeloma. Clinical trials do not represent fully the spectrum of patients who present with myeloma in this age group. The aim of this survey was to identify the number of patients that failed to receive a transplant, the reasons for this, what proportion of these patients might in different circumstances have been transplanted and to identify if there was unmet need for transplantation. From 2000 to December 2001, all patients with myeloma were identified in two regions (a) in Northern Ireland and (b) Northern Region (England) using the well established haematological malignancy data registries in these regions. A one-page questionnaire was completed for each of the patients under 65 years, who had not received peripheral blood stem cell transplantation. We found that only one third of patients were aged under 65 years and that 45% of these patients had received a transplant by December 2003. Co-morbid medical conditions (30%), early death (15%), refractory disease (12%), patient decision (11%) and failed mobilisation (11%), were the main reasons for non-transplantation, although there were variations between the two regions. The transplant rate of the NRHG (5.7 per million) and NIRHG (6.3 per million) was found to be lower than the UK as a whole using the EBMT activity survey for myeloma transplantation. The decreased transplant rate in these regions may be related to the overall decrease in the health status of these economically deprived areas, resulting in higher co-morbidity rates than the country as a whole. Nevertheless, the figures show less than 20% of all patients with myeloma will have peripheral blood stem cell transplantation. Multiple Myeloma (MM), is a hematological malign disease which can be cured by conventional chemotherapy (CT) only rarely. Although prospective randomized trials have proven that better and durable remissions are achived following single or double trnasplants, they lack certain aspects i.e they do not involve the outcome of ineligible patients. A more realistic approach is the evaluation of the results from a center where both CT and stem cell transplantation(SCT) can be performed. All 145 MM patients admitted to our center between 1991-2002 were analysed (F/M:55/90; Age: 56 (28-82); IgG / IgA / IgM / light chain / triclonal / other Ig / plasmacytoma; 88 / 28 /1 / 2 / 2 / 12 / 20; stage I / II / III ; 18 / 17 / 84 in 119 patients of whom the presentation data were avaliable. Risk groups; low (50.8%), intermediate (34.6%) and high (14.6%) were determined according to beta 2 microglobulin (3.5>,<),Albumin(3 >,<) and LDH (normal >,<) values in 75 patients. High dose therapy (HDT) was given to 31 ( auto: 26, allo: 3, tandem auto-allo: 2) patients (21.4 %). Clinical stage, sex and age was similar between HDT (+) versus (-) (p=0.46). Among the prognostic risk assesment avaliable patients 11 received HDT versus 32 patients who did not. Low risk patients constituted the HDT group whereas 31% of the HDT (-) patients were low risk (p= 0.004). When we analyzed only the low risk patients HDT did not improve the overall survival(OS) (p=0.1). Response evaluation to initial therapy was applicable in 68 of the patients(HDT:28,CT:40). According to response evaluation based on EBMT criteria refractorines before HDT was 32.2%. The ratio of complete response (CR) increased from 15% to 53%, and the overall response rate increased up to 87% following HDT. Estimated 3 year OS was 86% in HDT patients compared to 53% in the HDT (-) (p=0.008) in the cohort of patients with follow up (n=83). Fourty three (high (12%) / intermediate (37%) and low (51%)risk) patients of whom risk group was available estimated 3 year OS independent from HDT was 80%/27%/76%, respectively (P=0.016). In conclusion, there has been a tendency to perform SCT in low risk patients in our center. Regardless of prognostic factors, HDT compared to CT, increased CR and resulted with a survival advantage in MM.

The use of 166Ho-DOTMP followed by autologous stem cell transplantation (ASCT) has been reported in patients with multiple myeloma (MM). In MM, radiotherapy plays an important role both in ASCT and palliation of bone lesions. Application of bone-seeking radioisotopes has proven effective in treatment of osteolytic lesions. Malignant plasma cells in myeloma are located predominantely in areas of bone resorption. Thus targeting of bone lesions may be a suitable approach in high-dose therapy. When compared to 166Ho-DOTMP, 153 Samarium-EDTMP has some advantages: it is easier accessible in terms of production and logistics; has a longer physical half-life and shows higher skeletal retention. We treated two patients with refractory MM with myeloablative dosages of 153 Samarium-EDTMP up to 60 GBq. Patient 1 relapsed from CR 39 months and patient 2 had symptomatic relapse 9 months, each after one course of highdose melphalan. Both patients were refractory to 2 salvage chemotherapy regimens. They received 153 Samarium-EDTMP with a bone marrow dose of 28 (patient 1) and 35 Gy respectively. Doses to bone marrow were derived from scan data and the results of blood sampling and urine collection over 48 hours. Melphalan 140 mg/m² was given on days -3 and -2. Hemorrhagic cystitis was successfully prevented by continuous bladder irrigation. They experienced myeloablation already prior to high-dose melphalan and received ASCT 17 (patient 1) and 12 days after application of 153 Samarium-EDTMP respectively. During aplasia, the first patient developed pulmonary infiltrates (possible invasive aspergillosis) and unfortunately died on day +8 due to cerebral hemorrhage. The second patient experienced reversible renal failure and WHO °III liver toxicity. Following neutrophil engraftment on day +13, he recovered completely. The patient achieved partial remission for 6 months. Even though experience with application of 153 Samarium-EDTMP and highdose melphalan prior to ASCT is limited, we suppose it is an attractive treatment option in MM. The most important advantage of bone-seeking radiopharmaceutics when compared to external beam radiation is optimal lesion specific delivery, thus enabling the application of higher sceletal dosages. Distribution kinetics are required for each given patient to avoid toxicity to kidney, lung and liver. Toxicity of bone-seeking radionuclides is probably higher in extensively pretreated patients especially with regard to renal dysfunction. (Kuopio, Oulu, Tampere, Turku, Helsinki, FIN) Due to poor prognosis with conventional therapy, ASCT has been suggested for treatment of patients with AL. Only single centre series are available on the feasibility and efficacy of this approach. Altogether 20 AL patients (11 M, 9 F) with a median age of 54 years were included in HDT programs in five Finnish centres 1997-2003. Twelve patients were mobilised with G-CSF and eight patients with a combination of cyclophosphamide (CY) and G-CSF. Three patients (25 %) mobilised with G-CSF failed the minimum collection target (2 x 10 6 /kg CD34+ cells). The peak B-CD34+ counts were higher in patients mobilised with CY+G-CSF (76 vs. 38 x 10 6 /l) as well as the number of CD34+ cells collected (9.6 vs. 4.3 x 10 6 /kg). Sixteen patients received high-dose melphalan followed by PBSCT plus G-CSF. Four patients (25 %) died < 100 d from transplant-related reasons. Ten patients are evaluable for treatment response. Nine patients showed improvement or stable disease status after ASCT. With a median follow-up of 13 months from ASCT, the overall survival is 69 %. With a median follow-up of 26 months for the living patients after ASCT, two patients (18 %) have shown disease progression. In multicentre setting, TRM seems to be comparable to single centre series in this patient group. ASCT seems to be feasible in patients with less advanced AL giving stable responses in the majority of the patients.

Aiming at increasing allogeneic possibilities in multiple myeloma patients

Multiple Myeloma (MM) remains a worrying disease: rarely eradicable, with painful complications and often rapidly invaliding. Moreover median age onset sometimes makes intensive treatment hazardous. Up to day "cure" is obtainable only with allogeneic transplantation whose Transplant Related Mortality (TRM) has been recently reported as decreased . A tailored program is required and personal priorities of a well informed patient are to be taken into consideration. New roads which include new drug classes could also be searched. We report the policy (oriented in allogeneic direction) and the experience of our BMT Unit in MM pts who underwent allotransplantation (allo-tx). Materials: -Chemosensitive pts, younger than 55 yrs, are offered an allo-tx if a related or fully matched unrelated donor is available ( performed in 9 pts). -Patients with refractory disease with donor are revaluated for allo-tx after a "debulking" autotx ( performed in 2 pts).

-More recently advanced disease pts successfuly rescued by Thalidomide are also evaluated for allotx ( 1 tranplant performed) or autotx and pts older than 55 yrs. are offered a Reduced Intensity Conditioning procedure. Relapses after autotx are also considered.

-If no donor, a tandem double autotx is scheduled (performed in 67pts) Results: 12 pts (11 related, 1 unrelated donor) underwent conventional allotx (conditioning regimen CY-TBI, CSPA and MTX as GVHD prophylaxis). Status at transplantation: CR 3, PR 8 and stable disease 1. No early or late TRM was observed. Five pts (45%) experienced ac GVHD (grade II in all cases), which evolved in chronic in two of them. Responses to allotx were CR in 8 pts, PR in 3 pts; one patient did not show any change, progressed eight months after tx and died two months later. One patient ( transplantated in CR), relapsed after 20 m. is still alive 30 m. post-tx. At a median follow-up of 32 months (range: 6-101) we recorded: TRM 0%; OS 92% ; EFS 84%; Karnofsky >90% . Conclusions: acceptable TRM, good results in term of survival and quality of life, prompted us to extend the possibility of allotransplantation in MM to pts more advanced in age or in disease status. A tailored flow-chart can offer allogeneic procedure advantage to more MM pts given a careful evaluation of risks and an adapted treatment schedule.

Despite high-dose chemotherapy with stem cell support has extended event-free and overall survival in multiple myeloma patients, relapses still occur and the available options for a salvage therapy are limited. Our team investigated the combination Thalidomide, Doxil and Dexamethasone, named ThaDD, in order to asses its efficacy and feasibility. We enrolled 7 patients so far, 4 males and 3 females, relapsed after tandem high dose chemotherapy with stem cell transplantation. Among them, 3 patients received 5 lines of prior chemotherapy at least. All patients had advanced multiple myeloma: median age was 65 years (range 58-73); 3 had poor performance status and bone pain while 2 had extramedullary disease. Patients received 4 courses of ThaDD at least (4-6): none interrupted or decreased the dosages of Doxil and/or Thalidomide, while 3 of them delayed the administration of chemotherapy because of fever (2 patients) or severe neutropenia (1 patient). Two out of 7 patients (28,6%) achieved a complete response (absent M band), 2 had a partial response (>50% reduction in M band) and 1 had a minor response (25-50% reduction in M band). Therefore, overall response rate was 71%. Time to achieve response was rapid: after only one course, one patient had stable disease whereas the others achieved a PR (3 pts) or MR (1 pt). The commonest non-haematological side effects were constipation (5 pts), alopecia (5 pts), fatigue (4 pts) and peripheral neuropathy (2 pts). They usually were mild and didn't need Thalidomide withdrawal. Other non-haematological adverse effects were sedation (1pt), tremor (1 pt) and maculopapular skin rash (1pt). Leucopenia occurred in 3 patients and it was > grade 2, while 2 patients developed grade 2 anemia. Finally, thrombocytopenia was seen in 3 patients and it was < grade 2. There were 3 febrile episodes classified as FUO: two of them needed antibiotic therapy. Serious complications occurred in 2 patients (tumor lysis syndrome and suspected pulmonary embolism after one day of therapy) and both were fatal. Our preliminary data suggest that ThaDD combination seems an effective salvage regimen for patients who relapse after high dose chemotherapy although toxicity was not negligible, particularly infectious complications. Enrolment and follow-up are in progress.

Primary systemic (AL) amyloidosis is a rare condition in which deposits of monclonal immunoglobulin light chains cause damage to key tissues. Several features of immunoglobulin light chains are related with an aggregation-prone state, such as the lambda isotype and the lambda VI and kappa I subgroups. Amyloid deposition can occur in virtually every organ except the CNS, but the most common clinical manifestations of AL amyloidosis are proteinuria/nephrotic syndrome, cardiomyopathy, hepatomegaly, macroglossia and neuropathy. Prognosis of patients with primary systemic amyloidosis is poor with a median survival of only 12 months. Since AL amyloidosis is related to multiple myeloma (MM), treatment approaches of MM were investigated in amyloidosis. Halting production of amyloidogenic light chains by high-dose melphalan and autologous stem cell transplantation (ASCT) results in reabsorption of amyloid deposit and leads to improvement of organ function. However, transplantation-related mortality (TRM) is much higher than in patients with hematological malignancies due to visceral organ involvement, that is commonly absent in patients with MM. Here, we report on our experience with highdose melphalan followed by ASCT in patients with AL amyloidosis. We treated eight patients with primary systemic amyloidosis. Stem cell mobilisisation was performed by cyclophosphamide (2 or 4 g/m², depending on cardiac function). One patient died after mobilisation therapy. The other seven patients underwent high-dose therapy: six patients received 140 mg/m² and one patient with hepatic disease received 100 mg/m² of intravenous melphalan. The patients were between 32 and 60 years of age (median, 53 years). Two patients had only single organ involvement: one patient severe cardiomyopathy and one patient presented with isolated pulmonary amyloidosis. In six of the eight cases (75%) cardiac, in three (50%) advanced cardiac involvement (NYHA °III) was documented. Three patients (38%) had renal, three (38%) soft tissue manifestation, two patients were diagnosed with hepatic and two with pulmonary involvement. Currently, four of the eight patients are alive with a median follow up of 7 (1 -21) months. Nephrotic syndrome, as diagnosed in two patients, did not improve on therapy. They survived for 8 and 20 months. One patient with severe cardiomyopathy died on day + 13 after ASCT of multi organ failure. Thus, two patients died during therapy, accounting for a TRM of 25%.

Reverse transcriptase polymerase chain reaction (RT-PCR) was applied to evaluate the frequency of tumor cells contamination of peripheral blood stem cell (PBPC) products in 20 patients with Ewing´s sarcoma (ESFT) treated with VIDE induction regimen (vincristine 1,4 mg/m 2 d1, ifosfamide 3 g/m 2 d1-3, doxorubicine 20 mg/m 2 d1-3, etoposide 150 mg/m 2 d1-3 with mesna uroprotection). ESFT are characterized by non-random chromosomal rearrangements between the EWS gene on chromosome 22q12 and members of the ETS gene family of transcription factors. Tumor specific EWS-ETS gene rearrangements enables high sensitivity detection of ESFT tumor cells by means of RT-PCR in bone marrow (BM) and PBPC. At the time of diagnosis 34 BM samples obtained from 18 patients were analysed. BM ifiltration detected by light microscopy was found in two patients (4 samples) and by RT-PCR in eight BM aspirates from five patients. A total 37 aphereses were performed in 18 patients with Cobe Spectra separator after three to five cycles of VIDE via pre-existing permanent tunneled central venous catheter. PBPC were mobilised using filgrastim 5 µg/kg/day starting 24 hours after completion of chemotherapy. In all patients sufficient numbers of progenitors were collected (median CD 34+ yield 11.67 x 10 6 / kg, 3.05 -25.32). On average 2 harvests were needed (range 1 to 5). The bone marrow aspirates (n=15, 8 patients) sampled before the PBPC collection were all tested negative irrespective of the extent of BM involvement at the time of diagnosis. Contamination of the PBPC graft was confirmed in two patients (both with positive BM cytology at the time of disease diagnosis). The grafts collected after 4th and 5th VIDE, were tested reproducibly positive for the presence of contaminating tumor cells. Conclusion: The VIDE regimen with G-CSF is effective in mobilising of large numbers of hematopoetic progenitors. The frequency of PBPC graft contamination in ESFT patients treated with VIDE is low, but in patient with initial bone marrow infiltration more cycles of chemotherapy are needed to clean the graft. /Kg peripheral GCSF stimulated CD34+ cells were administered from an HLA-identical sibling after a reduced intensity conditioning regimen with thiotepa 10mg/Kg, cyclophosphamide 60mg/Kg and fludarabine 60mg/mq. Prophylaxis of reject/GVHD consisted of cyclosporine (CSA) and short course methotrexate. Infection prophylaxis with valaciclovir, fluconazole and thrimetoprim-sulfamethoxazole was given. Results: Neutrophiles and platelet engraftment was obtained on days 14 and 11 after NST. By PCR chimerism analysis, full donor chimerism (FDC) on total and myeloid cells was reached on day 28 after NST whilst FDC of lymphoid cells was obtained on day 60 after NST. A grade II acute graft-versus-host disease (GVHD) of the skin (overall grade I) was observed on day 14 after NST and was treated with 2mg/Kg methylprednisolone. On day 28 after NST a chest CT scan showed a minor response of disease. However, disease progression was then observed (on days 60 and 90) after NST. CSA was tapered from day 60 and withdrawn on day 98 after NST. On day 120, the patient developed a severe respiratory failure. Bronchial alveolar lavage and all the microbiological coltures were negative. The chest CT scan showed a diffuse interstitial involvement with multiple escavations and with a concomitant impressive disease regression. High dose methylprednisolone was succesfully administered. Low-dose oral prednisone and CSA were continued until 262 days after NST, when they withdrawn for disease progression. At the last follow-up, 285 days after NST, disease regression was observed again along with an improvement of the lung interstitium and a near complete resolution of lung escavations. Conclusions: This case report for the first time an evidence of a graft-versus-tumor effect in a patient with an advanced chemorefractory squamous tracheo-bronchial carcinoma. A powerful and promising graft-versus-lung response related to the immunosoppressive treatment changes was observed, resulting in a benefit on patient's outcome. Further studies need to confirm such effect in lung carcinomas.

Objectives: To assess efficiency and toxicity of the submyeloablative chemotherapy with Peripheral blood stem cell rescue in children older than 3 years with solid tumors. Methods: Between 1999 and 2003, 45 childrens were treated (in 30 pts. as first line treatment, in 15 pts. after progression of the disease). Diagnoses included embryonal brain tumors high grade (residual tumor greater than 1,5 cm 2 and/or amplification of cmyc oncogene and/or metastatic disease) in 36pts., germ cell tumor in 1pt., osteosarcoma in 7pts. and rabdomyosarcoma in 1pt. Results: After chemotherapy were 30 pts. (66 %) in CR, 2 pts. (4 %) in PR, 9 pt. (20 %) with DP. The hemato-and gastrointestinal toxicity were in all patients WHO grade III-IV, neurotoxicity gr. II-III (by 1 pt. gr. IV), 1 pt. finished the treatment after 3rd course of chemotherapy due to nefrotoxicity, 2 pt. showed ototoxicity gr. III, 1 pneumotoxicity gr. IV, 1 alergic reaction. 14 pt. died of progression. Conclusions: Initial evaluation show feasibility and promising results of this regimen in the treatment specially of high risk brain tumors (the crucial prognostic factors in children are complete resection of the tumor, no evidence of leptomeningeal disease and no evidence of amplification of c-myc oncogene) and in other worse prognostic solid tumors, despite high, but manageable toxicity. Further evaluation is necessary. To determine whether accelerated T-cell immune reconstitution is possible after dose-intensive CT, we explored the feasibility and toxicity of administering ex-vivo activated T cells as an adjunct to HDCT and autologous hematopoietic cell transplantation (HCT) for patients with breast cancer (BC). Sixteen patients, median age 46 (range31-56), with metastatic disease responsive to first line CT received consolidation with HD Melphalan/Thiotepa and HSC support. Patient lymphocytes obtained by apheresis procedures before stem cell mobilization were treated ex-vivo for 24 hours with IL2 (200U/mL) and transfused on day 14 and, depending on the amount of lymphocytes available, on day 21 and 28 following transplantation. Ex-vivo stimulation resulted in median 6-fold increase in the absolute number of CD25+ cells. Patients received a median of 1.26 x 10 8 CD3+ cells (range 0.17-2.1) divided in 1 (n=3), 2 (n=7) or 3 (n=6) infusions. Administration of activated lymphocytes was associated with fever and chills in the majority of patients. One patient developed capillary leak syndrome that remitted promptly with meperidine and acetaminophen. Data on immune reconstitution are reported in the Table. These data match favourably with historical controls. In conclusion, this pilot study demonstrates that adoptive transfer of activated T cells is feasible in patients with advanced BC undergoing autologous HCT, and is associated with a rapid immune reconstitution. Whether this novel approach will have a clinical impact in terms of disease control or reduction of infectious complications deserves a longer follow-up and a larger case series.

Introduction: Allogeneic hematopoietic stem cell transplantation (SCT) has become a treatment also for patients with metastatic solid tumours. Recent studies imply allogen effect in patients with renal cell, colon, ovarian, breast and primary liver cancer, following SCT. Clinical studies claim a variable response of the tumour load after SCT. Objective: We investigated if there is an association between variable response and free serum cytokines. Patients and methods: Two patients with colorectal and four with renal cancer, all with metastases, underwent allogeneic SCT. Conditioning included fludarabine (Flu) 30 mg/m 2 for 3 days, using sibling donors and 5 days to unrelated donors (MUD) followed by 2 Gy total body irradiation (TBI) or cyclophosphamide (Cy) 60 mg/kg for two days (n=1). Antithymoglobuline 4 mg/kg was given to patients with MUD (n=3). Immunosuppression was cyclosporine A, combined with mycophenolate mofetil (MMF) (n=4) or methotrexate (MTX). The tumour load was examined by computer tomography (CT) of the thorax and abdomen, before SCT and 3, 6 and 12 months after SCT. Free serum cytokines were analysed using ELISA. Results: Cytokine analyses were evaluated intra-individually concerning the balance between the anti-inflammatory (A-I) and the inflammatory (I) cytokines. Total assessment of the tumour burden in lung, lymph nodes and liver was performed. A correlation between a dominance of A-I cytokines (TGF-beta, IL-10) and tumour progression was found. Similarly, a dominance of I cytokines (TNF-alfa, IFN-gamma) was associated with tumour regression. Conclusion: An increased level of I cytokines in serum was associated with regression of the tumours, possibly mirroring tumour killing by type 1 T helper cells, Th1. In contrast, the dominance of A-I cytokines from type 2 T helper cells, Th2, might inhibit cytotoxic cells from the allogeneic anti-tumour effect. In two pediatric BMT centers in Poland, between 1996 and 2002, twenty one children with poor risk Ewing's tumor received high dose chemotherapy with autologus PBSCT. All but one patient have metastatic disease at presentation. There were eleven females and the median age at diagnosis was 12 years (range 4,5-18 years). Megachemotherapy consisted of melphalan 140 mg/m 2 / busulfan 16mg/kg in 12 pts, melphalan 140 mg 2 / treosulfan 10,0g/m 2 in 2 pts and melphalan with other drugs in 7 pts. Ten children were in I CR before transplantation, one girl in II CR, nine children had partial remission and one boy had progressive disease resistant to therapy. Eight of eleven patients transplanted in CR survived with a median follow up 17 months (range 7-53) and there were no severe regimen related toxicity in this group. Three other patients relapsed 9,13 and 16 months after PBSCT and died of progression of disease. Children transplanted without remission died: two of them due to transplant related mortality (MOF, VOD and HC) and eight from progression of disease in a median time 7 months after PBSCT.

We conclude that megachemotherapy with PBSCT is a save procedure in children in remission. Autologus transplantation in children with metastatic Ewing's sarcoma seems to improve their outcome, but results should be evaluated in a larger series of patients. Children with recurrent disease need new approaches. We retrospectively analyzed the therapeutic efficacy and toxicity of tandem high-dose chemotherapy (HDC) with autologous stem cell support (ASCT) in patients with advanced breast cancer. Twenty-eight patients with high-risk with >10 nodes positive (n=11) or metastatic (n=17) breast cancer received two successive courses of HDC after induction therapy. The first course, consisting of high-dose melphalan (HD-MELP) (140 mg/sm), was followed, 2-4 months later, by a second course consisting of ICE regimen (Ifosfamide 12 g/sm, carboplatin 18 AUC and etoposide 30 mg/Kg). Both HDC were supported by ASCT. Protocol induction and mobilization chemotherapy consisted of 4 cycles of FEC 90 (n=15) or docetaxel 75 mg/sm + epirubicin 120 mg/sm (n=13). A median of 4.7 (range 0.5-7.3) and 5.7 (range 3-9.1) x10 6 /Kg CD34+ cells were reinfused after each course of HDC respectively. G-CSF was administered from day +3 until neutrophils engraftment. The main toxicity was bone marrow suppression with neutrophils (>500/ul) and platelet (>20,000/ul) engraftment after a median of 10.5 and 11.5 days after the first and 10 and 12 days after the second course respectively. A median of 2 packed RBC and 2 units of platelets were reinfused after each HDC. No toxic death was observed. Non-hematological WHO grade 3 toxicities were relatively low after both HD-MELP (mucositis n=2, bleeding n=1) and ICE (infection n=2, mucositis n=2, diarrhoea n=2, hemorrhagic cystitis n=1, bleeding n=1) and always reversible. Other frequent toxicities no exceeding WHO grade 2 were: mucositis (68%), fever (43%), nausea/vomiting (36%), diarrhoea (32%), neurotoxicity (14%). Late complications were observed in 4 cases (pulmonary fibrosis, peripheral neuropathy, ototoxicity and HZV infection).No second malignancies occurred. At a median follow-up of 5.2 years (range 1.5-9.3 years) the progression-free (PFS) and overall survival (OS) rates for the 17 metastatic patients were 21% and 47% with a median of 35 and 72 months respectively.In the 11 patients with high-risk breast cancer the 5years projected PFS and OS rates were 72% and 69% respectively.In the relapsed patients salvage treatment was feasible without significant toxicity, and was able to induce prolonged OS. Our results suggest that two HDC with HD-MELP and ICE followed by ASCT are tolerable and effective in patients with advanced breast cancer. The low-toxicity profile and the clinical efficacy of this tandem HDC protocol warrant further investigations. Objectives: To explore possible graft-versus-host disease (GVHD) prevention and graft-versus-tumor (GVT) effect enhancement by the administration of an anti-HER2 monoclonal antibody after nonmyeloablative allogeneic stem cell transplantation (NST) in a patient with liver metastatic breast cancer overexpressing HER2. Methods: NST with a reduced intensity conditioning, based on thiotepa 5mg/Kg, cyclophosphamide 60mg/Kg and fludarabine 60mg/mq, followed by 5.0x10 6 /Kg peripheral GCSF-stimulated CD34+ cells from an HLA-identical sibling were administered. Prophylaxis of reject/GVHD consisted of cyclosporine (CSA) and short course methotrexate. Weekly trastuzumab 4mg/Kg (first dose) followed by 2mg/Kg was scheduled in case of progression disease after NST. Results: Neutrophiles and platelet engraftment was reached on days 10 and 11 after NST. Full donor chimerism on total and myeloid cells was reached on day 28 after NST (by PCR analysis), whilst 95% of lymphoid cells were of donor origin by day 132 after NST. Liver disease progression with radiological signs of neoplastic cholangitis and the occurrence of a controlateral breast relapse were observed 51 days after NST. Steroid therapy and CSA were tapered and they were withdrawn on day 100 after NST. Weekly trastuzumab was started on day 76 after NST and resulted in rapid decrease of bilirubinaemia, transaminasaemia and cholestasis parameters. The abdomen CT scan and MRI performed on day 98 and 132 respectively, showed a disease stabilization. CEA and CA15.3 tumor markers (previously increased) stopped growing and tended to decrease. Controlateral breast relapse disapperead. On day 132, a new slow increase in bilirubinaemia, transaminasaemia and cholestasis parameters was observed. On day 140, infusion of 1x10 6 /Kg donor lymphocytes was administered 24 hours after the 10th weekly trastuzumab administration. With a follow-up of 147 days after NST the patient is alive and no grade II-IV acute and/or chronic GVHD were observed. Conclusions: Stabilization of rapid progressive metastatic breast cancer after NST in a patient previously failed treatment to several lines of chemotherapy, ormonotherapy and trastuzumab, seems to suggest the possibility to enhance GVT effect by using antitumoral monoclonal antibodies. Antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) may have a role in this effect.

Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation is successful in the treatment of relapsed chronic myeloid leukemia. In acute lymphoblastic leukemia (ALL) however, graft versus leukemia (GVL) responses are rare while graft versus host disease (GVHD) is common. Since the CD4+ T cell subset is less associated with GVHD than the CD8+ T cell subset, infusion of purified CD4+ T cells may result in improved balance between GVL reactivity and GVHD. However, it is unclear whether CD4+ T cells alone can mediate GVL in ALL. We studied the anti-leukemic potential of purfied human CD4+ or CD8+ T cells in our NOD/scid mouse model of human ALL. In this model, the kinetics of T cell proliferation and GVL-reactivity can be monitored "real time" by sequential determination of absolute T cell counts and leukemic cell counts in the peripheral blood of individual animals. CD4+ or CD8+ T cell populations were isolated (mean purity 98%) by immunomagnetic bead selection and infused into leukemic mice. When obtained from HLA-disparate donors, infusion of CD4+ T cells resulted in emergence and subsequent regression of T cells (mean peak T cell count 3.0x10 6 /mL, all CD4+). During T cell emergence, complete remissions were induced in 3/3 animals. Infusion of CD8+ T cells also resulted in T cell emergence (1.5x10 6 /mL, all CD8+) and induction of complete remissions in 2/4 animals. We next studied the proliferative capacity and GVL potential of the T cell subsets in the clinically more relevant HLA-identical setting. When obtained from HLA-identical donors, only infusion of CD4+ T cells resulted in vigorous T cell proliferation (20x10 6 /mL, all CD4+) and complete tumor eradication in 8/10 animals. After infusion of HLA-identical CD8+ T cells, low numbers of T cells emerged (2.0x10 6 /mL, all CD8+) and complete remissions were not observed in any of 9 treated animals. Three CD4+ T celltreated animals survived long-term. In these animals leukemiareactive CD4+ T cells persisted (0.05-0.4x106/mL) during remission. However, rechallenge of these animals with leukemic cells resulted in limited secondary T cell expansion and leukemic cells were not rejected. We conclude that human CD4+ T cells harbor potent anti-leukemic reactivity. Considering their limited association with graft versus host reactivity, infusion of CD4+ T cells may be an attractive therapeutical approach, although development of T cell unresponsiveness in time may remain a major problem.

Patients with severe rheumatic autoimmune diseases have a poor prognosis after failure of conventional therapy. In these advanced disease stages with multiorgan dysfunction, high dose chemotherapy followed by autologous hematopoietic stem cell transplantation (ASCT) is increasingly being studied. The concept of stem cell transplantation is a resetting of the immune system. Chemotherapy and immunoablation destructs the immune system (autoreactivity) of the patients followed by a T-cell depleted autologous stem cell transplant with development of a tolerant immune system. Remissions of systemic lupus erythematosus (SLE), relapsing polychondritis (PC), systemic sclerosis (SSc) are reported. The duration of these clinical and serological remissions after ASCT is unknown. In Berlin 11 patients with severe autoimmune diseases refractory to conventional therapy underwent intensive immunoablation and ASCT. With a follow up of more than five years we report here on clinical, serological and immunological data of these patients. 8 patients are alive and 5/8 are in clinical and serological remission (relapsing polychondritis n=1, systemic lupus erythematosus, SLE n=4). Progressive disease occurred in 3/8 pts. (systemic sclerosis, SSc n=2, small vessel vasculitis n=1) with unchanged autoantibody titers (ANA, anti Scl 70) in SSc pts. after conditioning and ASCT. 3 out of 11 pts. are dead, 1 pt. with SSc due to a acute deterioration of a chronic cor pulmonale. The other 2 pts. with SLE went into clinical and serological remission after ASCT. On died on d+82 due to cerebral infarction with Aspergillus emboli and the other relapsed 17 months after ASCT and succumbed due to active intractable SLE 37 months after ASCT. All pts. in remission are autoantibody negative. With reappearance of SLE and clinical relapse, change of autoantibody profile occurred. Immunological studies on the remission pts. showed early reconstitution of the Th cell compartment characterized by the appearance of activated memory/effector Th cells expressing HLA-DR. No autoreactive Th cells could be detected, while increased frequencies of virus specific Th cells were detected in 1 pt with reactivation of varicella. Approximately 5 months after ASCT naïve B cells appeared. At 12 months after ASCT the T cell receptor repertoire was normalized and we could observe a reactivation of thymic function after ASCT. In conclusion: In vivo and ex vivo immunoablation and ASCT S169 creates a new immune system and stops the rheumatic inflammation in pts. with relapsing polychondritis and SLE. If transplantation in early stages will improve the results in other rheumatic autoimmune diseases remains to be studied. The transplantation of highly purified CD 34 + stem cells from haploidentical donors is a well established method in our institution for the treatment of children with high risk leukemias. However, relapses represent a major problem after transplantation. In these patients the treatment of minimal residual disease (MRD) by infusion of donor NK cells immediately after transplantation might be an approach to minimize the occurance of relapse without inducing graft-versushost-reactions, but expression of HLA class I inbhibits NK cell mediated cytotoxicity on tumor cells through killing cell inhibitory receptors (KIR). Therefore we measured HLA class I expression using quantitative FACS analysis in leukemic cells from pediatric patients with acute lymphatic leukemia (ALL) and investigated the impact of HLA class I expression and several adhesion molecules on NK cell mediated lysis. Blasts with reduced HLA class I expression were effectively lysed by donor NK cells after stimulation with IL2 , whereas blasts with high expression of HLA class I were more resistant NK lysis. We found a linear correlation between number of HLA class I molecules and specific lysis of NK cells in an Europium release test (r2=0.54). The lysis could be strongly enhanced by blocking of HLA class I. Taking KIR-ligand-mismatch into account we found a strong correlation (r2=0,95, p<0,0001) between HLA class I expression and specific lysis of NK cells for leukemic blasts without KIRmismatch. In a model with regard to both HLA expression and KIR-ligand-mismatch a r2 of 0,9 (p<0,0001) was observed. Different patterns of adhesion molecules (ICAM 1-3, LFA 1+3) and CD95 were found without significant influence on NK cell lysis.

In these experiments NK cell lysis was mainly dependent on HLA class I expression and in addition on KIR-mismatch. We therefore suggest that screening leukemic blasts for HLA class I expression is valuable to choose a suitable immunotherapeutic strategy. Post transplant stimulation of the regenerating NK cells with IL2 in combination with donor NK cell infusions and selection of a NK-alloreactive-donor might be promising in the reduction of relapse rate.

Objectives: Cyclin D1 participates in cell-cycle control and is over-expressed in a wide range of human solid tumors, but is not detectable in normal differentiated adult cells. In a significant proportion of myeloma samples and more importantly, in all mantle cell lymphomas (MCL) Cyclin D1-protein is strongly overexpressed. Methods: To investigate whether Cyclin D1-specific cytotoxic T lymphocytes (CTL) are capable of killing Cyclin D1+ hematological targets, CTL were generated from healthy HLA-A*0201-positive volunteers by incubating T cells with autologous dendritic cells (DC) transfected with Cyclin D1-RNA. Results: Cyclin D1-specific CTL lysed HLA-A2+ Cyclin D1+ tumor cells including several lymphoma and myeloma cell lines, as well as malignant targets from MCL-and myeloma patients. Normal Cyclin D1-negative target cells (CD14+ monocytes, activated B cells) were not attacked by Cyclin D1-specific CTL. Conclusion: Cyclin D1 can be used as a target for specific immunotherapeutic strategies against hematological malignancies.

Co-administration of apoptotic cells with a bone marrow graft to enhance marrow engraftment induces a TGF-betadependent expansion of CD4+CD25+ regulatory T-cells P. Saas, S. Perruche, H. Bittard, J.M. Chalopin, P. Tiberghien, F. Kleinclauss, INSERM E0119/MEN EA2284, Urologie CHU, INSERM E0119 Néphrologie CHU (Besançon, F) Apoptotic cells are endowed with immunomodulatory properties. Such dying cells can be used as a cell therapy product to control inflammation or favor hematopoietic engraftment. Here, we examined how infusion of apoptotic cells can favor bone marrow (BM) engraftment. Recipient BALB/c mice received a 6Gy total body irradiation (TBI) and were grafted with a low number (10e6) of allogeneic FVB BM cells and irradiated (40Gy) apoptotic FVB cells. Engraftment was assessed by flow cytometry 6 weeks after BM transplantation (BMT). Since TGF-beta can be released during apoptosis (by dying cells or by cells phagocyting apoptotic bodies), an anti-TGF-beta antibody (Ab) (5mg/kg) was administrated at the time of apoptotic cell and BM infusion. Such treatment completely abolished the graft facilitating effect induced by apoptotic cell infusion (0% engraftment vs 40% in the absence of Ab, P<.05). Analysis of spleen T cell populations in engrafted mice given apoptotic cells revealed an expansion of donor CD4+CD25+cells (9+0.7% vs 4+0.4% in control mice, n=10, P<.05) with a regulatory T cell (Treg) phenotype (CD62L/CTLA-4+). Such expansion was restricted to engrafted mice and specific to apoptotic cell infusion: no Treg expansion was observed in mice engrafting with a higher number of BM cells (3x10 6 , 1+0.2%, n=6) or after an increased TBI dose (7 Gy, 2+0.2%, n=5) without apoptotic cells (P<.05). Anti-TGF Ab significantly reduced splenic Treg (3+0.9%, n=5), showing that Treg expansion after apoptotic cell infusion was TGF-dependent. The role of Treg after apoptotic cell infusion was studied by infusing an anti-CD25 Ab (8.5mg/kg) at day 0 and 3 after BMT. Despite significant reduction of Treg, similar number of engrafted mice was observed in mice receiving apoptotic cells with a BM graft (42%) or in mice that also received anti-CD25 Ab (50%). However, CD25 depletion significantly decreased the levels of circulating donor-derived cells in the chimeric mice (23+5% after anti-CD25 Ab treatment vs 66+7% in control mice). This is not surprising since CD25 is not a Treg specific marker. CD25 can be expressed by host activated T cells. Overall, apoptotic cell infusion enhances BM engraftment by TGF-beta-dependent mechanism. A novel link between apoptotic cells and Treg is reported. This can contribute with other mechanisms to prevent deleterious responses against dying cells during physiological turnover. Such mechanism may be used therapeutically to favor engraftment after BMT.

H. Roelofs, R.M. Egeler, W.E. Fibbe, LUMC (Leiden, NL) In various animal models it has been shown that cotransplantation of mesenchymal stem cells (MSCs) enhances the engraftment of hematopoietic stem cells. Also in a clinical study co-transplantation resulted in reduced graft versus host disease and increased survival. In order to introduce the advantages of co-transplantation in the clinic, our aim is to develop expansion procedures that result in clinically relevant numbers of MSCs (>1E8) and that allow clinical application with respect to the regulations for application in humans. We have expanded mesenchymal stem cells from potential clinically applicable sources. Bone marrow aspirates were harvested under local anesthesia. The mesenchymal stem cells were expanded -based on their adherent properties -under standard laboratory culture conditions and under conditions that will allow clinical application. Because of the adherent nature of MSCs, we also tested whether these cells could be isolated and expanded more efficiently from bone marrow biopsy material (Table) . There was no clear difference in the efficiency with which MSCs could be expanded from either bone marrow aspirates or biopsies. Because of logistic reasons, we chose bone marrow aspirates as the preferred source of adult MSCs. For the expansion of adult bone marrow aspirate-derived MSCs we have been able to develop a clinically applicable expansion protocol requiring cell cultivation in a GMP facility in low-glucose DMEM supplemented with 10% of an approved gammairradiated batch of characterized FCS and passaging of the cells using a batch of irradiated trypsin/EDTA (="GMP"-condition). Using this method a number of 1E8 expanded MSCs could be reached in a period of 3-4 weeks, starting from a 50 ml bone marrow aspirate, indicating that this is a feasible strategy for clinical application in the setting of allogeneic stem cell transplantation.

R. Peceny, A.H. Elmaagacli, R. Trenschel, H. Ottinger, D.W. Beelen, University of Essen Medical School (Essen, D) In the setting of our phase II protocol of transplantation using highly purified peripheral blood CD34+ cells from HLA-identical sibling donors in chronic myeloid leukemia, we studied the development of chimerism in lymphocyte subpopulations using fluorescence in situ hybridization for the X and Y chromosomes. Up to date, we transplanted 51 adult patients without any prophylactic immunosuppression. Severe acute graft-versus-host disease (GvHD) did not occur post transplant. Forty two patients at least 3 months after the first donor lymphocyte infusion (DLI) received a median of 3 DLI starting on day 93 (44-986) in a median maximal dose of 3.3x10 6 CD3+/kg body weight (0.17x10 6 -100x10 6 ). In the beginning, DLI were given to treat cytogenetic relapse. In the last 28 patients a T-cell-addback (TCA) was applied on days +90 and +135 with doses of 0.33x10 6 and 1x106 CD3+ per kg body weight. NK-cells recovered rapidly within the first month. Numbers of Bcells and CD8+ cytotoxic T-cells normalized in the 2nd month post transplant whereas T-helper cell numbers stayed low beyond the first year (Beelen et al., 2000) . Twenty one patients receiving DLI had a donor-patient gender disparity, 12 of these received donor lymphocytes as TCA. In contrast to a rapid complete chimerism of the NK-and B-cells, Tcell chimerism developed slowly: 5.2% at 3 months, 39.7% at 6 months, 53.8% at 9 months, 62.2% at 12 months, reaching just 87.4% at 18 months (see graph). Early T-cell addback did not make a difference; the T-cell chimerism in the groups with TCA vs. late DLI was 39.7% vs. 31% (p=1.0) at 6 months and 48.4% vs. 59.8% (p=0.64) at 9 months. A rapid increase in T-cell chimerism could be shown, if GvHD occurred and needed systemic treatment. On the other hand, the transition to complete T-cell chimerism was not always associated with the onset of GvHD. At least if T-cell doses > 5x10 6 CD3+/kg were given, a major increase in T-cell chimerism (> 85%) did not always lead to remission, nevertheless further DLI or alpha-Interferon were successful. In summary, expansion of patient clones is responsible for the early recovery of CD8+ T-cells. This might contribute to the low incidence of infections, but also to the known increased risk of secondary graft failure. And as was shown in mice already by Y.-M. Kim et al. (Blood, 2003) , early manipulated DLI -especially CD8-depleted -might increase the risk of secondary graft failure in this setting substantially.

A phenotypic marker for thymic activity: recurrence of CD31+ CD45RA+ ''thymic'' naive Th-cells after stem cell transplantation indicates reactivation of thymic activity A. Thiel, C.A. Schmidt, G. Przybylski, T. Alexander, S. Kohler, S. Kimmig, F. Hiepe, R. Arnold, A. Radbruch, DRFZ Berlin, University Hospital Greifswald, University Hospital Charité (Berlin, Greifswald, D) Aim of the study: Methods to quantify Th-cell reconstitution after hematopoietic stem cell transplantation (HSCT) or highly immunosuppressive therapies are becoming a key issue since persistent Th-cell deficiencies may result in severe therapy complications and adverse events. So far only the recently introduced examination of T-cell receptor excision circles (TRECs) can be used as a correlate for thymic activity, while phenotypic markers for the assessment of thymic function were not available. We aimed here to evaluate whether reactivation of thymic activity can be monitored by the reappearance of peripheral CD31+ ''thymic'' naive Th-cells. Methods: We assessed here the Th-cell reconstitution in patients that had received highly purified autologous CD34+ stem cell transplants (CD34-ASCT) after cyclophosphamide/ATG conditioning for the treatment of various severe autoimmune diseases. We performed mulitparameter cytometry for phenotypic analysis of CD4+ T-cells and quantitative real-time PCR analysis to determine TREC numbers. Results: In every patient (n=6) allmost all CD45RA+ Th-cells recurring during the course of immune reconstitution after CD34-ASCT co-expressed CD31, and thereby resembled ''thymic'' naive Th-cells. In 3 patients with a follow-up time of more than 4 years we were able to perform PCR analysis of TRECs in highly purified naive Th-cell subsets. TRECs were detected almost exclusively among CD31-expressing ''thymic'' naive CD45RA+ Th-cells and as high as compared to aged matched controls. Conclusion: We show here for the first time that CD31-expressing CD45RA+ ''thymic'' naive Th-cells reappearing in the course of immune reconstitution after CD34-ASCT are indicative of a thymic rebound, since rising numbers of post ASCT TRECs are highly enriched in this Th-cell compartment. M. Koldehoff, N. Steckel, R. Peceny, R. Trenschel, D. Beelen, B. Opalka, A. Elmaagacli, University Hospital of Essen (Essen, D) Gfi-1B is a zinc finger protein which is expressed exclusively in hematopoietic cells. Gfi-1B regulates transcription during erythropoiesis and is also involved in regulating the process of hematopoietic cell differentiation and megakaryopoiesis. We studied the effect of transfection with small interfering RNA (siRNA) targeting Gfi-1B in leukemic cells and normal CD34positive cells in regard of proliferation, inducing apoptosis, and cell differentiation. Further, we evaluate if the post-transcriptional gene silencing of Gfi-1B mRNA can be augmented by use of two further siRNAs targeting the bcr-abl hybridgene or targeting the wt-1 gene expression in the cell line K-562. A reduction of Gfi-1B gene expression measured by real-time RT-PCR to amounts between 12% and 45.4% (mean) was observed in the K562 and Hel cell lines compared to controls ( controls were set up to 100%). We found a two-fold increase of induced apoptosis in MV4-11 cells, NB4 cells and a four-fold increase of apoptosis in Hel, Kasumi-1, K-562 cell lines 24 hours after transfection with Gfi-1B siRNA versus controls. Proliferation was strongly inhibited (of about 80%) in cell lines: Hel, Kasumi-1, MV4-11 by Gfi-1B siRNA and moderate decreased (of about "d50%) in NB4 and K-562 leukemic cells. In normal CD34-positive cells, the proliferation was inhibited too (by about 70%). In contrast to leukemic cells, no induced apoptosis was found in CD34+ cells after Gfi-1B siRNA transfection. Cotransfection with Gfi-1B siRNA, wt-1 siRNA, and bcr-abl siRNA inhibited the proliferation rate not more effectively than transfection with Gfi-1B siRNA alone. The rate of induced apoptosis was constant to the transfection with each siRNA. No synergistic effects of Gfi-1B siRNA with bcr-abl siRNA or wt1 siRNA was measured. The transfection of Gfi-1B siRNA in CD34+ cells had no influence on differentiation markers CD13, CD14, CD33, CD34, CD45, CD64 and glycophorin A. Glycophorin A expression increased after transfection with Gfi-1B siRNA in the Hel cell line, whereas CD64 positive monocytes increased in the K-562 cell line after the transfection with Gfi-1B siRNA. These findings suggest that Gfi-1B seems to be a promising target for new therapeutic strategies with siRNAs in the treatment of erythroleukemic cells. It has been previously demonstrated that purified CD34+ cells induce allogeneic T cell proliferation and generation of cytotoxic T cells. In this study, we addressed the hypothesis of whether CD34+ cells may activate also NK cells. A 51Cr assay was used to determine the ability of unfractionated mnc to lyse HLAnegative, NK-sensitive (K562 cells) target cells, before and after culture with irradiated purified CD34+ cells or CD14+ monocytes from allogeneic G-CSF mobilised donors. Stimulation by CD34+ cells or monocytes resulted in an increased ability of allogeneic mnc to lyse K562 cells (26±9% before MLC and 49±26% and 40±13%, respectively, after culture, at a 10:1 E/T ratio (n=3). However, while the generation of monocyte-induced cytotoxic activity was prevented by costimulatory blockade (with CTLA4-Ig at 2 µg/ml) by 53±18% (n=3), CD34+ cell-induced cytotoxic activity was only slightly affected (by 21±15%, n=3). Stimulation by CD34+ cells was associated with the recovery of more NK cells as compared to monocytes (85±19 % vs 36±21%) (n=3). To test whether CD34+ cells could directly activate NK cells, CD3-CD56+ NK cells were immunomagnetically purified. CD34+ cells induced significantly greater NK than monocytes (50±14% vs 15±17% K562 lysis, respectively) (n=4, p=0.02). We then tested whether coculture of purified NK cells with CD34+ cells would result in the lysis of NK-resistant Daudi cells (LAK activity). While LAK activity was negligible before culture, it substantially increased upon culture with CD34+ cells, but not monocytes (53±11% and 22±16%, respectively) (n=6, p=0.01). Interestingly, CD34+ cell priming was comparable to the addition of high dose IL-2 (at 1000 U/ml) that induced a lysis of 49±3% at a E/T ratio of 10:1 (n=4) (p=0.22). NK cells cultured with CD34+ cells produced greater amounts of IFNg and TNFa as compared to monocytes (500 pg/ml and 125 pg/ml vs 50 pg/ml and less than 5 pg/ml) and were induced to express higher levels of activationrelated molecules, such as CD69 (that increased 5-fold and 2fold, respectively, above baseline) and CD25 (that increased 10fold as opposed to 2-fold) (n=2). Moreover, CD34+-induced NK activation was prevented by anti-NKG2D (reduction of the lysis of Daudi cells from 47% to 15%, at E/T Ratio of 50 to 1, but not by anti-IL-2 antibodies. Therefore, our data show that CD34+ cells activate NK cells in vitro possibly by direct stimulation of the NK activating receptor NKG2D.

E. Rohde, D. Thaler, G. Lanzer, W. Linkesch, D. Strunk, Klinikum Graz (Graz, A) Regenerative stem cell therapy (SCT) is currently tested in clinical trials. The ideal type, number and source of cells needed for stem cell mediated tissue repair is not yet defined. Lineage (Lin)-depletion is an experimental procedure capable to enrich all recently recognized SC types with regenerative potency. This study was performed to define a practicable mAb cocktail for good manufacturing practice (GMP)-grade Lin-depletion and to test whether large scale Lin-depletion is possible. CD2 (T cell), CD19 (B cell), CD14 (monocyte), CD15 (granulocyte), CD56 (NK cell), CD41 (platelet), and Glycophorin A (RBC) specific mAb were selected to specifically mark seven mature blood cell lineages (Lin7). Lin7-cells were analyzed in peripheral blood (PB, n=9), mobilized PB (MPB, n=5), umbilical cord blood (UCB, n=5) and bone marrow aspirates (BM, n=4) by flow cytometry. Mononuclear cells were collected from nonmobilized peripheral blood of four healthy volunteers by leucapheresis. Pre-clinical Lin-depletion was tested with four different Lin-cocktails containing CD2/14/15/19/56 (Lin5), CD2/19/14 (LIN3 A), CD2/19/15 (LIN3 B) and CD2/19/56 (LIN3 C) following GMP principles. Lin-products were analyzed by flow cytometry and cultured in cytokine-stimulated liquid cultures for 2-6 weeks. In PB, MPB, UCB and BM, 0.23±0.04%, 0.27±0.03%, 0.53±0.07% and 0.49±0.03% nucleated cells (NC) were Lin7-, respectively. CD34+ cells, dendritic cells (DC) and basophils constituted the major Lin7-subpopulations (together 84±2%, 90±3%, 40±3%, 80±3%, in PB, MPB, UCB and BM, respectively). Minor populations included CD7-and CD45-cells. Pre-clinical Lin5 and Lin3-depletion after automated RBC and platelet reduction resulted in a 10 to 20-fold enrichment of CD34+ and CD34-/Lin-cells. Cultured Lin-cells showed hematopoietic as well as fibroblastoid and endothelial differentiation characteristics after 2-6 weeks of culture. A seven mAb cocktail is sufficient to label >99% NC in PB, MPB, UCB and BM. Lin-depletion results in a significant enrichment of CD34+ and CD34-candidate regenerative blood cells and can be performed under GMP conditions. Functional data indicate a sizable enrichment of cells with endothelial and fibroblastoid differentiation indicating regenerative potency. Thus, by avoiding artificial loss of CD34-regenerative SC due to positive (CD34/133) selection, Lin-depletion may represent a valuable alternative approach to enrich SC for regenerative SCT. . Experiments were performed as triplicates and repeated once with similar results. The Granzyme B ELISPOT for NK cells expresses adequately the activation and inhibition due to appropriate KIR-ligand interaction of allogeneic and autologous NK cells. This is the first time that allo-reactivity of NK cells could be detected in bulk NK cell population without the need for limiting dilution cloning of NK cells and consecutive testing of cytotoxicity of the clones against recipients B-LCL. The Granzyme B ELISPOT assay for NK cells is currently used for further investigation in ongoing allogeneic HSCT. Frequencies of allo-reactive NK cells could be correlated with KIR/KIR-ligand mismatch of donors and recipients and the occurrence of considerable clinical events like e.g. relapse, GvHD, engraftment failure and survival.

A. Yoshimi, S. Matthes-Martin, U. Duffner, J. Starý, P. Bader, D. Dilloo, T. Klingebiel, F. Locatelli, C 

Objective: Juvenile myelomonocytic leukemia (JMML) is a rare clonal disorder of early childhood. Currently, only allogeneic stem cell transplantation (SCT) offers long-term cure, although the relapse rate remains high. There is evidence that a graft-versusleukemia (GVL) effect plays a role in controlling JMML after SCT, but the benefit of donor leukocyte infusion (DLI) following SCT in JMML is currently unknown. Therefore, we report the results of a retrospective study on DLI in 21 patients (pts) with JMML. Patients: Twenty-one pts (M:F= 13 : 8) registered in EWOG-MDS who underwent SCT for JMML (BMT:15, PBSCT:5, CB:1) from 1/1995 -8/2002 and received DLI were studied. Age at SCT was 9 -94 mo (median 44). Four pts who received DLI after a 2nd SCT were included. Eight pts were transplanted from a matched family, 2 from a 2 or 3 loci mismatched family, and 11 from an unrelated donor. Hematological relapse was diagnosed in the presence of an increased number of blast cells in PB or BM, or clinical signs of relapse with BM hypercellularity and absence of megakaryocytes. DLI and patient outcome: Two pts with complete chimerism (CC) received DLI as pre-empiric therapy and are alive in remission. Of the other 19 pts, 4 received DLI for mixed chimerism (MC) only and 15 for hematological relapse. Six of the 19 pts received a single DLI, 13 pts 2-6 infusions (med.3). The T cell dose of each infusion varied from 1x10 4 to 2.4 x10 8 /kg. Six of 19 pts achieved CC: 2 of 4 pts with MC and 4 of 15 pts with hematological relapse. Five of these pts received DLI from an URD and 1 from a matched cousin. However, only 1 pt maintained in CR and is alive, 2 relapsed, and 3 died of complications (1 acute GVHD, 1 hyper-eosinophilic syndrome, 1 BM failure). Acute GVHD following DLI occurred in 3 of 6 responding and 1 of 13 non-responding pts, chronic GVHD in 2 responding pts. None of 8 pts who received less than 1x107/kg T cells responded, and 6 of 11 pts who received equal and more than 1x10 7 /kg T cells achieved CC (2 of 2 with MC and 4 of 9 with relapse). Conclusion: One-third of the pts with relapsed JMML achieved CC after DLI. However, outcome of the responding pts was poor due to complications and late relapses. At least 1 1x10 7 /kg T cells were required for the achievement of CC. Early intervention before hematological relapse, modification of the administration of DLI to reduce complications including GVHD may improve outcome for pts with relapsed JMML.

M. Weber, W. Guenther, A. Kohn, H.-J. Kolb, GSF -National Research Center (Munich, D) Minor histocompatibility antigens (mHA) are presented by hematopoietic cells, including leukemia cells and they may serve as targets for graft-versus-leukemia reactions. Adoptive immunotherapy with mHA-specific T cells provides a promising way to treat leukemia relapse. Here we report the production of mHA-specific CTLs in DLA-identical canine littermates as a preclinical model. mHA-specific CTLs could either be generated in vitro by coculture of donor T cells with dendritic cells of a DLA-identical littermate or by immunization in vivo of the donor with skin or dendritic cells of the host. The ability of mHA-specific CTLs to suppress the growth of hematopoietic progenitor cells was tested in vitro in a Delta-Assay using lineage-negative bone marrow as target cells and in vivo using the conversion of mixed chimerism as an indicator of suppression of host progenitor cells. In the Delta-Assay mHA-specific CTLs resulted in a suppression of hematopoietic progenitor cell growth in 57% (12/21) of DLAidentical littermate combinations, ranging from 25% to 98% inhibition. From one littermate combination mHA-specific CTLs were produced both in vitro and in vivo using host-derived dendritic cells. The resulting CTLs were then compared in a Delta-Assay. We observed a growth suppression of 62% using in vivo immunized CTLs and a growth suppression of 44% using in vitro generated CTLs. A DLA-identical donor was immunized against the recipient by a skin graft, followed by transfusion of blood leukocytes. After adoptive transfer of donor T cells mixed chimerism converted to complete donor chimerism within 30 days and it remained complete for 4 years. Finally in vitro co-culture of donor T cells with dendritic cells of the host effectively immunized the T cells to suppress growth of hematopoietic progenitor cells in vitro in the Delta assay (97 % at an effector to target ratio of 5:1). Moreover the in vitro immunized donor T cells were able to convert mixed to complete chimerism within 30 days after infusion of 3.4 x 10 6 /kg mHA-specific CTLs. Neither animal receiving immunized cells showed graft-versushost disease (GvHD). Further transplants will be performed to determine the rate of acute and chronic GvHD associated with this treatment. Both in vivo and in vitro immunization against mHA is feasible in DLA identical littermates. The conversion of mixed chimerism in the dog is a valuable preclinical model of the graft-versusleukemia reaction in human patients.

Lymphocyte subset reconstitution after allogeneic haematopoietic stem cell transplantation from unrelated donors V. Vavilov, T. Zabelina, N. Kroeger, B. Fehse, N. Fehse, A. Pugachov, A. Zander, B. Afanasyev, State Pavlov Medical University, University Clinic Eppendorf (St. Petersburg, RUS; Hamburg, D) The retrospective analysis of 54 unrelated haematopoietic stem cell transplantations (HSCT) was provided in order to determine the influence of transplant procedure characteristics on the quantitative reconstitution of peripheral blood lymphoid subsets. A cohort of 53 adult patients (median age -42 years, range 19-61 years) received allogeneic HSCT from alternative fully HLA matched or 1 locus-mismatched donors in the BMT Unit of the University Clinic in Hamburg. Patients suffers from different haematologic malignancies: acute leukaemias -14, CML -26, MDS -4, multiple myeloma -8, and other malignant lymphomas -3. Bone marrow and peripheral blood were used to receive stem cells in 31 and 23 cases respectively. Median CD34+cells dose was 3,5x10 6 /kg. 33 transplantations were provided with conventional, and 21-with reduced-intencity conditioning. All patients received antilymphocyte globulin (ALG) in different doses to prevent GvHD. Blood samples for lymphoid cells immunophenotyping were obtained approximately 1 month (mean D+35) and 3 months (mean D+101) after transplantation. Cell-line specific markers (CD3, CD4, CD8, CD19, CD56, CD45RA) and as well an absolute lymphocyte count (ALC) were defined. We observed strong decline of all (except NK-cells) lymphoid subsets with their following elevation and normalisation of CD8+ and B-cells. Lymphocyte levels 1 month after HSCT were lower in HLA-mismatched transplantations (for CD4+cells P < 0,05), and in transplantations with CD34+cell dose lower than median (ALC and CD4+cells P < 0,05). They were not affected by stem cell source and preparative regimen intensity. Lower ATGdosage was associated with significantly higher levels of ALC, CD3+, CD4+ and CD8+ cells 1 month after HSCT. There was no association between lymphoid recovery and disease relapse. Some insignificant elevation of all cell levels was observed prior to the development of chronic GvHD. Estimated 5-years overall and progression-free survival was significantly higher in those patients, who reached ALC level 500 cells/mkL or higher 1 month after HSCT (82,7% and 72,2% versus 52,2% and 40,6%, P < 0,05). We are concluding that allogeneic HSCT from unrelated donor associated with severe decrement of lymphoid cells number in early posttransplant period. The rate of this immunosuppression does not depend from conditioning regimen intensity, but from ATG-dosage. The ALC is more predictive for treatment outcome as any lymphoid subset level alone.

J. Park, Y. Jung, J. Choi, H. Lim, Y.J. Kim, H.C. Kim, Ajou university school of medicine, Lifecord Biomedical research center (Suwon, KOR) Dendritic cells(DCs) can be generated either from monocytes or hemopoietic stem cells. We established the ex vivo culture system of DCs from autologous hematopoietic stem cells with GM-CSF and interferon-gamma, and applied autologous DCs in various malignant disorders for the responses and the side effects. 24 patients(4 patients for adjuvant setting) including 9 cases of metastatic renal cell carcinoma, 3 cases of stage III multiple myeloma, 2 cases of metastatic breast cancer and single case each of lung cancer, stomach cancer, ovarian cancer, malignant lymphoma, malignant fibrosing histiocytoma and Kaposi's sarcoma were enrolled in this study. For the stem cell collection, G-CSF (filgrastim, 300ug/day) was given subcutaneously for consecutive 5 days and the leukapheresis started from 4th day of mobilization. 1 x 10 6 of CD34+ cells were collected and cultured in X-vivo media. Cytokines were added every 3 days (GM-CSF for 1st week, Interferon-gamma for 2nd week). On 14th day of culture, the cells were injected intravenously or intradermaly and Interleukin-2 was injected for 2 weeks immediate after DCs injection. DCs injection was given at 4 weeks interval. The characteristics of dendritic cells were assessed by the morphology and functional assay. We also assessed the side effects and the response in tumor size. At the end of 2 weeks of culture, the cells expressed CD11c, CD40, CD80, CD86 and HLA-DR on flowcytometry. Interestingly, about 25% of the cells expressed CD8. IFN-gamma production as measured by permeabilization method was remarkable as well as IL-12 productivity that was augmented with antigen pulsation. The mean age of the patients was 41.5years(ranges 3-73). The mean(± S.D.) counts of injected cells was 30.4(±35.94) x 10e7/kg and the median cycles of DCs therapy was 2(ranges 1-9). 1 case of complete response, 5 cases of partial response, 3 cases of stable disease and 13 cases of progressive disease were observed (the overall reponse rate of 27.3%). 4 cases of adjuvant therapy maintained disease free state. Thrombocytopenia, fever(>38.4oC), headache and chest discomfort were transient and easily controllable. Autologous DCs generated from CD34 positive stem cells infused in patients with malignancies produced negligible side effect. Although the clinical response with reduction of tumor size was modest due to the advanced stage of diseases in majority of patients, further studies are warranted to elucidate the efficacy of the therapy.

Pharmacokinetics of treosulfan in a myeloablative conditioning combination with cyclophosphamide prior to allogeneic haematopoietic stem cell transplantation R.A. Hilger, M.E. Scheulen, R. Trenschel, S. Seeber, D.W. Beelen, Universitätsklinikum Essen (Essen, D) Treosulfan (TREO), a water-soluble bifunctional alkylating agent is currently evaluated in clinical trials for conditioning of patients with hematological malignancies. Due to the advantageous clinical toxicity profile high-dose TREO in combination with cyclophosphamide (CY) has recently been administered to patients (pts) with an increased risk for organ toxicities precluding standard myeloablative conditioning regimens. We have now completed the pharmacokinetic (PK) analyses for intravenous TREO days -6, -5, -4 (12 g/qm: n= 8 or 14 g/qm: n=12) within a dose escalation trial. For PK analyses, a minimal sample strategy was performed. Therefore, at day -6 and -5 of therapy five blood samples were collected and on day -4 six blood samples were taken. In addition, urine samples were collected over 72 hours. A total of S174 316 blood samples and 374 urine samples were analyzed. A two compartment model was chosen for PK fit. Data sets of two patients in the 14 g/qm group and additional two urine data sets within each group were excluded due to faults in sample collection or time course documentation. A comparison of the PK parameters terminal half life, volume of distribution, clearance, and renal clearance revealed no dose dependent differences. After dose escalation of TREO from 12 to 14 g/m², there was a significant increase in the area under concentration vs. time curve and in the peak plasma concentration. This increase was significant comparing both, the PK parameters calculated from a multiple dose model as well as the comparison of the parameters of first TREO dose of each dose group. There was detected a low intra and inter patient variability in the day to day PK calculations. Additionally, in one patient with brain metastases, we were able to analyze low, but relevant TREO concentrations in liquor, taken from an ommaya reservoir. We conclude that the TREO/CY regimen is reasonably well tolerated and provides excellent disease control particularly in early disease stages (Abstract: Trenschel R. et al). Together with the reliable pharmacokinetic profile of high-dose intravenously administered TREO, these data strongly support clinical valuation of the TREO/CY regimen as alternative option for myeloablative conditioning.

Immunotherapy using rIL-2 activated mismatched donor lymphocytes positively selected for CD56+ for the treatment of resistant haematologic malignancies after stem cell transplantation S. Slavin, S. Morecki, M. Shapira, S. Samuel, A. Ackerstein, Y. Gelfand, I. Resnick, M. Bitan, R. Or, Hadassah University Hospital (Jerusalem, IL) DLI cannot be used safely following transplant of haploidentically mismatch stem cells due to the risk of acute and chronic GVHD, which has been associated with significant morbidity and mortality. Alloreactive NK cells may result in anti-tumor effects as well as promote engraftment without causing GVHD. Allogeneic rIL-2 activated NK cell-mediated immunotherapy with donor lymphocytes was pioneered at our Center in the late 80's for patients with HLA matched donors. We have now extended these studies, aiming to maximize anti-leukemia effects while minimizing GVHD using rIL-2 activated NK cells following transplantation of donor stem cells. This approach was done in 8 patients age 4-63 (median 25) years, 3 females and 5 males with hematological malignancies who relapsed or were at very high risk for relapse after SCT. Patients received rIL-2 activated DLI, positively selected for CD56+ with immunomagnetic beads (Miltenyi's CliniMACS). SCT donors were haploidentically mismatched in 3 patients; matched siblings in 4 patients and matched unrelated in 1 patient. Disease categories included MDS in 2; acute leukemia in 3 (AML, ALL and biphenotypic); Hodgkin's Disease in 1; NHL in 2. Method: Donor lymphocytes were incubated for 4 days with rIL-2 6,000 IU/ml at 37C and then positively selected for CD56+. Percent CD56+ after incubation and positive selection ranged 30-71% (median 39%); % CD3+ ranged 2-21% (median 3%). Number of CD56+ cells infused ranged between 10-600 million (median 120 million) cells. Cell infusion was uneventful. No GVHD was observed in 8/8 patients. One patient with relapsed ALL demonstrated CR post of 25 million CD56+ cells/kg but died 8 months later due to pulmonary aspergillosis that was already present at the time he was admitted for SCT. A second recipient of haploidentically mismatched stem cells from KIR nonalloreactive mother for refractory MDS/RAEB-T is >6 months posttransplant in CR with no GVHD after 2 infusions, a total of 4.5 million CD56+ cells. Currently, 4 patients are alive; 1 with disease; 3 with no evidence of disease 9, 13 and 22 months post SCT; 4 patients died. We conclude that rIL-2 activated allogeneic CD56+ can be given post SCT and may result in beneficial anti-leukemia effects with no GVHD.

U. Hartwig, M. Nonn, S. Khan, W. Herr, C. Huber, University Medical School (Mainz, D) Depletion of T-cells from allogeneic bone marrow transplants (BMT) ameliorates graft-versus-host disease (GvHD) but is associated with impaired engraftment, immunosuppression, and abrogation of the beneficial graft-versus-leukemia effect (GvL). We, therefore, explored the possibility of separating alloreactivity from T-lymphocytes mediating GvL-responses ex vivo by selective depletion of GvH-reactive T-cells using CD95/CD178mediated activation-induced cell death (AICD) in HLAmismatched as well as HLA-matched settings. Initially, PBMC derived T-cells were restimulated with fully HLA-mismatched stimulators in an allogeneic mixed lymphocyte culture (MLC) in the presence of agonistic anti-CD95 monoclonal antibody (mAb). Proliferative responses of alloreactive T-cells were consistently found to be reduced to 65-90% in the presence of anti-CD95 reagents depending on the mAb used. ELISpot analysis conducted to evaluate the specificity of depletion by monitoring CMV-pp65 reactive T-cells present in PBMC from seropositive donors before and after AICD revealed the retainment of pp65specific T-cells in the residual allogeneic T-lymphocyte population comparable to frequencies detected in normal controls. Based on the results obtained in HLA-mismatched settings we then performed equivalent experiments in HLAmatched situations in the context of an ALL BMT by stimulating donor T-cells with recipient derived DC. As expected, proliferative responses were quite weak due to low frequencies of alloreactive precursor T-cells, but revealed a 2-2,5 fold increase compared to autologous DC used as stimulators. Addition of agonistic anti-CD95 mAb into MLC again resulted in 95% inhibition of these alloimmune responses. Investigations on residual GvL-and antiviral-specificities of MLC responders after depletion of alloreactivity by AICD are currently in progress. Our studies will ultimately aim at the manipulation of allogeneic T-cell repertoires towards a more effective GvHD prophylaxis without compromising the immunotherapeutical benefits of allogeneic BMT. D. Jeong, N. Chung, B. Choi, S. Park, C. Han, Catholic HSCT Center (Inchon, Seoul, KOR) Donor specific transfusion may induce tolerance due to immune regulatory cells in kidney transplantation. There are reverse immunological situation between graft and host in hematopoietic stem cell transplantation (HSCT), compared with in organ transplantation. Also, early IL-2 injections after allogeneic murine HSCT were shown to prevent lethal GVHD due to induction of CD4+ cells. We investigated induction of regulatory CD4+CD25+ cells after transfusion of irradiated recipient cells with IL-2 into donor. We obtained spleen cells(SP) from 6 week-old Balb/c mice(H-2d) and prepared irradiated single cell suspension. Donor mice(C3H/He, H-2k) received 5E6 irradiated SP and 5,000 IU IL-2 injected intraperitoneally on the day prior to HSCT. We analyzed CD4+CD25+ cells in C3H/He SP injected with recipient cells. Donor SP treated with recipient SP and IL-2 contained more CD4+CD25+ cells(5.4±1.5%) than untreated mice SP(1.4±0.3%). In mixed lymphocyte proliferation assay, Balb/c SP was used as a stimulator, and donor SP with or without treatment. Inhibition of proliferation was 30.0±13% compared to control. Also, we evaluated the proliferation of CD25+ or CD25-cells, isolated by MidiMacs. There was profound inhibition in CD4+CD25+ cells(-60±6%), but marked proliferation in CD4+CD25-cells(129.8±65.2%). Lethally irradiated Balb/c mice were received 1E7 donor BM with 5E6 SP treated with irradiated recipient SP and IL-2 without any separation. Survival in mice with SP treated was longer than in control, although all mice died until post-HSCT day 13. Also, lethally irradiated Balb/c were transplanted with 1E7 donor BM and 5E6 CD4+ CD25+ cells. Other recipient mice received 1E7 donor BM with 5E6 CD4+CD25-cells or untreated SP. There was mild weight loss in CD4+CD25+ group during early postHSCT period, in contrast to severe weight loss in control and CD4+CD25-groups. There showed low GVHD scores and slow progression in CD4+CD25+ group from post-HSCT day 4 to day 9, but a high score and rapid progression in control and CD4+CD25-groups. The probability of survival was 83.3% in CD4+CD25+ group until post-HSCT day 35, and all mice in control and CD4+CD25-groups died on post-HSCT day 8 or 9. We suggest that recipient specific transfusion with IL-2 into donor may be able to induce abundant regulatory CD4+CD25+ cells for preventing GVHD.

The role of donor parity has not been evaluated as extensively as the mismatch (MM) at HLA loci. Hypothetically, allosensitization of maternal T cells to fetal minor HLA or the persistence of fetal cells after childbirth may contribute to an increase in GVHD. We hypothesized that recipient (R) with a female donor (D) having male child (ren) or female recipient having male child (ren) should have an increased incidence of GVHD. Patients and method: We were able to collect R and D pretransplant obstetrical data from database and by phone call survey of 334 patients (med age 30, range:15-58 , M/F:200/134) from our center. They all received HLA identical sibling's stem cells (PBSC/BM:177/157) following ablative conditioning regimen (92%) for CML (41%) or acute leukemia (45%). The overall incidence of acute and chronic GVHD was 45.6% and 55.2%. The contributing factors analyzed for the incidence of GVHD were sex MM (sMM), ABO-MM, donor and recipient age, diagnosis, stem cell source, conditioning regimen (ablative vs reduced intensity (RIC)), parity, male children and G-CSF use in a univariate and multivariate Cox-proportional risk assessment analysis. Results: The univariate parameters influencing overall aGVHD and cGVHD were R age (p<0.05), D age (p=0.006), and sMM (47.5% vs 65.5%, p=0.007), use of PBSC (p<0.0001), recipient age (p<0.0001) and donor age (p<0.0001). In multivariate analysis the factors remaining significant for aGVHD and cGVHD were D age (p<0.05), and sMM (p=0.003), stem cell source (p<0.0001) and R age (p<0.05). Within the sMM group which consisted of 161 patients (79 M to F and 82 F to M), pretransplant parity was observed in 50 cases: 28 F/R with 22 sons (median: 1, range 1-4) and 22 F/D with 15 sons (median: 1, range 1-3). In sMM group, regardless of parity, the incidence of aGVHD and cGVHD was 41% and 59%. The incidence of aGVHD and cGVHD in parous R or D were similar, 36% vs 66%, respectively. Whereas, R with pretransplant male parity have an increased incidence of aGVHD compared to female parity (46% vs 5.8%, p=0.014). When PBSCT was compared to BMT, incidence of cGVHD was more frequent in nulliparous but not multiparous R/D pairs (75.9% vs 21.1%, p=0.04).

Conclusion: Pregnancy and having male children may be contributing but not independent factors for the increased incidence of cGHVD following sMM transplants. In addition, R age and use of PBSC are independent prognostic factors for cGHVD and D age for aGVHD.

Graft-versus-host disease of the liver: 109 cases from a single-centre R.F. Duarte, J. Delgado-Gonzalez, A.F. Quaglia, B. Shaw, D. Wrench, M. Ethell, A.P. Dhillon, S. Mackinnon, M.N. Potter, Royal Free Hospital (London, UK) Despite large numbers of studies assessing prophylaxis, very few have focused on the outcome of liver graft versus host disease (GVHD-L). We have reviewed all cases of GVHD-L included in our data base since 1978 (109 in 767 allogeneic HSCT). 96 cases were included in the analysis after those with concomitant causes of liver dysfunction other than GVHD-L (n=13) were excluded (62 male/34 female; median age 27, range 3-50; Acute: n=80, median onset day 31, range 4-99; Chronic: n=16, median onset day 143, range 103-545; 32 AML, 26 ALL, 20 CML, 7 MM, 4 MDS, 3 NHL, 4 others). Retrospective review included medical history, standard screening tests and, in 45 (47%) cases, liver biopsies. Disease stage at HSCT was CR1/CP1 in 35 cases and more advanced in 61. The source of HSC was bone marrow (n=79) or peripheral blood (n=17) from 74 related (62 matched/12 mismatched) and 22 unrelated (16 matched/6 mismatched) donors. 88 (92%) cases received TBIbased conditioning, while 8 received chemotherapy only. GVHD prophylaxis consisted of cyclosporin plus methotrexate or other immunosuppressive drugs (n=39), T-cell depletion (TCD) alone (n=36) or TCD plus cyclosporin (n=21). First line therapy was glucocorticoids in 98% of cases. Secondary treatment was heterogeneous, with more than 10 single drug or combination protocols used. Median overall survival (OS) was 3.1 months (range 0. 2-130.3) . Causes of death were infection (62%), liver failure (14%), haemorrhage (9%), relapse (6%) or others (9%). Multivariate Cox regression analysis identified the following independent covariates associated with OS: response to treatment (HR=4.3, p<.001), peak bilirubin level >/<6mg/dl (HR=2.5, p=.035), concomitant gastro-intestinal GVHD (HR=2.4, p=.009), GVHD prophylaxis (HR=2.2, p=.029) and HSCT before vs after 1993 (HR=1.87, p=.037). Acute vs chronic onset (p=.005) and underlying disease (p=.017) were significantly associated with OS in the univariate analysis but not in the multivariate model. Other variables known to influence the incidence of GVHD and HSCT outcome, such as age, donor type (un/related, HLA-match, female to male HSCT) and disease status at HSCT were not associated with OS after diagnosis of GVHD-L, nor were the peak levels of AST, ALT, GGT and ALP. The outcome of patients with GVHD-L is extremely poor. The factors we found to be associated with OS should be considered in the selection of candidate patients for new experimental therapies and in the design of future trials.

Patient and donor polymorphisms within the HLA class III genes associate with the outcome of allogeneic haematopoietic stem cell transplantation K. Bogunia-Kubik, A. Lange, L. Hirszfeld Institute, Lower Silesian Center for Cellular Transplantation (Wroclaw, PL) Objectives: In the present study the role of matching for the genes located within human MHC class III region was investigated. Polymorphic features of HSP70-hom, TNF-alpha (TNFA) and TNF-beta (TNFB) genes of patients and donors were analysed and correlated with the outcome of allogeneic haematopoietic stem cell transplants (alloHSCT). Methods: 95 patients (49 transplanted from sibling and 46 from family haploidentical or matched unrelated donor) who had undergone non-T-cell-depleted alloHSCT were typed for HSP70hom (+2763 G/A), TNFA (-308 G/A) and TNFB 1 intron (G/A) alleles. HSP70-hom alleles were detected with the use of ARMS technique. TNF polymorphisms were analysed by PCR amplification followed by digestion with NcoI restriction enzyme. In addition 40 donors of alternative transplants were investigated. Results: Generation of grade II-IV toxic lesions associated with recipient TNFB heterozygosity (28/39 vs 29/56, p=0.05) and was more frequent among HSP-AA homozygous patients (10/11 vs 47/84, p=0.02). A tendency was observed for a higher incidence of aGvHD in patients homozygous for HSP-A than those carrying HSP-G allele (8/11 vs 43/84, p=0.17). Donor TNFA and TNFB genotypes associated with the risk of aGvHD. Patients grafted from family haploidentical or unrelated donors having A in -308 pp. of TNFA (TNFA*2; a marker of a higher TNF-alpha production) less frequently developed aGvHD than those transplanted from GG homozygotes (TNFA*1,1) (5/14 vs 17/24, p=0.034). All 22 donors homozygous for TNFB*2,2 were also TNFA*1,1 positive (p=0.000). Due to the strong linkage between TNFA*1 and TNFB*2, patients who received transplants from donors carrying TNFB*1 allele (G in polymorphic position) less frequently presented with aGvHD than patients grafted from TNFB*2,2 homozygous donors (6/16 vs 16/31, p=0.013). When the outcome of transplants from A, B, DRB1 matched family haploidentical or unrelated donors (n=30) were analysed, a tendency was observed towards the higher incidence of aGvHD among patients transplanted with class III mismatched donors as compared to those transplanted with A, B, DRB1, TNFA, TNFB, HSP matched donors (10/14 vs 8/16, ns) (mostly due to the TNF mismatches). Conclusion: These results imply that polymorphic features of class III genes of patients and donors of alloHSCT influence the risk of post-transplant complications and, that matching for HLA class III alleles may affect the development of aGvHD.

L. Cannella, R. Laylor, H. Dewchand, E. Simpson, F. Marelli-Berg, F. Dazzi, Imperial College London, MRC (London, UK) Donor T cells administered with allogeneic stem cell transplantation (SCT) can cause graft-versus-host disease (GvHD) but they are also responsible for eradicating residual leukaemic cells through the graft-versus-leukaemia (GvL) effect. Although GvHD and GvL recognise an overlapping range of antigenic structures on host cells there is clinical evidence that it is possible to produce a GvL effect without GvHD. GVHD seems to be a consequence of the cytokine storm induced by the pretransplant conditioning regimen. Cytokines such as TNFa, implicated in the pathogenesis of GVHD, can also regulate lymphocyte extravasation by activating the vascular endothelium. We tested the hypothesis that the cytokine storm at transplantation influences donor T cells homing and extravasation into tissues. We addressed this question into an animal model in which donor T cells were administered intravenously in lethally irradiated (800 cGy) syngeneic recipients. In this system the migration of T cells is not influenced by the alloantigens but only by the tissue damage induced by the conditioning treatment. Donor T cells, stained with carboxyfluorescein diacetate N-succinimidyl ester (CFSE), were detected at different time after infusion in subcutaneous and mesenteric lymph nodes, spleen, bone marrow, and blood by flow cytometry or confocal microscopy. Donor T cells exhibited different tissue infiltration patterns following total body irradiation as compared to untreated recipients. Donor T cells were already measurable at 4h after infusion but were maximally detectable 48h after the injection; at 96h cells CFSE-labelled T cells disappeared in the untreated animals, whereas still persisted in the irradiated recipients. T-cell infiltration was mainly visible in lymph nodes and spleen in the irradiated mice, whilst lymph nodes and blood were the major target tissues in non-irradiated recipients. Since naïve and memory T cells display distinct recirculation patterns, we analysed the migration of primed and unprimed T cells injected into irradiated animals. Primed T cells preferentially infiltrated spleen and bone marrow, while the unprimed T scell migrated especially in the lymph nodes and the spleen. Our data suggest that the conditioning regimen pretransplant strongly influences the migration of donor T cells. This different pattern might determine the occurrence of GvHD.

HLA-DPB1 mismatch is associated with acute GvHD after HLA-identical sibling donor stem cell transplantation D. Gallardo, S. Brunet, A. Torres, M. Alonso-Nieto, C. Vallejo, A. Jiménez, M. González, G. Sanz, D. Serrano, I. Espigado, S. Osorio, E. Carreras, C. Martín, C. Sanz-Rodríguez, J. Sierra, J. Zuazu, M.F. González-Escribano, J. R. González, J. Román, J. Pérez de Oteyza, R. 

The role of HLA-DPB1 as a transplantation antigen is controversial. A higher incidence of acute graft-versus-host disease (aGvHD) has been described in recipients of an unrelated bone marrow transplant when both HLA-DPB1 alleles were mismatched.The incidence of HLA-DPB1 mismatch (generated by haplotype crossover) between HLA-A-B-DRB1 matched siblings is 4-6%. The study of the association of this mismatch and the incidence of aGvHD after HLA-A-B-DR identical sibling donor transplantation should clarify the biological role of HLA-DPB1 in allogeneic SCT. We investigated the impact of a single HLA-DPB1 mismatch after HLA-A-B-DRB1 identical sibling donor transplantation on aGvHD. We analyzed 627 adult patient-donor pairs and identified 30 pairs without HLA-DPB1 identity (4.78%). In all cases, only one allele was mismatched. In 17 cases, the patient had an allele that was not shared by the donor. The cumulative incidence of grades II-IV aGvHD was 66.7% in the HLA-DPB1 mismatched group and 35.7% in the matched pairs (p: 0.012). When analyzing the relationship between HLA-DPB1 mismatch and aGvHD in the patients receiving a non-T-cell depleted graft, this association was even more evident (cumulative incidence of grades II-IV aGvHD: 80.0% versus 36.7%; p: 0.005). The HLA-DPB1 mismatch was identified by multivariate analysis as an independent risk factor for aGvHD (RR: 2.68; 95%CI: 1.73 -3.62; p: 0.020). HLA-DPB1 mismatch was not associated with a higher risk of chronic GvHD. Relapse incidence and overall survival were similar in both groups. In conclusion: HLA-DPB1 can mediate alloreactive responses. A single HLA-DPB1 mismatch increases the risk of aGvHD after sibling donor stem cell transplantation, but it does not affect transplant outcome. Financed by a grant Fundació Josep Carreras and by grant FIS PI02-0148.

GvHD after allogeneic peripheral blood stem cell transplantation: comparison between conventional and reduced-intensity conditioning regimens J.A. Perez-Simon, E. Ocio, M.D. Caballero, V. Mateos, F.M. Sanchez-Guijo, L. Vazquez, J.F. San Miguel, Hospital Clinico de Salamanca (Salamanca, E) Introduction:

Chemotherapy-induced toxicity significantly decreases after reduced intensity conditionig regimens (RIC). This decreased toxicity would also reduce the inflamatory cytokine production associated with the high doses of chemotherapy, thus disminishing "cytokine storm" which contributes to the development of graft-versus-host-disease (GVHD). Patients And Methods: In the current study we have compared the incidence and characteristics of GVHD between patients receiving RIC allo-transplant (n=60) vs myeloablative conditioning regimen (MCR) (n=49) in the same period of time. In terms of GVHD, cumulative incidence of acute graft-versushost-disease (aGVHD) was lower in RIC as compared to MRC patients (45 % vs 63 % respectively, p=0.01), but no differences in TRM directly attribuited to aGVHD were observed (p=0.54). Cumulative incidence of chronic graft-versus-host-disease (cGVHD) was similar in both groups (76 % vs 72 % in RIC vs MRC patients, respectively, p=0.84). Response to GVHD (both acute and chronic) first line treatment was similar in RIC and MRC patients. Conclusion: This study shows that RIC significantly decrease incidence of aGVHD as compared to MRC using the same GVHD prophylaxis even through RIC patients are significantly older.

J. Shankari, M. Ortin, S. Patel, R. Ridwan, P. Fomasagaram, R. Sinha, S. Ball, J. Treleaven, St. George's Hospital Medical School, The Royal Marsden Hospital (London, Sutton, UK) Campath is widely used for in vivo T-cell depletion in patients undergoing haemopoietic progenitors transplant (HPT) as prophylaxis for graft versus host disease (GvHD). However, concerns regarding an increased incidence of viral infections has been raised. In this study we evaluate our experience with Campath in children receiving allogeneic HPT, where GvHD and viral infections are known to have a lower incidence than in adults. Patients and methods: Records from all the allogeneic paediatric HPT performed at our centre between 1996 and 2003 (n=108, median age 11±4.6 years) were considered. 60 patients received grafts from unrelated donors (UD) and 48 from related donors (RD). Indications for HPT included ALL (75 cases), AML (12), MDS (6), CML (10), undifferentiated (3), Lymphoblastic NHL (1), HLH (1) . Campath was administered to 54 patients as part of the conditioning. Endpoints for this study were incidence of viral infections, incidence of GvHD, overall outcome (alive/dead) and cause of death (relapse/toxic). Overall, children receiving Campath experienced a significantly higher incidence of viral infections (20/45 vs. 10/53, p<0.04), including CMV reactivations (9/19 vs. 4/22, p<0.05). Children having RD HPT, the administration of Campath did not influence the incidence of GvHD or toxic deaths. The incidence of GvHD in children having UD transplants remained unaffected by Campath. However, children having UD HPT experienced a significantly (p<0.02) lower incidence of severe forms of chronic GvHD, so the need for its treatment was also significantly (8/42 vs. 4/7; p<0.05) lower. No influence on acute GvHD was seen. A non significant higher survival in children having Campath was found in this group (32/52 vs. 2/7). No significant differences in the cause of death were seen. When mismatched UD HPT recipients (n=19) were compared with matched UD, an overall worse performance (p<0.04) in terms of deaths was found. However, these differences disappear when only children receiving Campath are considered. Conclusion: Campath has a beneficial effect in UD HPT in children by reducing the incidence of severe forms of chronic GvHD. In mismatched UD grafts, it improves the overall performance, making it comparable to matched grafts. The increased incidence of viral infections seen in this study does not seem to influence the overall outcome of these patients.

R. Garland, S. Groves, J. Hallinan, S. West, K. Winship, S. Robinson, A. Oakhill, J. Cornish, D. Pamphilon, D. Marks, N. Goulden, C. Steward, University of Bristol, Bristol Royal Hospital for Children (Bristol, UK) Antibodies CAMPATH-1H and anti-thymocyte globulin (ATG) are used as part of the conditioning regimen prior to SCT to prevent graft rejection and for GVHD prophylaxis. CAMPATH-1H is a humanised antibody recognising cell surface marker CD52, whilst ATG (Fresenius) is a rabbit polyclonal raised to the Jurkat T cell line. The objective of this study was to monitor the persistence of these T cell depleting antibodies in SCT recipients. Firstly, a flow cytometry-based assay was performed to determine the levels of CAMPATH-1H or ATG in patient serum samples compared to a standard curve. The binding of these antibodies to the cell lines Hut-78 or Jurkat respectively was assessed after the addition of FITC-conjugated secondary antibodies. Thirty-two patients were treated with CAMPATH-1H and 7 with ATG. For CAMPATH, the maximum serum level recorded for the 12 patients with samples available within 24hours of treatment was 3.56 (1.78-8.18 ) (microg/ml, median and range). The highest serum sample level achieved for each of the 32 patients was 3.56 (0. 1-22.8) . At least 28 days after CAMPATH treatment, serum levels were detectable in 17 patients at 0.6 (0.12-7.72) and undetectable (<0.1microg/ml) in 4/21. For ATG the maximum serum level was 9.98 (2.19-29.27) and it persisted at levels of 0.45 (0.11-2.70) at least 28 days after treatment in 5/7 patients. Secondly, a functional Chromium release antibody dependent cellular cytotoxicity assay was performed. CAMPATH levels similar to those persisting in patients around 28 days after treatment (~0.1microg/ml) were able to sensitise normal PBL to lysis by autologous effectors. Knowledge of the levels and persistence of such antibodies may allow us to optimise antibody dosing schedules and to avoid donor lymphocyte infusions at times when T cell depleting antibodies still persist at functional levels.

F. Patriarca, A. Sperotto, S. Prosdocimo, D. Damiani, G. Morreale, A. Olivieri, F. Bonifazi, G. Milone, J. Peccatori, P. Corradini, R 

Infliximab (Remicade) is a chimeric human and murine antibody that inhibits TNF-alpha, a key cytokine in the inflammatory cascade of acute GVHD. We evaluated efficacy and toxicity of infliximab in 29 patients with acute GVHD: 6 were children, 23 were adults with a median age of 39 years (range 25-61). Twenty were male and 9 female. Diagnoses were: acute myelogenous leukaemia (11), acute lymphoblastic leukaemia (5), chronic myelogenous leukaemia (2), lymphoma (4), multiple myeloma (4), Fanconi anaemia (1), solid tumour (2) . Eighteen patients received a conventional myeloablative treatment, which included total body irradiation in 9 cases, whereas the other 11 patients received a reduced intensity regimen. Stem cells came from 16 sibling donors and from 10 matched unrelated donors. GVHD prophylaxis included: cyclosporine (28), methotrexate (24), ATG (12), mofetil mycofenolate (MMF) (1), tacrolimus (1) . Median time between BMT and occurrence of GVHD was 22 days (range 10-161). GVHD at the initiation of infliximab was grade II (3 ), grade III (8) and grade IV (18). Organ involvement included: skin (7), gastrointestinal tract (29), liver (13). Fifteen patients had 2 or more organs involved. All patients had previously received treatment with 2 to 5 mg/day prednisone in addition to cyclosporine (27), ATG (14), tacrolimus (7), MMF (3), azathioprine (2) . Patients received infliximab at the dose of 10 mg/Kg weekly for a median of 3 doses (1) (2) (3) (4) (5) (6) (7) (8) . There was no infusion-related side effects. Five patients (17%) had a complete response, 14 (48%) had a partial response, whereas 10 (35%) were refractory. Age below 35, predominant gut involvement and occurrence of GVHD later than 30 days after BMT significantly correlated with a favourable response to infliximab. At present 14 patients were alive with a median follow-up of 340 days (range 31-930) after BMT, one of them had a recurrence of the haematological disease, 5 of them had signs of chronic extensive GVHD. Fifteen patients were dead at a median of 44 days (20-330) after the occurrence of GVHD. Causes of deaths were infections (4), thrombotic thrombocytopenic purpura (1), refractory GVHD (10). Fatal infections occurred in 4 complete or partial responders to infliximab. We conclude that infliximab induced clinical improvement in 65% of patients with severe steroid-refractory GVHD; the greatest benefit was reported by patients with intestinal involvement and late onset of GVHD.

The IL2R is a heterotrimeric receptor consisting of the a-chain (CD25) and the two signal transducing b-,g-chains. Anti-CD25 monoclonal antibody (Daclizumab) binding to the a-chain, blocks high affinity IL2 binding, thereby preventing complete T cell activation. This opportunity to hinder T cell triggering is of ample importance in transplantation medicine and the treatment of autoimmune disease; e.g. for the prevention of an acute graft versus host reaction during allogeneic hematopoietic cell transplantation. However, gene targeting experiments revealed, that CD25 has an important role in mediating activation induced cell death (AICD) thereby maintaining T cell homeostasis. Thus, CD25 antibodies may not only block T cell activation but may also prevent AICD attributing a dual function to IL2, which has been described by the term AICD paradoxon. The molecular mechanisms of AICD remain to be elucidated. In this study, the modulation of the genomic expression profile of human peripheral blood mononuclear cells (PBMC) with therapeutic concentrations of anti-CD25 mAb was investigated with the aim to identify genes that are involved in T cell activation or in AICD. PBMC were stimulated with OKT3 together with recombinant IL2 in the absence or presence of 30 µg/ml Daclizumab. Cells were incubated for 16 hrs, RNA extracted and subjected to microarray analysis on U133A gene chips (Affymetrix). Gene chip profile revealed up regulation of 60 genes and down regulation of 36 genes respectively, by Daclizumab. Anti-CD25 treatment inhibitied cytokine genes typically expressed during T cell activation including CD40L, IL9, LT and IFN-g as previously shown (Burdach et al., JCI) . Surprisingly, Daclizumab also blocked expression of several genes important for susceptibility to apoptosis as well as IL2-mediated repression of anti-apoptotic genes. Microarray analysis of these apoptosis related genes was confirmed by RT-PCR and functional assays. In conclusion, CD25-mediated induction of pro-apoptotic as well as repression of anti-apototic gene clusters should be considered for future drug development of CD25-antibodies in the clinical arena: these apoptosis related gene products may represent new pharmacologic targets in hematopoietic cell tranplantation as well as in the treatment of autoimmune diseases.

E. Nadal, S. Marktel, R. Szydlo, E. Olavarria, E. Kanfer, A. Rahemtulla, J. Apperley, J. Goldman, F. Dazzi, Imperial College (London, UK) The incidence and severity of acute GVHD in patients receiving donor lymphocyte infusions (DLI) is much reduced when cells are administered using an escalating dose regimen (EDR). We have investigated factors for GVHD in patients undergoing EDR-DLI. We analyzed 60 pts treated for chronic myeloid leukemia in relapse after conventional allogeneic SCT. The median time between relapse and transplant was 9 months. Twenty-one patients were in molecular, 22 in cytogenetic and 17 in haematologic relapse. Patients transplanted with a sibling (SIB) donor (n=26) received 10 7 -> 5x10 7 -> 10 8 donor CD3+ cells/Kg. Patients transplanted with a volunteer unrelated donor (VUD) (n=34) received 10 6 -> 10 7 -> 5x10 7 -> 10 8 donor CD3+ cells/Kg. The median time between relapse and DLI was 7.8 months; the median interval between doses was 22 weeks. Fifty five patients (91%) achieved molecular remission. The overall incidence of acute (grade II-IV) and chronic GVHD was 21% and 13%, respectively. We observed a correlation between GVHD and response to DLI: all infusions resulting in acute GVHD grade II-IV also produced molecular remission (12/12), whereas 43 of the 121 infusions (36%) not associated with GVHD (< grade II) resulted in molecular remission (p=0.00001). Accordingly, none of the 12 patients who developed acute GVHD grade II-IV relapsed compared to 4/43 (9%) of those with GVHD < grade II post-DLI. Among the factors influencing the severity of GVHD the interval between transplant and initiation of DLI was the most important one. Thus 43% of the patients who commenced DLI less than 1 year from transplant developed acute GVHD (II-IV) as compared to 15% who received the first dose at least 1 year after transplant. A higher cell dose was also related with GVHD II-IV. The donor type affected incidence but not the severity of both acute and chronic GVHD(VUD 47% and 17% vs SIB 23% and 7%). We conclude that donor type, accumulated cell dose, and interval between transplant and first DLI are important risk factors for GVHD post-DLI. The presence of GVHD >II was strongly associated with a prolonged remission.

Extracorporeal photochemotherapy for acute and chronic GvHD, a single-centre report of 43 patients E.J. Dann, E. Badian, I. Avivi, N. Haddad, T. Stravets, R. Fineman, L. Bonstein, A. Oliven, E. Kertsman, T. Zuckerman, J.M. Rowe, Rambam Medical Center (Haifa, IL) Acute and chronic GVHD remain the leading cause of morbidity and mortality of allogenic stem cells transplantation. Immunosuppressive therapy is the common practice to combat GVHD, however it heavily impacts on post transplant rate of infection and in associated with secondary malignancies affecting both morbidity and mortality. Exracorporal phototherapy (ECP) has been previously reported to be a useful modality treating patients with coetaneous T cells lymphoma, various autoimmune diseases and GVHD. We report here our five-year experience of ECP for acute and chronic GVHD patients from 11/1992. Mononuclear cells were collected using Cobe -Spectra. One and half to 2 blood volumes were circulated through the cell separator with a product of approximately 5X10 9 mononuclear cells. When product hematocrit was less then 3%, volume adjusted to 300ml and to 500ml if higher. 8 Methoxy psoralen (8MOP) was added to final concentration of 0.2ug/ml. The product was irradiated at 2 Joule /cm2 of UVA (365nM) (Biogenetic Vilber Lourmat). Cells were then re transfused. The treatment protocol consisted of 2 consecutive ECP performed at bi weekly intervals until clinical improvement. Forty-three patients 27, males and 16 females, age 1-55y (median 31 yrs), were treated. Patients underwent allogeneic BMT for the following indications. AML, ALL, CML, MDS, NHL. Eight patients were treated for acute GVHD and 36 for chronic GVHD. Diffuse GVHD of skin was diagnosed in 32, gastro-intestinal tract 5; joint involvement 10, and liver involvement 26 patients. Patients had from 2 to 66 ECP (median 25). Results: 15 pts succumbed, 7 with acute GVHD and 8 with chronic GVHD. Improvement of GVHD was reported in29 patients. While 2/10 (20%) pts with acute GVHD responded, the response in chronic GVHD was much higher 27/ 36 (75%). Conclusions: ECP is a useful modality of therapy and is most effective in patients with chronic GVHD, for both liver and skin involvement, and less effective for patients with scleroderma. It is suggested that this modality should be used in early management of these patients concomitantly with immune suppressive therapy.

S. Vidal, M. Morante, J. Briones, R. Martino, J. Sierra, J. Rodriguez-Sanchez, Institut Recerca Hospital Sant Pau (Barcelona, E) Acute graft-versus host disease (aGvHD) is a major complication after allogeneic stem cell transplantation with donor T cells being involved in the effector phase. In addition to the antigen-specific signal mediated by the T-cell receptor, costimulatory signals provided by antigen-presenting cells are required for a complete T cell activation. The expression of costimulatory molecules involved in aGvHD is of interest since blockade of such molecules may be considered as a part of novel therapeutic strategies. In the present study, we analyzed by multicolor flow cytometry the expression of a number of activation and costimulatory T cell molecules such as CD25, CD69, HLA-DR, CD154 (CD40L) and CD134 (OX-40) on CD4+ and CD8+ T cells during aGvHD. Twenty-four patients receiving HLA-identical stem cell transplants were studied. Nineteen patients received peripheral blood stem cells after reduced intensity conditioning; three received a myeloablative CD34+-selected graft from a related donor and two received an unrelated myeloablative non-manipulated bone marrow transplant. Expression of activation and costimulatory molecules from patients with stage II-IV aGvHD (n=9, 4 skin, 3 intestine, and 2 skin plus intestine) were compared with patients without aGvHD (n=15). Serial analysis showed that peripheral blood lymphocytes of patients with aGvHD presented a 2 to 3-fold increase of CD4+CD69+ (p<0.05), CD4+DR+ (p<0.05) and CD4+CD25+ cells (p<0.03) as well as CD4+CD134+ cells (p<0.01). Additionally, there was a significant increase of CD8+ cells expressing the costimulatory molecules OX40+ (p<0.01), and CD40L+ (p<0.02). After resolution of aGvHD, the increased expression of these molecules returned to values comparable to those observed in patients without aGvHD. At several points, only two of the fifteen patients without clinical signs of aGvHD presented a high percentage of CD4+ T cells expressing activation markers that could not be attributed to the development of aGvHD. In summary, our data show that multiple activation molecules are preferentially upregulated on CD4+ and CD8+ cells from patients with aGvHD. Of note, these patients had a significant increase in the expression of the costimulatory molecules OX40 and CD40L. These data may be useful for designing novel strategies in the treatment of aGvHD patients. Graft-versus-host disease (GvHD) can be a major complication after allogeneic stem cell transplantation (SCT) especially when donor and recipient are unrelated. Both the selection of the best stem cell donor and prediction of the development of acute GvHD prior to SCT in one simple test system is still an attractive prospect. The destruction of recipient's tissues and cells during GvHD is mediated by donor-derived cytotoxic T lymphocytes and natural killer cells either by an apoptotic mechanism which involves the pore-forming protein perforin and granzymes or by the interaction of the Fas/APO-1 molecule with its ligand. We measured GrA and GrB production levels in the supernatants of 96 hours pretransplant mixed lymphocyte cultures (MLC) of 26 sibling and 31 unrelated patient/donor pairs by enzyme linked immunosorbant assay (ELISA). The GrA and GrB production levels of 37 potential patient/donor pairs were correlated with relative responses (RR) of MLC and with HLA class II mismatches and with the development of acute GvHD in a cohort of 20 sibling SCT recipients. In vitro measurement of GrA and GrB production levels significantly correlated with the RR of pre-transplant MLC (r=0.492, p<0.01 and r=0.853, p<0.01, respectively) and increased with the number of HLA class II mismatches between patient and donor. Pre-transplant GrA production levels were significantly associated with the in vivo development of acute GvHD grades II-IV in patients transplanted with an HLA-identical sibling donor (p<0.001).

In conclusion, in vitro GrA and GrB production levels can be measured by a quantitative and sensitive ELISA. This method may be used for functional selection of unrelated stem cell donors and for the identification of patients who are at risk for acute GvHD grades II-IV. Klein, G. Bug, B. Waßmann, D. Hoelzer, H. Martin, Klinikum der Johann Wolfgang Goethe-Universität (Frankfurt, D) Steroid refractory intestinal acute Graft versus Host Disease (aGvHD) is a life threatening complication after allogeneic hematopoietic cell transplantation. No therapy has been proven to increase the survival rate in steroid refractory aGvHD so far. A new strategy is the use of Pentostatin, a purine nucleotide analogue and inhibitor of Adenosine deaminase, which is known to decrease function and number of lymphocytes. Here we report the follow-up of a pilot study on salvage therapy of aGvHD by Pentostatin. We treated 15 patients with steroid refractory acute intestinal GvHD stage III or IV with Pentostatin (6 female and 9 male patients). The mean age was 45 (range: 25-61 years). The underlying diseases were ALL (4), AML (8), Multiple Myeloma (1), low grade NHL (1) and CML (1) . Eight patients were allografted with HLA-identical sibling donors, one with a related donor with a HLA A mismatch and six with unrelated donors. All patients were allografted with peripheral blood stem cells. As GvHD prophylaxis Ciclosporine A and Mycophenolate mofetil were used. Ten patients had an acute GvHD stage III, five stage IV. All patients had a severe GI-tract involvement. After failure of steroid treatment (Prednisolone >2 mg/kg) Pentostatin was applied as a salvage therapy (1 mg/m 2 for three consecutive days). Six patient received between one and three further courses of Pentostatin every three to four weeks. Therapy was well tolerated. No severe neutropenia or impairment of renal function were observed. Nine patients achieved a complete, four a partial remission of aGvHD. The time until a clinical improvement could be observed was 12-14 days. Two patients died before day 12 without improvement of GvHD symptoms. Six patients are alive (240-1350 days post transplantation), two with an extensive chronic GvHD. Nine patients died (two due to relapse of leukaemia, seven transplant related). In summary, in this pilot 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. By a multicenter prospective randomised study we are investigating whether Pentostatin can improve the outcome in steroid refractory intestinal aGvHD.

A.S. Hassan, M.A. Elawad, G. Davies, K. Rao, P. Amrolia, P. Veys, Great Ormond Street Hospital (London, UK) Objective: Gut graft versus host disease (g-GVHD) is a major cause of morbidity in paediatric haemopoietic stem cell transplantation (HSCT). The aim of the study was to assess the incidence, risk factors and outcomes of graft versus Host disease of the gut (g-GVHD) in paediatric HSCT. Methods: 130 children (74M/56F) who underwent 139 allogeneic HSCT between January 2000 and July 2003 were evaluated retrospectively. Median age at transplant was 4.1 years (range 0.13-17.74), the mean duration of follow-up post HSCT was1.8 years (range 0. 3-3.8) . We examined the prognostic significance of gastrointestinal (GI) disease prior to HSCT, donor match, the presence of GI infection both pre and post HSCT, type and intensity of conditioning, donor/recipient sex match, and T-cell depletion. The diagnosis, staging of g-GVHD and overall GVHD grade was made according to Glucksberg criteria. Results: 94/130 (72%) developed GI symptoms following HSCT. 52/130 (40%) developed g-GVHD, of which 35/52 had grade I-II and 17/52 had grade III-IV GVHD. 26/52 underwent gut biopsy of which 15 (58%) showed histological features of GVHD. The median time from onset of symptoms to a positive biopsy was 14 days compared to 50 days for children with negative biopsies. 24/45(52%) children with pre-HSCT GI symptoms developed g-GVHD compared to 28/85(32%) children with no pre-existing GI disease (P = 0.02). MMUD 12/19(63%) had significantly higher incidence of g-GVHD compared to other donor types (P=0.05), MFD 7/13(53%), MUD 20/54(37%), haplo 5/15(33%) and MSD 7/27(25%). 62/94 children (66%) had proven infection on stool examination post HSCT. The commonest infection encountered was adenovirus 38/62 (61%).

The incidence of g-GVHD amongst this group was 29/62(46%) compared to 23/68(33%) without GI infection (P=0.13). There was no significant difference between donor/recipient sex match, type or intensity conditioning, or T-cell depletion in those developing or not developing g-GVHD. The overall survival rate was 106/130(82%). Mortality was 7/35(20%)in patients with grade I-II g-GVHD and 3/17(18%)in patients with grade III-IV this was comparable to mortality in patients without g-GVHD 14/78(18%)(P=0.8) Conclusion: This is the largest report to date examining risk factors for g-GVHD in the paediatric population. The major risk factors appear to be the presence of pre HSCT GI disease and the type of donor used with MMUD being at highest risk. Timing of biopsy appears to be important in obtaining histological confirmation; we recommend that biopsies are obtained within 14 days of onset of symptoms. Although mortality is not increased significantly improved GVHD prophylaxis and or early intervention may reduce morbidity in these patients.

Objective: Steroid refractory GVHD is a major cause of morbidity and mortality after allogeneic transplantation. The response rates of grade III-IV GvHD to second line therapy are 20-30%, while long term survival is less than 10% (Arai et al. 2002) . Patients: Thirty five patients with steroid refractory acute GvHD of the intestine grade III-IV were treated with a combination of Daclizumab (anti-CD25) and Sirolimus. Twenty two patients had concurrent grade III-IV GvHD of the liver. Daclizumab (1 mg/kg) was given on days 1, 4, 8, 15, 21 and 28 . Sirolimus was given orally. Blood levels were targeted at 10-15 ng/ml. The underlying diseases were AML (18), ALL (9), CML (2), NHL and MMY (6). Twenty patients had an unrelated donor, 9 had a matched sibling donor and 6 an haploidentical sibling donor. The stem cell source was bone marrow (6), peripheral stem cells (21) and both (6). In two patients GvHD developed after donor lymphocyte infusion. Diagnosis of intestinal GvHD was confirmed by total colonoscopy in most cases. GvHD of the liver was diagnosed clinically. Results: No adverse reactions related to infusion of the antibody were observed. FACScan analysis of peripheral blood revealed that CD25 was undetectable on T-lymphocytes after the first antibody infusion. Overall response to therapy was 48% (17/35) with 37% complete responders (13/35). The median time to response was 21 days. Those patients with concurrent liver GvHD responded poorly to therapy (1 CR and 4 PR) . Parallel viral reactivations occurred in 19 patients (8 x HHV6, 8 x EBV, 2 x CMV, 1 x Adeno). Despite the high response rates twenty six patients died. Five patients died of relapsing acute leukaemia, 8 of GvHD and 13 of infective complications -most often sepsis and pneumonia. Conclusion: The combination of Daclizumab and Sirolimus provided potent immunosuppression and was highly effective in intestinal GvHD. GvHD of the liver was refractory to therapy. Prospective trials are warranted to define the role of Daclizumab and Sirolimus in the second line therapy of acute GvHD. Considering the high incidence of infective complications prevention of severe GvHD without hampering the GvL effect remains the major obstacle in allogeneic stem cell transplantation.

M. Mohty, C. Faucher, S. Bagattini, V. Bardou, K. Bilger, B. Gaugler, C. Chabannon, P. Ladaique, C. Lemarie, N. Vey, J.A. Gastaut, D. Maraninchi, D. Olive, D. Blaise, Institut Paoli Calmettes, INSERM U119 (Marseille, F) We previously reported that the incidence of acute GVHD (aGVHD) was significantly associated with the ATG dose (Sangstat*) infused during RIC using fludarabine, busulfan and ATG (Mohty et al., Blood, 2003) . The impact of ATG is likely related to a dose-dependent in vivo T cell depletion. Here, we investigated whether a correlation exists between graft composition (CD34+, CD4+, CD8+, CD19+ and NK cells) and transplant-related events. 71 consecutive pts from a single center receiving HLA-identical RIC allogeneic transplantation, were analyzed. Pts and graft characteristics are: median age 50 y. (18-60), diagnosis: 34 myeloid malignancies (48%) and 37 lymphoid malignancies (52%). 51 pts (72%) were considered as high risk. 38 pts (53%) received a low ATG dose (2.5 mg/Kg), while 33 received the highest dose (7.5 or 10 mg/Kg). 40 pts (56%) received a PBSC graft and 31 (44%) received BM. All pts received CsA alone for GVHD prophylaxis. Median times for ANC and platelet recovery were 15 (10-23) and 14 d (0-99). Increasing CD34+ cell doses were associated with accelerated neutrophil engraftment (P=0.001). 42 pts had aGVHD [13 grade I (18%), 12 grade II (17%) and 17 grade III-IV (24%)]. Chronic GVHD (cGVHD) occurred in 36 [8 limited (11%) and 28 extensive (39%)] out of 55 evaluable pts. Univariate analysis showed that stem cell source, ATG dose, CD4+, CD8+, CD19+ and CD56+ cells infused, significantly influenced the risk of aGVHD. However, in multivariate analysis, CD8+ T cell dose (>69x10 6 /Kg) was the only parameter significantly associated with the risk of aGVHD (P=0.0002; RR=3.5; 95%CI, 1.8-6.7). The impact of CD8+ cell dose on aGVHD could be also confirmed if restricting the analysis to PBSC recipients (P=0.02). In contrast, in multivariate analysis, the risk of cGVHD was not statistically associated with lymphoid subsets, but was associated with grade II-IV aGVHD (RR=2.5; 95%CI, 1.2-5.0) and stem cell source (PBSC; RR=2.35; 95%CI, 1.1-5.0), suggesting that other cell subtypes (e.g. dendritic cells…) might contribute to long term graft-versus-host reactions. With a median FU of 31 m (5-58), 37 pts are alive with OS being significantly higher in pts experiencing aGVHD and/or cGHVD as compared to pts without GVHD (P=0.004). Overall, these results suggest that graft manipulation, subtle changes such as in the ATG dose or the choice of the stem cell source may have a significant impact on the probability of a favorable outcome after RIC transplantation.

R. Stocchi, D. Damiani, P. Masolini, A. Michelutti, A. Geromin, M. Cerno, A. Sperotto, M. Baccarani, R. Fanin, University Hospital (Udine, Bologna, I) In peripheral blood (PB) two subsets of dendritic cells (DC) have been identified: DC1, myeloid, lineage negative, HLADR positive,CD11c positive; DC2, lymphoid, lineage negative, HLADR positive and CD123 positive. DC1 activate T lymphocytes, while DC2 induce antigen-specific tolerance. We used a 3-colour flow cytometric assay to assess DC1 and DC2 reconstitution in 87 patients undergoing allogeneic transplantation for haematological malignancies: 23 patients (group 1) received CD34 positive selected PBSC plus 1times 10(e)7 CD3 positive cells from an HLA identical sibling donor, 31 patients (group 2) received unmanipulated bone marrow stem cells from a matched unrelated donor, 33 patients (group 3) received unmanipulated PB stem cells from a matched related (n=18) or unrelated (n=15) donor. 58 autologous transplant patients were used as control group. Before conditioning regimen the mean DC1 and DC2 number/microliter was 2.9 and 3.1 (group 1), 2.0 and 2.5 (group 2) and 2.1 and 2.4(group 3): these numbers were similar to those of autologous patients (2.9 and 2.8), but lower than in normal donors (6.0 and 6.7). In autologous patients at day 0 and +7 the DC1 and DC2 number was lower than 0.02while in allogeneic patients DC1 and DC2 could be not detected. All autologous patients recovered to the pre-transplant number of DC1 and DC2 within day plus 20 (3.0 and 2.7), reaching to normal numbers after 6 months from transplant (6.2 and 5.7). In allogeneic patients, pretransplant values were reached at day +90 in group 1 (2.7 and 3.5) , at day +180 in group 2 (3 and 3.1) and at day +270 in group 3 (4.5 and 2.1. One year after transplant mean DC1 and DC2 number was still lower than in normal subjects in every group. The delay of recovery in group 2 and 3 could be related to major incidence of acute and chronic GVHD. DC1 and DC2 absolute number was lower in patients who developed acute and chronic GVHD. Moreover in these patients the DC1/DC2 ratio was always more than 1.

In conclusion, DC1 and DC2 recovery is markedly delayed following allogeneic transplant if compared with autologous: this may be linked to a delay in immune system reconstitution and/or to the onset of acute and chronic GVHD. We used a MHC mismatched mouse model to investigate the possibility of pretreatment of donors with G-CSF plus/minus methylprednisolone before bone marrow harvest as a new GVHD prophylactic strategy. In the present study, male C57BL/6 (H-2b) was used as donors and female BALB/c (H-2d) as recipients. Donors received a daily injection of 250ug/kg G-CSF for 4 days with or without a single intraperitoneal injection of 100mg/kg methylprednisolone on day 3. Mice given PBS instead of G-CSF were used as controls. Bone marrow cells (5x10 6 ) with splenic cells (2x10 7 ) were given to each lethally irradiated (800 cGy) recipient by tail vein injection. The severity of GVHD was assessed with a clinical GVHD scoring system and a semiquantitative scoring system for histological examination. The WBC counts in groups of G-CSF and G-CSF+MP were 6.9 and 4.6 times higher than PBS controls. The numbers of nucleated cells and CFU-GMs harvested in G-CSF groups were 2~5 times higher than PBS controls. The ratio of CD4/CD8 was 1.4±0.05 and1.47±0.03% in the bone marrow of G-CSF and G-CSF+MP group, compared to 2.80±0.59 in PBS group. Neutrophils in all animals recovered post BMT, and there was a trend toward faster engraftment in the animals received G-CSF mobilized grafts. The mean survival time for mice in G-CSF, G-CSF+MP and PBS groups were 29.5±6.8, 32.8±4.8 and 22.6±8.1 days, respectively, post transplantation. The clinical signs of GVHD, such as marked weight loss, hunched back, hair loss, diarrhea, etc. appeared at day +14 post BMT in PBS group. The assessed clinical score were 5.0±1.5, 3.0±1.0 and 9.0±2.0 for the three groups of mice at +35 day post BMT. The histological scores assessed by a semi-quantitative scoring system for the above groups were 6.0±1.5, 4.0±1.0 and 13.0±2.5 at day +14, 18.0±3.0, 9.0±1.5 and 33.0±3.5 at day +35, respectively. Conclusion G-CSF primed donor bone marrow can accelerate donor chimerism and reduce the incidence and severity of aGVHD, while methylprednisolone pretreatment had no additional effect on aGVHD compared with G-CSF only. The mechanism of these effects might be related to the decreased CD4+/CD8+ ratio and the predominance of type 2 cytokine profile.

A comparison of the number of informative short tandem repeat loci between HLA identical donor-recipient pairs and the frequency of graft-versus-host disease K. Dalva, M. Arat, E. Serbest, M. Beksac, Ankara University Faculty of Medicine (Ankara, TR) Detection of the disparities within the short tandem repeat (STR) loci in different parts of the chromosomes enables specific and sensitive monitorization of the chimeric status. Rationale: The relationship between HLA allele disparities and GVHD is well known but there is not any satisfactory data on the association between STR disparities and GVHD. With this aim we analyzed STR data collected from patients who were grafted from their HLA identical related donors (n=32) or from an HLA identical unrelated donor (n=1) during the year 2003, in our center (F/M: 13/20, diagnosis CML/Acute Leukemia/other: 10/18/5, median age: 34(16-47), sex mismatch 21%, ABO mismatch 67%) grafts (BM/PB: 11/22) following a myelo-ablative (n=23), or a reduced intensity conditioning regimen (n=10). All patients engrafted and were included for acute GVHD (aGVHD) whereas 21 remained the analysis of chronic (cGVHD). Method: After the isolation of genomic DNA,11 STR loci were amplified by Amp FlSTRâ SGM Plus PCR Amplification Kit (Perkin Elmer Biosystems,USA). The amplified products were analyzed by Gene Scan Analyzer following capillary electrophoresis. Statistics: All categorical and cumulative data were sequentially analyzed by chi-square and t-test analysis. Log-rank analysis was applied to compare the differences within the Kaplan-Maier survival curves. Multivariate analysis performed by logistic regression analysis. Results: Number of disparate STR loci ranged 3-11 (median:6). The disparity frequency observed was similar in all STR loci. The number of informative loci correlated positively with the frequency of aGVHD (r=0.7, p=0.025), and cGVHD (r=0.389, p=0.049) only in patients with CML. When the number of disparities were categorized as below (Table) the incidence of aGVHD (X2:0.721) or CGVHD (X2:0.150) didn't seen to be effected. In the multivariate analysis none of the parameters known to be important in the pathogenesis of GVHD could be shown to be significantly important. Although >5 disparities survival>cGVHD seemed to be more frequent in pairs with more (analysis revealed longer (36.7mo) outcome compared to recipients with disparity at 1-5 loci (32.5mo) (p=0.05). Conclusion: Since the size of the population is the limiting factor, advanced results with accrual of more patients from transplants before 2003 will be presented at the meeting. To our knowledge this is the first report comparing the effect of STR disparities on GVHD incidence.

H. Huang, Z. Cai, M.F. Lin, W.Z. Xie, L. Li, Y. Luo, J.S. He, W.Y. Zheng, J. Zhang, X.J. Ye, B. Liang, The First Affiliated Hospital, Zhejiang University (Hangzhou, CHN) Materials and methods: From November 1998 to December 2002, 69 patients with chronic myelogenous leukemia, acute leukemia, myelodysplastic syndromes and multiple myeloma received unrelated donor bone marrow transplantation (URD-BMT) at our center. Two patients received their URD-BMT as a second BMT after graft failure and four patients received CsA and MTX for GVHD prophylaxis were excluded from further analysis. The median age was 28 years (range 8-52). All cases were facilitated by Tzu Chi Marrow Donor Registry (TCTMDR). The high resolution DNA testing for class I and II method was employed in HLA typing of all donor-receiver pairs. HLA allele matched in 41 cases, mismatched with one locus in 16 cases and with two loci in 6 cases. 62 patients were prepared with cyclophosphamide (CY) 60mg/kg/day for 2 days (total dose 120mg/kg) and busulfan(Bu) 1mg/kgx4/day for 4 days (total dose 16mg/kg). One patient with CML in blast phase was prepared with CY 60mg/kg/day for 2 days (total dose 120mg/kg) and 7.5 Gy of single fraction TBI. GVHD prevent regimen was MMF, CsA, and short course MTX. CsA: 3mg/kg/d continuous i.v. infusion, oral 6mg/kg/d later, blood CsA concentration 200-400ng/ml, MTX, 15 mg/m2 on d 1 and 10 mg/m 2 on d 3, 6 and 11. MMF:0.5g/d, from day 0 to day 120. Results: 59 of 63 (93.7%) patients engrafted successfully. The median time to achieve ANC>0.5x10 9 /l was 15 days (range 10-30 days), platelets >20x10 9 /l was 19 days (range 8 to 90 days). Acute GVHD of grade II-IV were seen in 18 of 59 (30.5 %) patients who engraftment successfully and the severe acute GVHD of grade III-IV were seen in 6 of 59 (10.2 %) patients. Chronic GVHD developed in 24 (40.7 %) patients, three limited and 21 extensive. Twenty of 63 (31.7 %) patients died of transplant-related causes between 2 and 32 months posttransplant (median 6), the cause of death were interstitial pneumonitis (n=10), pneumonitis (n=1), primary graft failure (n=3), multiorgan failure (n=3), acute GVHD (n=2) and extensive chronic GVHD (n=1). Clinical relapse were detected in three patients(4.8%). After a median follow-up of 12 months, the overall and disease free survival at 2 years are 70.4% and 66.5 %, respectively. Conclusion: This study showed MMF, CsA, MTX can be used effectively and safely for prevention of aGVHD in URD-BMT.

Primary PUVA phototherapy can be effective for acute graftversus-host disease of the skin T. Wetzig, H. K. Al-Ali, S. Kirstner, U. Hegenbart, M. Sticherlin, D. Niederwieser, University of Leipzig (Leipzig, D) Introduction: Acute Graft-Versus-Host Disease (GvHD) occurs frequently after allogeneic stem cell transplantation (SCT). Steroids form the backbone of most treatment regimens of moderate-to-severe acute GVHD. Responses are usually at the expense of catabolic damage and an increased risk of infection. Photochemotherapy with 8-methoxypsoralen plus ultraviolet light irradiation (PUVA) has immunomodulatory effects and is used to treat a variety of immune-mediated dermatologic diseases. We present our 6 year experience with PUVA as primary therapy for acute GvHD of the skin. Patients and methods: 58 pts, median age 42 years (range 21-66) were transplanted from HLA-MRD (n=27), HLA-MUD (n=29) and HLA-mismatched UD (n=2). Conditioning consisted of TBI 12 Gy/Cyclo (n=19), TBI 12 Gy/Cyclo/ATG (n=19) or TBI 2 Gy/Fludarabin (n=20). GvHD prophylaxis consisted of CSA/MTX (n=45) or CSA/MMF (n=13). All developed histologically proven grade II-III acute GvHD of the skin at a median of 36.5 days (range 15-126). PUVA was given initially without systemic steroids in 28/58 pts (48%) with acute GVHD confined to the skin. 30/58 pts (52%) received PUVA as adjunct therapy to steroids because of additional gut and/or liver GvHD. Results: PUVA was well tolerated with minimal side effects (vomiting, nausea). PUVA was applied for a median of 44 days (range 15-86 days). The median number of treatments given was 18 (range 2-132) at a mean cumulative exposure of 24 J/cm 2 (range 3-67). 18/28 (64%) pts with acute GvHD confined to the skin, had a complete response at the end of therapy and 3/28 (11%) pts a partial response. There was no need for additional steroids. Steroids had to be added in 4/28 (14%) pts because of acute GvHD of the liver after a complete response to PUVA (1 pt) and because of progression of skin GvHD (3 pts). After one year, 3/28 (10.7%) pts developed chronic skin GvHD after PUVA. Conclusions: PUVA can be highly effective as primary therapy for acute GvHD of the skin in a substantial proportion of pts after SCT. Systemic steroids can thereby be avoided. Phototherapy is indicated as primary therapy in pts with acute GvHD confined to the skin. These results should be confirmed in prospective studies and the effectiveness of PUVA compared to other treatment modalities.

A. Indovina, R. Mannino, A.M. Cavallaro, S. Tringali, R. DiBella, R. Scimè, A.O. V.Cervello (Palermo, I) Incidence of chronic GVHD (cGVHD) after allogeneic stem cell transplantation (ASCT) is likely to rise with the increasing use of HLA-disparate and unrelated donors and availability of peripheral blood stem cells (PBSC). Skin involvement > 50%, platelet count < 100 x 10 9 /L and the presence of progressive cGVHD are predictors of reduced survival. cGVHD is often steroid-refractory and resistant to second-line immunosuppressive therapies. Recently extracorporeal photopheresis (ECP) has been introduced as treatment of refractory cGVHD. ECP is an immunomodulatory therapy based on exposure of MNC collected by apheresis to UVA light in the presence of the photosensitizing agent 8-methoxypsoralen (8-MOP). ECP in cGVHD seems to be able to attenuate the Th1-mediated cytokine secretion and has demonstrated promising results in several uncontrolled case series. 18 pts.(14 males, 4 females), median age 51 (19-63) with extensive refractory cGVHD following ASCT (15 PBSC, 3 BM from MUD) started on ECP at our Center during a 21-month period.6 patients had one of the features associated with reduced survival, 4 had two features, 1 three and 7 none. 15 pts had a mucocutaneous disease. Cutaneous cGVHD was extensive (>50%) in 8 pts and was accompained by visceral involvement in 10. Eye involvement was present in 11 pts, and 2 pts had a platelet count < 100 x 10 9 /L. ECP was begun at a median of 42.6(3.3-76.7) months after ASCT and 38.2(0.0-73.3) months after onset of cGVHD. Treatments were given on 2 days with various schedules (1-4 per month) depending upon the clinical response. A total of 399 ECP procedure were performed utilizing either the Therakos UVAR XTS system (267) or a threestep technique employing the PUVA COMBI LIGHT irradiator (132). CR and PR rates were 29.4% (n=5) and 47% (n=8) respectively, yielding a 53% (n=9) steroid-sparing rate.Looking to response rate for different organ, best results were observed in oral (75%) and ocular (72%) involvement. No major complications were observed and no patient died during the ECP treatment. In 5 responding patients ECP was started again after a median stop of 9 (5-10) months due to a relapse of cGVHD. Comparing the 2 ECP techniques adopted, no differences were observed in terms of efficacy and safety, but irradiation with the COMBI LIGHT device was less expensive. ECP has a low toxicity profile and may be helpful in management of steroidrefractory cGVHD but its real role and optimal schedule is to be defined.

In bone marrow and stem cell transplantation photopheresis is used to treat acute and chronic graft versus host disease (GvHD) (Greinix et al, Blood 2001) . However, no controlled and prospective studies have been published so far. Extracorporeal photochemotherapy (ECPC) is a variant introduced by Andreu et al to the original photopheresis as it was first published by Edelson. A continuously operating cell separator, Spectra (Gambro) is used to collect the mononuclear cell concentrate (MNC). 8-MOP is added directly into the collection bag immediately after MNC harvest. Irradiation is performed in a specially designed device ( Biogenic A (Vilber-Lourmat)). This leads to higher MNC yields and lower red blood cell contamination in the concentrate and to lower extracorporeal blood volume. The cells are exposed to an exactly defined irradiation dosage. At the start of aphereses, two ECPC sessions per week were performed, after 2 -4 weeks, the number of treatments was reduced according to the patient's clinical improvement. During each treatment cycle the patients´ blood volume was processed 1.5 to 2.5 times. So far, we treated 18 adult patients with GvHD after allogeneic stem cell transplantation. Of 8 patients with late acute or early chronic GvHD (GvHD occurring in the first 6 months after SCT = group 1) 37% showed a partial response, 13% a mixed response, 25% stable disease and 25% progression. Among 10 patients (group 2, GvHD > 6 months) suffering from late extensive chronic GvHD we saw partial remissions in 50%, mixed responses in 20% and progressive disease in 30%. Responses were most frequently observed in lichenoid and scleroderma-like skin GvHD, but also in some patients with visceral GvHD and BO. In group 1 we observed 3 viral infections and 1 thrombocytopenia in the course of ECPC. In group 2 bacterial infections were seen twice and in one patient hepatitis B virus load increased. 5 patients from group 1 are still on ECPC, one patient from each group does not need any further immunosuppressive treatment. No relapses of underlying disease were observed. ECPC is a very valuable second line treatment with low toxicity and treatment related mortality in patients with GvHD. Response rates were comparable to data reported in the literature.

The effect of conditioning therapy on Langerhans' cell density in human bone marrow transplantation

Graft versus host disease (GvHD) after bone marrow transplantation is mediated by donor lymphocytes. Their initial point of contact in the recipient is likely to be a recipient antigenpresenting cell (APC) and the response of these cells to conditioning is a potential determinant of the severity of GvHD following engraftment. Previous studies have shown a decline in tissue APC, in the early post-transplant period, but the kinetics of this, effect of different conditioning regimes and precise relationship with GvHD are not well-defined. We have studied human Langerhans Cells in the immediate post-transplant period, taking advantage of the confocal scanning laser microscope to image intact epidermal sheets. This provides much more accurate information of the LC network than conventional immunohistochemistry, allowing up to 1mm2 epidermis to be examined for cell density and morphology. In normal breast skin, we find 705 +/-94 LC per mm 2 . Transplant patients have a similar density of cells, despite previous exposure to chemotherapy (645 +/-273 / mm 2 ). At day 0, after completion of conditioning therapy most LC remain in situ ( 593 +/-143 / mm 2 ) regardless of the preparatory regime, including CAMPATH and total body irradiation. Although LC do not significantly decrease in the first week post-transplant, morphological activation is prominent. There is then a progressive decline to reach a nadir at day 21 (117 +/-93 / mm 2 ), significantly later than the neutrophil nadir occurring in the first seven days post transplant. Acute GvHD and steroid exposure is associated with a rapid loss of LC from the epidermis, as observed in previous studies. By day 100 post-transplant, LC density is restored (822 +/-332 / mm 2 ) and may even exceed pre-transplant levels, although again, variation with GvHD is seen. These studies show LC kinetics after transplantation in great detail and illustrate that significant recipient antigen-presenting cells remain in the early post-transplant period, regardless of conditioning therapy. Strategies to reduce APC numbers in the recipient may therefore have beneficial effects in reducing acute GvHD.

Mycophenolate mofetil in combination with cyclosporine-A (CsA) ± prednisolone -the new standard for GvHD prophylaxis?

D. Linck, K. Schaefer-Eckardt, M. Kroeger, M. Bornhaeuser, I. Blau, H. Wandt, A. Jenke, J. Kienast, V. Armstrong, N. Basara, H. Schroeder, A. Fauser, M.G. Kiehl, Klinik für Knochenmarktransplantation, Klinikum Nuremberg, University Hospital, Benjamin Franklin Hospital (Idar-Oberstein, Nuremberg, Muenster, Dresden, Berlin, Goettingen, D) Risk of acute GvHD after HLA-mismatched related (MMRD), HLA-matched unrelated (MUD) and HLA-mismatched unrelated (MMUD) transplantation is significantly higher than after HSCT from an HLA-matched sibling. Despite administration of immunosuppressive drugs GvHD still occur in 30-90% of those patients (pts) with a mortality up to 50%. Regimen containing MMF have shown promising results but randomized studies to evaluate the efficacy and safety are still missing. In this trial the combination of CSA, MTX ± Prednisolone (pred) was compared to CSA, MMF ± Pred after HSCT. The aim was to evaluate the efficacy, safety and optimal dose of MMF on incidence and severity of aGvHD and to compare TRM and OS between groups. Patients: 45 pts. (16F/29M; ALL 15, AML 16, CML 8, others 6) receiving an allogeneic HSCT (11BM/34PBSC), MMRD(n=6), MUD(n=29) and MMUD(n=10) of 52 were available for analysis. 24 pts. were transplanted in CR. Methods: Pts were randomized to receive either CSA + MMF 2g/d (Group A, n=14), CSA + MMF 3g/d (Group B, n=18) or CSA + MTX (Group C, n=13). MMF started at day 0 and was administered i.v. bid daily until day +21, followed by orally administration until day +60. MTX consisted of 15mg/m 2 on day +1, 10mg/ m 2 on days +3 and +6. Pred in a dose of 0.5mg/kg bw. from day +7 to +14 and 1mg/kg bw. from day +14 to +28 was allowed. Results: AGvHD occurred in 34 pts.(76%; grade I/II 18 pts. (40%), grade III/IV 16 pts. (36%)

Cause of study termination were severe aGvHD (14 pts.), death (2 pts.), toxicity (1 pt.) and infection (1pt.).No difference in incidence and severity of aGvHD comparing group A and C was observed, but group B was significantly worse (p=0.018). Severe mucositis was more common in the MTX group (61%; p<0.001). Time to engraftment of neutrophils and thrombocytes was faster in the MMF groups (neutrophils >500/µl: p=0.062; thrombocytes >20,000/µl: p=0.057). Kaplan-Meier estimates of OS demonstrates no difference comparing group A and C (p=0.52). Conclusions: Our data demonstrate that MMF is an effective and save drug after allogeneic HSCT and might be capable to replace MTX as standard GvHD prophylaxis.

Oral beclomethasone dipropionate for the treatment of gut acute graft-versus-host disease C. Castilla, J. A. Perez-Simon, F. M. Sanchez-Guijo, M. D. Caballero, M. C. Cañizo, M. Diez, E. M. Ocio, L. Vazquez, J. F. San Miguel, Hospital Universitario de Salamanca (Salamanca, E) Introduction: Treatment of gut GVHD relies on systemic immunosuppressive therapy such as glucocorticoids which increase the risk of infections after transplant, among other complications. Beclometasone dipropionate (BDP) is a topically active corticosteroid with a low absorption thus limiting systemic effects. Previous studies suggest its efficacy in the treatment of gut GHVD in association with systemic steroids and even as a single agent, although this latter option has been assayed in a limited number of patients. Patients and methods: DBP was administered po at a dose of 2 mg q6h for 28 days in 18 patients undergoing allogeneic peripheral blood stem cell transplantation which had a biopsy proven gut GVHD in all cases. Eleven received reduced intensity conditioning regimen based on fludarabine plus melphalan or busulphan, 4 received CyTBI and 3 BuCy. Median age was 42 years (14-67). Median follow up was 420 days (159-1154) . GVHD flared at a median of 38 days after transplant (18-90). Ten patients had gut aGVHD, 7 had skin and gut and 1 liver and gut aGVHD. Gut aGVHD symptoms consisted of nausea (n=10), diarrhea (n=1), both (n=5) and anorexia plus skin involvement (n=2). Results: DBP was started at a median of 46 days after transplant. Patients received a median of 28 days of DBP. Thirteen patients reached complete remission (CR), 2 partial response (PR) and 3 had no response. The only patient who had liver GVHD did not respond. By contrast 86% of patients who had skin and gut aGVHD reached CR. No differences in terms of response were observed between patients who had upper (nausea, vomiting) or lower (diarrhea) aGVHD symptoms. Moreover, among the six patients who had diarrhea five reached RC (n=5) or PR (n=1). From the 17 patients who responded to DBP 4 relapsed later on. Thus, 7 patients out of 18 required systemic glucocorticoids.

Conclusions: The present study shows that BDP is well tolerated and has a marked efficacy in the treatment of gut GVHD, with a high percentage of patients reaching complete or partial response. In these patients systemic glucocorticoids are avoided thus decreasing toxicity related to immunosupresive therapy.

Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells K. Le Blanc, I. Rasmusson, B. Sundberg, C. Götherström, M. Hassan, M. Uzunel, O. Ringdén, Huddinge University Hospital (Stockholm, S) Pluripotent adult bone marrow derived mesenchymal stem cells (MSC) retain the capacity to differentiate along osteogenic, chondrogenic and adipogenic lineages. MSC are not inherently immunogenic but suppress alloreactivity induced by cellular antigens and prolong the rejection of mis-matched skin grafts in experimental animals. We transplanted haploidentical MSC in a patient with severe therapy-resistant grade IV acute graft-versushost disease (GVHD) of the gut and liver. A 9-year old male with ALL in 3rd remission was transplanted with blood stem cells from an HLA-identical unrelated donor after full myeloablative conditioning. Immunosuppression included thymoglobulin, cyclosporin (CsA) and methotrexate. On day +11, he had a skin rash. By day +22, the patient developed diarrhoea >1000 ml/day and morphine-requiring abdominal pain. Bilirubin and ALT increased and the GvHD was unresponsive to steroids, PUVA therapy, Infleximab, Daclizumab, mucophenolate mofetil and methotrexate. By day +70, the patient had grade IV acute GVHD with diarrhoea twenty times daily and a bilirubin of 250 mmol/l. Bone marrow derived MSC were isolated from the mother and expanded ex vivo. On day +73, the patient received 2 x 10 6 MSC/kg as an i.v. infusion. Four days later, the frequency of diarrhoea decreased to twice daily. A decline in total bilirubin was noted five days after the MSC transplantation and two weeks later the patient resumed oral food intake. By day +143, DNA analysis of the patient's bone marrow showed the presence of minimal residual disease (MRD). Immunosuppression was therefore discontinued. By day +150, the patient again had diarrhoea, but no abdominal pain. Colonbiopsies showed mild GVHD and 4% female epithelium by FISH. He was transplanted with the same batch of MSC (1 x 10 6 /kg) on day +170. After one week, the stools were normal and the patient started to eat again. Bilirubin normalized. He was discharged on day +220. He is now 14 months after transplant, well and with a bilirubin of 8 mmol/l. He is MRD negative in blood and bone marrow. In our experience of 1000 allogeneic stem cell transplantations, 25 patients developed grade IV acute GVHD. This is the only patient with grade IV acute GVHD alive more than 6 months after ASCT. T We postulate that MSC have a potent immunosuppressive effect in vivo. This case encourages prospective controlled studies using MSC as prophylaxis and treatment of GVHD.

Unconventional treatment of steroid-refractory acute GvHD: pulse cyclophosphamide and/or intra-arterial steroid administration J. Mayer, M. Doubek, Y. Brychtova, M. Krejci, University Hospital Brno (Brno, CZ) Objectives: The response to salvage therapy after failure of initial corticosteroid therapy is usually very poor. The most common second-line drugs are ATG and monoclonal antibodies. However, despite some clinical improvement, the survival of patients (pts) is very low. Pulse cyclophosphamide (CP) is the established treatment for some autoimmune disorders. Because of several similarities between GvHD and autoimmune diseases, we decided to try pulse CP as a salvage treatment in severe GvHD pts non-responding to corticosteroids. Because of the lowest response rate of pulse CP in pts with GIT GvHD, we also adopted the strategy of GIT intra-arterial (i.a.) steroid administration. Methods: Pulse CP: an i.v. infusion at the dose of 15-20 mg/kg. If needed, further pulses were planned at the time intervals 3-6 weeks. Other immunosuppression (steroids, cyclosporin A, mycophenolate mofetil) was maintained or gradually tapered. I.a. administration: 40-60 mg of methylprednisolone/vessel (mesenteric and/or gastro-duodenal artery), or 75mg/m 2 ± MTX 10mg/m 2 into the hepatic artery. Results: There were 13 CP and 7 i.a. administrations (19 vessels in total) in 13 pts. 5 pts were treated with both methods, either concomitantly, or in succession, in the case of incomplete response after one method. The best response rates were seen in liver (100%) or skin (100%) GvHD. In 1 patient with oral mucosa involvement, very good response was seen. The worse response was seen in intestinal GIT (7pts, 3 good responses, 1 stabilization). Concerning all pts, evident signs of clinical and laboratory improvement (sometimes very dramatic) occurred in the time interval of 1 week upon the drug administration. Surprisingly, pulse CP was accompanied only by no or manageable, mild, and self-limiting hematological toxicity. 5 pts died, 3 directly from GvHD or its complications (fierce intestinal involvement), 2 pts from leukemia. Conclusion: Pulse CP and/or i.a. steroid administration seem to be cheep, effective and non-toxic treatment of steroid-refractory GvHD. In severe intestinal involvement, the effectiveness seems to be limited, however.

Patient outcome after Haemopoietic Stem Cell Transplantation (HSCT) is governed by time to engraftment, tumour relapse and development of Graft versus Host Disease (GvHD). Myeloablation induced by radiotherapy and chemotherapy is vital for HSC engraftment, however this also activates Dendritic Cells (DC), and initiates GvHD. The purpose of this study is to develop novel strategies to manipulate DC to control GvHD and improve outcomes for HSCT recipients. The aims of this project are: (1) to determine DC numbers and subset composition in mice at different time points post irradiation and (2) to determine whether delayed transplantation post irradiation can attenuate GvHD. C57BL/6 and B10.BR mice were irradiated at time zero and sacrificed 6, 24, 48, 72 and 96 hours post irradiation. We evaluated the numbers and subsets of DC in the bone marrow (BM) and spleen of these animals at the time of sacrifice by flow cytometry. A significant increase in CD11c+MHC-II+ cells was noted and two DC populations, plasmacytoid DC (CD11c+, MHCII+/-, CD45R+, Gr1+) and modified regulatory rDC (CD11c+, MHCIIhi, CD40lo, CD80 lo) that have been implicated in the attenuation of GvHD were identified. We then transplanted lethally irradiated B10.BR mice with BM and spleen cells from C57BL/6 mice 0, 6, 24, 48, 72 and 96 hours post irradiation. Further study is expected to determine whether manipulating the time of transplantation post irradiation can attenuate GvHD. Optimal distancing of transplantation from conditioning could reduce GvHD without subjecting patients to additional complications associated with delayed haematopoietic reconstitution.

The impact of human leucocyte antigens in graft-versushost disease and survival after haematopoietic stem cell transplantation in patients with leukaemia K. Ozdilli, F. Savran Oguz, H. Bilgen, S. Anak, M.N. Carin, G. Gedikoglu, Halic University, Istanbul University, Our-Leukemia Children Foundation (Istanbul, TR) Graft Versus Host Disease (GvHD) is one of the life-threatening complications after hematopoietic stem cell transplantation (HSCT). Any studies have attempted to define factors predictive of GvHD beyond the degree of histocompatibility. Recent reports have suggested a probable association between certain Human Leucocyte Antigens (HLA) and GvHD. The association between various HLA antigens and the occurence of acute and chronic GvHD was evaluated in 57 HSCT patients with HLA identical sibling donors. There were 37 boys and 20 girls with a median age of 10. All of the patients had leukemia (AML=33, ALL=15, CML=9). As conditioning; patients with AML received Busulphan (16mg/kg) + Cyclophosphomide (120 mg/kg), ALL patients received TBI + VP16 (1500g/m 2 ) and CML patients received Busulphan (16mg/kg) + Cyclophosphomide (120 mg/kg). Among patients who underwent HSCT GvHD incidence was found to be 17,5%, the frequency of HLA-DRB1*04 (p=0,024) allele was higher, but that of HLA-DRB1*11 (p=0,0031) allele was lower and HLA-B38 (p=0,002, Fisher's=0,028) and HLA-B41 (p=0,002, Fisher's=0,028) antigens were found only in the patients who had experienced GVHD. The frequency of HLA-DRB1*11 allele (p=0,037) and HLA-A26 (p=0,012) antigen were higher, whereas that of HLA-A2 antigen (p=0,033) was lower in the patients who relapsed after HSCT. The survival in 57 patients with leukemia was found to be 57,4% in five years and the frequency of HLA-A26 (p=0,001) and HLA-B8 (p=0,012) antigens were higher in the patients who had succumbed to death, whereas that of HLA-A24 (p=0,037) was higher in those who survived. As a conclusion, this study showed that HLA antigens and alleles led to a predisposition in occurance of GvHD after HSCT in 57 cases taken from the genetic pool of The Turkish population but larger groups of studies must be done in future. M Zaraiski, N. Mikhaylova, N. Ivanova, A. Vitrischak, A. Pugachov, V. Vavilov, L. Zubarovskaya, B. Afanasyev, Pavlov SPb State Med University (St. Petersburg, RUS) Acute graft-versus-host-disease (aGvHD) is a serious complication of hematopoietic stem cell transplantation (HSCT) in hematological patients. Pathophysiology of aGvHD is rather complex, including activation of all immune cell subsets caused by MHC mismatches in donor-patient pairs. Numerous cytokines play crucial role in these events. Objectives: The aim of our study was to estimate the levels of gene expression for interleukin-1beta (IL-1b), -2, -6, and -10 before and after HSCT (up to D+50) in cases with/without aGvHD, to evaluate clinical significance of these findings. Materials and methods: Fourteen leukemia patients underwent either myeloablative (Bu+Cy+/-ATG) or nonmyeloablative (Flud+Mel+/-ATG, or Flud+Bu+/-ATG) conditioning regimens before HSCT. Total RNA was extracted with phenol-chloroform from peripheral blood mononuclear cells (PBMNC) before conditioning, and weekly upon hematopoietic recovery. The levels of cytokine RNA production were analyzed by original semi-quantitative RT-PCR assay using specific primers for exons 4-5 or 5-6 of each separate cytokine. Beta-actin gene was coamplified in each PCR, as a reference signal. The amplification products were visualized in agarose gel, scanned and examined by GelPro 31 software. Results: The applied protocol allowed simultaneous analysis of the non-spliced nuclear cytokine RNA, as an early marker of cell activation (EMCA), along with mRNA, thus reflecting proteinsynthesizing ability of the cells (PSAC). In the cases of aGvHD, EMCA for IL-1b, -2, and -6 showed no regular increase, as compared to the pre-HSCT levels. Meanwhile, this parameter in aGvHD was significantly increased for IL-10 (P=0.003). On other hand, the indexes of PSAC in aGvHD patients were highest for IL-1b (P=0.048), whereas mRNA levels for IL-6 and -10 also tended to increase in these cases. Expression of IL-2 gene in the course of aGvHD was decreased, probably, due to adequate immunosuppressive therapy by cyclosporin A. Conclusions: The results of our pilot study suggest that: (i) monocytes/macrophages could play the more important role than lymphocytes, at least, at the stage of aGvHD initiation, (ii) detection of IL-10 nuclear RNA expression level in PBMNC may be used for early prediction of aGvHD, (iii) additional therapy of aGvHD could be targeted for suppression of both IL-1b mRNA synthesis and/or cellular production of IL-1b.

Graft-versus-host disease is associated with a lower relapse incidence after haematopoietic stem cell transplantation in patients with acute lymphoblastic leukaemia A. Nordlander, J Mattsson, O. Ringden, K. Le Blanc, B. Gustavsson, P. Ljungman, P. Svenberg, J. Svennilson, M. Remberger, Huddinge University Hospital (Stockholm, S) To determine the graft-versus-leukemia (GVL) effect after hematopoietic stem cell transplantation (HSCT), we studied 199 patients with acute lymphoblastic leukemia (ALL) transplanted at Huddinge University Hospital between 1981 and 2001. Seventy-four patients were in first complete remission (CR1) and 125 in later stages of the disease. Most patients had an HLAidentical sibling donor. Conditioning consisted mainly of total body irradiation and cyclophosphamide and graft-versus-host disease (GVHD) prophylaxis with cyclosporine (CsA) and methotrexate (MTX). Acute GVHD developed in 143 patients and chronic GVHD in 67. The five-year probability of relapse and relapse-free survival (RFS) were 32% and 49% in patients in CR1, as compared to 53% and 33% in those with more advanced disease. In the multivariate risk factor analysis of relapse, we found that the absence of chronic GVHD (p<0.001), absence of herpes simplex virus (HSV) infection after HSCT (p=0.003), combination prophylaxis with MTX+CsA (p=0.01) and >6 weeks from the diagnosis to CR (p=0.025) were independent risk factors for relapse after HSCT. Factors associated with a better RFS were: chronic GVHD (p<0.001), AB0 blood-group mismatch (p=0.006), younger patient age (p=0.01) and an HLA-matched donor (p=0.01). The association between HSV infection and a low frequency of relapse is a new observation and may indicate that viral antigens play a role in the induction of an anti-leukemic effect.

Blocking ICOS: ICOSL interaction before allogeneic bone marrow transplantation may increase acute graft-versushost disease

Graft versus host disease (GVHD) remains a major complication after allogeneic bone marrow transplantation. Acute GVHD is initiated by alloreactive donor T cells that recognize HLA disparities on host cells as well as peptides presented by them. The infiltration of several target organs such as gut, liver, and skin by donor leukocytes including T cells is thought to be one of the key processes in the early phase of a GVHD. The activation and expansion of the donor T cells, leading to the secretion of proinflammatory cytokines and the recruitment of additional inflammatory effector cells to these sites, further damages the affected tissues. Costimulatory receptors on T cells have a key role in this T cell receptor/CD3 dependent activation process. The inducible costimulator (ICOS) is a new member of the CD28/CD152 receptor family that regulates T cell activation and function. The apparent involvement of ICOS/ICOSL interaction in several T cell effector functions and the abundant expression of ICOSL in a variety of tissues also affected in a GVHD provided the rationale to investigate its role in GVHD development. C57Bl/6 mice were lethally irradiated and reconstituted with allogeneic spleen cells (sc) in the absence/presence of an recombinant ICOSIg construct. Controls were reconstituted with syngeneic sc. Reconstituted mice were monitored for weight loss and survival. 3x10 E7 sc were sufficient to reconstitute irradiated mice. Mice reconstituted with allogeneic sc experienced 12-16% weight loss around day 4 after transplantation and died of acute GVHD within 7-10 days after transplantation. Unexpectedly, mice reconstituted with allogeneic sc together with ICOSIg showed increased signs of GVHD as restricted movability and increased morbidity. However, weight loss and overall survival was identical to isotype treatment groups. Flow cytometry of spleen from reconstituted mice revealed a dramatic T cell expansion and reduction of host B cells in allogeneic treatment groups with no signs of influence of ICOSIg on overall cell numbers. Collected data suggest that ICOSIg therapy has no prophylactic potential in an acute GVHD, and requires further investigations on the underlying mechanisms.

Chimeric and humanized CD25 antibodies (ch/anti-CD25) are specific for the alpha-chain of the interleukin-2 (IL-2) receptor and blocks IL-2 dependent activation of human T lymphocytes. Murine, chimeric and humanized anti-CD25 had immunsuppressive activity in treatment of acute GVHD after allogeneic stem cell transplantation (SCT) in adults. We assessed the safety of ch/anti-CD25 in pediatric recipients of unmanipulated allogeneic peripheral hematopoietic grafts (n=11) under treatment with chimeric (7) or humanized (4) anti-CD25. In addition, we assessed the duration and efficacy of CD25 blockade (n=9). In 7 ALL, 3 AML, 1 MDS patients ch/anti-CD25 (1 mg/kg) was given 6 hours before SCT and on day 4 (d+4), +28, +56 and +84 from SCT. All patients received myeloablative conditioning and GVHD prophylaxis with Cyclosporin A. Ch/anti-CD25 was well tolerated without adverse events related to the antibody application. 6/11 patients completed the treatment protocol. 3/6 showed complete CD25 blockade (<1% CD25+ T cells by FACS detected in peripheral blood) until d+100. In the other 3 patients duration of CD25 blockade was 13 ± 2, 16 ± 3 and 23 days after last antibody application. 5/11 patients suffering from chronic GVHD showing CD25+ cells received another 2 to 5 anti-CD25 applications after d+100. CD25 blockade in these patients ranged from 21 ± 3 days [19; 23] to 55 ± 11 days [46; 64] 95%CI (mean, SD, [95%CI]) and was 37.3 ± 12.8 days for all patients. Interpatient variability was greater than intrapatient variability. Time to engraftment was 14 days (11 to 25) after peripheral blood (n=9) and 36 days after cord blood (n=2) SCT. The incidence of GVHD grade II-IV and III+IV with prophylactic ch/anti-CD25 (n=11) was 6/10 and 5/10, median time to GVHD was 17 days (9 to 26). Incidence of chronic GVHD calculated on patients surviving beyond d+90 was 7/8 in all patients, 1 patient showed extensive disease. In spite the high rate of chronic GVHD in our cohort 7/11 patients suffered from relapse. Probability of overall survival was 0.22 (3/11 patients) . Ch/anti-CD25 is efficaious in receptor blockade and well tolerated in children after allogeneic SCT. Engraftment was within normal range. Our findings may also suggest a role for CD25 in the balance between T cell activation and activationinduced cell death (AICD). The complex role of CD25 in regulatory and effector T cells to achieve allo-recognition and leukemia control warrants further investigation.

L. Savino, M. Carella, G. Beltrami, M.M Greco, M. Centra, A. M. Carella, IRCCS CSS (San Giovanni Rotondo, I) We report clinical outcome and results of chimerism analysis in 53 patients (32 male, age: 30-60, mean: 47 yrs) with different malignancies (6 HD, 16 NHL, 11 MM, 3 CML, 4 AML 1 CLL, 6 Kidney and 6 Breast cancer) that received different non myeloablative conditioning, followed by allogeneic SCT: 18 Flu/Mel 16 Flu/TBI 19 Flu/Cy. In all cases unmanipulated PBSC from fully matched HLA-identical siblings were used; median number of infused CD34+ cells was 3.30x10 6 /kg (range 0.90-10.61). GVHD prophylaxis consisted of CSP/MTX. Chimerism analysis was performed using multiplex PCR amplification of shorth tandem repeat markers and fluorescence detection. Fifteen STR loci and the Amelogenin locus are co-amplified in a single reaction. Donor engraftment was evaluated on unfractionated peripheral blood (PB), bone marrow samples and on immuno-magnetically selected PB CD3+ cells. Median followup was 225 days (34-912) . The rates of complete donor chimerism (CDC=>95% donor cells) at day +15,+30, +90, +180, +270, +360 and beyond +360 were 23%, 36%, 60%, 86%, 93%, 93% and 93% respectively. Kinetics of engrafting donor cells was different in the three conditioning regimens. The rate of CDC at days +30 were 73%, 20%, 0%, respectively; at days +90 were 92%, 57%, 0%. Acute GVHD occurred in 5/18 (28%) patients (pts) undergoing Flu/Mel, three of them were grade > II (2 died), while 7/18 (39%) developed chronic GVHD, none of them died of it. In two (12%) pts treated with Flu/TBI protocol, grade III-IV acute GVHD was observed (1 died), chronic GVHD occurred in 7/16 (44%) pts and one of them died of it. Finally in Flu/Cy protocol acute GVHD was absent, while 4/19 (21%) pts developed chronic GVHD. Thirty-two patients (60%) are alive to date. Our study shows that Flu/Mel protocol has unincreased transplant related mortality, mainly due to higher incidence of acute GVHD, while chronic GVHD occured in all three protocols. We cannot evidentiate acute GVHD in patients that underwent Flu/Cy conditioning regimen because at day +90 none of them has achieved complete donor chimerism.

H. Biersack, R. Trenschel, R. Peceny, A. Elmaagacli, D.W. Beelen, University Hospital Essen (Essen, D) A major contribution to transplant related non-relapse mortality is represented by acute graft-versus-host disease (GvHD). In the treatment of steroid refractory GvHD monoclonal antibodies hold increasing promise and represent therapies which are more or less specific against activated T-cells or signaling pathways involved in cell activation. Among these, Campath-1H represents a humanized monoclonal antibody that is directed against the CD52 antigen of lymphocytes. After allogeneic stem cell transplantation ten patients developed severe grade III and IV GvHD which had already been treated with high dose steroids without success. The patients were transplanted with peripheral stem cells or bone marrow from an HLA-identical sibling (1), HLA-identical unrelated donors (7) or unrelated donors with missmatch (2) . Underlying diseases were AML (5), ALL (1), CML (3) and once a myelofibrosis. Patients had skin GvHD grade III and IV (9), intestinal tract GvHD grade III and IV (5) and liver GvHD (1) . The overall grade of GvHD was severe (3) and life-threatening (7). Our treatment regimen contained 10mg Campath-1H per day, given intravenously for five consecutive days and followed by 10mg given once every week. Patients achieved complete remission (3), partial remission (3), mixed remission (1) or progressive (3) GvHD. One of the patients with partial remission was later shown to have an additional CMV enteritis which was successfully treated by ganciclovir. Seven of ten patients died of infectious complications (3), early relapse (1), progressive GvHD (2) or EBV-associated lymphoproliferative disease (1) . Two are still on therapy with a sustained therapeutic response, one is alive with relapsed and progressive GvHD. The overall response seems to be excellent but patients often are at the start of the treatment in such bad condition that GvHD does not represent the only hitch. Mortality still remains high in this group of patients and it requires additional investigation whether supportive therapy can protect patients from fatal outcome.

A. Prudenzano, G. Palazzo, L. Stani, G. Pricolo, G. Pisapia, B. Amurri, P. Mazza, P.O. "S.G. Moscati" Hospital (Taranto, I) Donor CD8+ lymphocytes, as recently emphasized, are thought to play a major role in mediating GvHD in patients undergoing allogeneic bone marrow transplantation; some dissociation of GvHD from GvL effect in CD8+ depleted grafts was also reported by some authors. In order to test the hypothesis of a correlation between CD8+ cell dose in the graft and the development of acute GvHD (grade>III), we retrospectively analysed transplanted doses of donor CD34+ cells and of T-lymphocyte subsets (CD3+,CD4+,CD8+,CD56+ cells) in relation to incidence of aGvHD. 39 patients (pts) received bone marrow allografts (BMT pts) while 25 pts received peripheral blood progenitor cells as source of CD34+ cells (PBPCT pts). We also evaluated CD4+/CD34+, CD8+/CD34+ and CD56+/CD34+ ratios in relation to aGvHD incidence. As yet described in the literature, we observed a lower incidence of aGvHD in the BMT group (6/39 pts, 15%) than the PBPCT group (8/25 pts, 32%). In the BMT group, median CD34+, CD3+,CD4+,CD8+,CD56+ cell doses were respectively 3. ; however, considering the ratios CD4+/CD34+, CD8+/CD34+, CD56+/CD34+ we found P=.421, P<.05, P=.853 and P=.343, P<.05, P=.505 in the BMT and PBSCT group, respectively. It thus appears that the absolute number of CD8+ relative to that of CD34+, in particular a higher ratio between CD8+ and CD34+ cell dose, rather than the absolute count of CD8+ alone, could correlate with the incidence of aGvHD. We conclude that probably a higher count of CD8+ could favour aGvHD if not counterbalanced by an adequate CD34+cell number. This was observed in both BMT and PBPCT groups. Instead, no correlation was found between aGvHD and CD4+/CD34+ or CD56+/CD34+ ratios. However, these data should be confirmed in largest series and other variables which influence the development of GvHD should be taken into account.

Extracorporeal photoimmune therapy with uvadex® for the prevention of acute graft-versus-host disease in patients undergoing standard myeloablative conditioning and allogeneic haematopoietic stem cell transplantation P.J. Shaughnessy, B.J. Bolwell, K. van Besien, S. Abhyankar, M. Mistrik, O. Ilhan, A. Grigg, H.M. Prince, T. Dodds, A. Machado, J. Wang, U. Thienel, F. Foss, J. Apperley, Texas Transplant Institute, The Cleveland Clinic Foundation, University of Chicago, Kansas City Cancer Center, Klinika hematologie a transfuziologie, Ankara University Medical School, Royal Melbourne Hospital, Peter MacCallum Cancer Institute, St. Vincents Hospital, Instituto Portugues de Oncologia de Francisco Gent, Therakos, Inc, New England Medical Center, Hammersmith Hospital (San Antonio, Cleveland, Chicago, Kansas City, Exton, Boston, USA; Bratislavia, SK; Ankara, TR; Melbourne, East Melbourne, Sydney, AUS; Lisbon, P; London, UK) Host antigen presenting cells (APCs) may be important for alloantigen presentation and stimulation of donor T cells in acute graft versus host disease (aGVHD). Extracorporeal photoimmune therapy (ECP) has shown benefit in some patients (pts) with aGVHD and is associated with a decrease in host APCs. We report preliminary results of the first multi-institutional phase II study examining ECP with a standard myeloablative preparative regimen and allogeneic stem cell transplantation. ECP was given on 2 consecutive days from D-10 to D-6, followed by cyclophosphamide 60 mg/kg/d for 2 days and TBI 1200 cGy delivered over 3 days. Prophylaxis for aGVHD was cyclosporine 3-5 mg/kg daily beginning D-1 and then adjusted to keep levels between 200-600 ng/ml and methotrexate 10 mg/m 2 on D1, 3, 6, and 11 for pts who had matched unrelated donors (MUD) or HLA class I one-antigen mismatched related donors (MMRD); or 10 mg/m 2 on D1 and 5 mg/m2 on D3, 6, and 11 for pts who had matched related donors (MRD). Sixty pts have been enrolled in the study and data are available on 49 (33 male) pts with a median age of 40 (range 20-58) years. Diagnoses included: ALL (n=12), CML (n=13), AML (n=12), MDS (n=3), NHL (n=4), CLL (n=2) and other (n=3). The 23 pts who received bone marrow engrafted at a median of 20 (range 15-25) days and the 26 pts who received peripheral blood engrafted at a median of 15 (range 10-32) days. Grade II-IV and III/IV aGVHD developed in 7 (30%) and 2 (8%) pts, respectively, who received MRD transplants (n=23), and in 9 (36%) and 4 (16%) pts, respectively, who received MUD transplants (n=25). No aGVHD occurred in 1 pt who received an MMRD transplant. One pt experienced mild hypotension related to ECP, but continued with therapy. Five pts relapsed, 4 with ALL and 1 with NHL. Overall, 44 pts (90%) are alive with a median follow up of 111 (range 13-347) days. Five pts who received MUD transplants have died (2 pts with multi-organ-system failure, and 1 pt each with pseudomonas infection, aGVHD, and relapse of NHL). No pts who received MRD or MMRD transplants have died. Preliminary results of this study reveal no adverse effects of ECP on regimen related toxicity or engraftment after a standard myeloablative preparative regimen and ASCT. The incidence of acute GVHD warrants further study and correlation with measurement of APCs. Further follow up is needed to assess malignancy control and survival. occurring beyond three months after allogeneic stem cell transplantation (all-SCT) have became recognized as lifethreatening complications. We retrospectively analyzed the incidence and the outcome of LONIPC among 55 allo-SCT recipients from unrelated donors, with the aim of identifying risk factors and optimal treatment. Conditioning included TBI and cyclophosphamide (50 patients) or busulfan and cyclophosphamide (5 patients). Graft-versushost disease (GVHD) prophylaxis consisted of short-term methotrexate and cyclosporine. A diagnosis of LONIPC was based on clinical symptoms, thorax computer thomography abnormalities and abnormal results of pulmonary function tests (PFT) and was confirmed by a histological finding in 70% of the patients. Among the 43 patients surviving at least 3 months, 12 (28%) fulfilled the diagnostic criteria of LONIPC and were further subclassified as having bronchiolitis obliterans (5 patients), bronchiolitis obliterans with organizing pneumonia (5 patients), and interstitial pneumonia (2 patients). Advanced stage of disease at transplant and chronic extensive graft-versus-host disease (GVHD) were significantly associated with the development of LONIPC. PFT results before transplantation were similar in all patients, but patients with LONIPC had a significant decrease in PFT indexes at the third month after BMT compared with controls. Moreover, the rate of cyclosporine taper during the fourth and fifth month after BMT was significantly more rapid in patients with LONIPC than in controls, suggesting that the risk of LONIPC may be influenced by a faster reduction of GVHD prophylaxis. At a median follow-up of 6 moths after the onset of LONIPC (range 3-26), 4 patients had a durable partial response to immunosuppressive treatment, the remainder 8 did not respond and 5 of them died of respiratory failure. Only 1 out of 12 LONIPC patients (8%) in comparison with 15 out of 31 non-LONIPC patients (48%) had a relapse of their primary malignant disease (p=0.017). These results indicate that the development of LONIPC was strongly associated with chronic GVHD and produced simultaneously a graft-versus malignancy effect. Because of the high mortality due to LONIPC, careful monitoring of patients, especially those with chronic GVHD, with PFT is warranted. Prevention may be achieved by slowly tapering cyclosporine and prolonging its administration beyond 6 months. Objective: A majority of patients with chronic graft versus host disease (CGVHD) have oral lichenoid lesions which resemble lichen planus both clinically and histopathologically. There has been some reports on a relationship between lichen planus and certain human leukocyte antigen (HLA) types. Although there is no consensus between the investigations, HLA-DR1, DR2, DR3 and DR9 are the antigens which have been observed more frequently among such patients. In this study, it is hypothesized that certain HLA-DR antigens may play a role in predisposition of allogeneic stem cell transplant (AHSCT) recipients to develop CGVHD related oral lichenoid lesions. Such an association could represent a predictive criterion for developing CGVHD related oral lichenoid lesions, alarming the physician before the transplantation procedure. Methods: A triple comparison of HLA-DR1-10 and HLA-DR11-18 expression, identified by micro-lymphocytotoxicity or molecular (SSP) methods, was undertaken among 22 patients with oral lichenoid lesions of CGVHD, 40 patients with CGVHD who had visceral involvements other than skin or mucosa and 91 healthy controls. Patients had received AHSCT from HLA matched sibling donors. All the HLA work-up has been performed in our EFI accredited HLA Laboratory. The patients are representing a single center cohort, who was followed up by always the same dermatology consultant (HS).

Results: Frequencies of HLA-DR1-10 antigens were not different among the three groups. The only difference was observed in the frequency of HLA-DR17, which was increased in patients who had oral lichenoid lesions of CGVHD when compared with healthy population 12.5% vs 0% (p=0.03). However, the frequency was similar between patients who had CGVHD with or without skin-mucosa involvement (p=0.28). Similarly HLA DR 17 was more frequently observed among patients with CGVHH compared to those without GVHD (p<0.05). Conclusion: We were able to show a linkage between HLA-DR 17 antigen expression and the development of CGVHD. However this finding is not confirmed by the comparison between patients with and without lichenoid lesions. We were not able to show a definitive impact of HLA antigens in chronic GVHD associated lichenoid oral mucosal lesions unlike their native counterpart lichen planus.

A. Zander, T. Zabelina, F. Ayuk, H. Kabisch, R. Erttmann, N. Fehse, B. Fehse, C. Wolschke, N. Stute, A. Kratochwille, H. Schieder, J. Panse, T. Eiermann, N. Kroeger, University Hospital Eppendorf (Hamburg, D) Allogeneic bone marrow transplantation of matched unrelated donors carries an increased risk of Graft-versus-Host-Disease and transplant related mortality. We introduced ATG Fresenius [median dose 90mg/kg bw] as part of the conditioning for prevention of serious GvHD. We compared 48 patients with mismatched transplant with 170 patients with an HLA matched transplant. The mismatch involved of 1 or 2 loci. The groups differed in age. HLA match 33y (0,9-61), HLA mismatch 21y (0,9-51), and graft source BM vs. PBSC 67 / 33% in match vs. 83 / 17% in mismatch. They were comparable in diagnoses, stage of disease and conditioning Conclusion: Addition of ATG Fresenius permits hemopoietic stem cell transplantation from mismatched donors with results comparable [regarding GvH control, TRM and survival] to HLA matched donors.

Clinical impact of early complete donor chimerism after allogeneic stem cell transplantation M. Bieniaszewska, J. Balon, M. Reichert, K. Halaburda, M. Zaucha, R. Pawlowski, A. Hellmann, Medical University School (Gdansk, PL) Monitoring of hematopoietic chimerism after allogeneic SCT has been shown to be predictive for graft failure or relapse. However, the impact of complete chimerism (CC) as well as the relation between time to reach CC and emergence of cGvHD remains unclear. There are different methods of chimerism detection employed by different transplant centers. Those methods vary in their sensitivity and therefore the results are usually not comparable. Chimerism is often defined as complete if more than 95% donor DNA is detected. That definition is by far not enough to establish the impact of chimerism on cGvHD. Commercial multiplex STR-PCR assay, originally designed and validated for forensic purposes, has recently been introduced for chimerism detection. It gives quantitative information with low variability and high reproducibility which makes this method one of the most valuable for monitoring of chimerism. The aim of the study was to find if CC within 100 days post SCT is associated with increased risk of cGvHD. Chimerism analysis was performed in blood of 45 allogeneic transplant patients (43 matched related, 2MUD). There were 42 myeloablative (37-BuCy120, 5-TBI/Cy) and 3 reduced conditioning procedures. 37 patients received PBSC and 8 BM. Median age of the patients was 37 (18-54). Chimerism was assayed on the following days: 30, 100, 180, and then every three months. 9 STR markers and the amelogenin locus were co-amplified in a single reaction. Separation of the PCR products and fluorescence detection were performed on capillary electrophoresis instrument. Results:16 patients achieved CC within 100 days after SCT, while only 3 of them on day 30. 15 of those patients developed extensive chronic GvHD. 10 of them needed 3 or more drugs to control cGvHD. 3 patients died of cGvHD. CC after day 100 was observed in 25 patients in the course of tapering immunosupression. In this group 16 patients developed cGvHD (9 extensive, 7 limited). Only one patient needed 3-drug therapy. In 4 patients persistent MC was observed and due to aim of the study this group was excluded from further analysis. Conclusions: Chimerism analysis with multiplex STR PCR assay and fluorescence detection revealed that minority of patients achieved CC before day 30 post SCT. 25 of 41 patients showed minimal amounts of host DNA after day 100. In the group of patients with early CC the incidence of severe extensive cGvHD was more frequent(p<0.01)

The identification of patients at risk for GvHD by means of a more sensitive mixed lymphocyte culture A. Müller, R. Häfner, U. Schrey, H. Vogelsang, F. Zintl, University of Jena (Jena, D) In spite of the improved HLA-typing GvHD remains a severe complication in HLA-identical transplantation. The common mixed lymphocyte culture (MLC) in those donor/recipient pairs is not useful to predict the risk of GvHD. We modified this method to detect activation by addition of SCF and IL2 and compared the results with the common method without those growth factors. Material and Methods: Frozen samples of nucleated cells of 12 unrelated HLA-different healthy donor pairs ( control group), 12 HLA-identical and 9 haploidentical donor /recipient pairs were used. Half of the cells were irradiated with 30 Gy to prevent proliferation. The cells were cocultivated in 5-fold repeats in a 96 well plate in RPMI+15% inactivated human normal serum supplemented with 100 ng SCF/ml. Four days later 5 ng/ml IL2 was added. On day 7, the wells were marked with 37 kBq H3 Thymidin. After a 16 hours incubation time the incorporation was measured by means of a Beta-counter. The maximum stimulation was induced with an irradiated cell pool from 5 healthy blood donors. Results: Table 1 : The stimulation index (SI) after 7 days MLC p-value < 0.05 is considered as statistically significant Conclusions: The modified MLC is a useful method to detect patients at risk for GvHD. We could only investigate a small number of transplanted donor/recipient paires. In additional experiments we investigated the possibility to deplete donor cell samples for donor lymphocyte infusion from HLA-reactive cells. In this serie of experiments only the modified MLC was useful to detect the remaining HLA-reactive cells. Similar results can be achieved with the non-radioactive method of BrdU-incorporation.

Surveillance of nosocomial sepsis and pneumonia in patients after haematopoetic stem cell transplantation. A multicentre project H. Bertz, M. Dettenkofer, S. Wenzler, R. Babikir, W. Ebner, H. Rüden, P. Gastmeier, F. Daschner, University of Freiburg, Charité, Department of Hospital Epidemiology and Infection (Freiburg, Berlin, Hannover, D) Despite broad spectrum antiobiotic prophylaxis and therapy, infections, especially sepsis, are still a main cause of treatment related morbidity and mortality during HSCT. In 2000 a multicenter study (called ONKO-KISS) was initiated in Germany for the surveillance of nosocomial infections (NI; sepsis and pneumonia) during neutropenia in adults after autologous and allogeneic BMT or PBSCT. NI were identified using CDC definitions (laboratory-confirmed blood stream infection; BSI) and modified criteria for pneumonia in neutropenic patients. Trained infection control practitioners visited the wards once a week. Beside demographic data the general data collected included, type of central venous catheter and antimicrobial/antineoplastic treatment. Nursing notes, medical notes, microbiology reports, temperature charts and antibiotic treatment charts were reviewed to de-termine whether a patient had any signs or symptoms of infection. Over the first 26-month period, 1,071 patients with 16,184 neutropenic days were investigated. Of these, 698 (65%) had undergone allogeneic and 380 (35%) autolo-gous BMT or PBSCT. The mean length of neutropenia was 15 days. In total, 231 bloodstream infections and 114 cases of pneumonia were identified. Site-specific incidence densities (pooled mean) were: 14.3 BSIs and 7.0 cases of pneumonia per 1,000 neutropenic days, respectively. Differences between autologous and allogeneic transplantation: BSI/1000 neutopenic days: allo+auto(14.3); allogeneic(12.7), autologous(19.7) Pneumonia/1000 neutropenic days: allo+auto(7,0); allogeneic(7,4); autologous (5, 9) The main pathogens associated with BSI were coagulasenegative staphylococci (55%), followed by streptococci (10,9%). Conclusions: For the first time the ONKO-KISS project provides reference data, showing the importance of gram-positive pathogens. Thus it serves to improve the quality of care provided to BMT and PBSCT patients. This project is supported by the Federal Ministry of Health, Germany

Caspofungin is highly effective as secondary prophylaxis in allogeneic stem cell transplantation in patients with prior systemic or invasive fungal infections N. Stute, A. Kratochwille, T. Zabelina, N. Fehse, W. Hassenpflug, J. Panse, C. Woltschke, F. Ayuketang, H. Schieder, H. Renges, R. von Hinüber, H. Kabisch, A.R. Zander, N. Kröger, University Hospital (Hamburg, D) Background: Fungal infections of Aspergillus and Candida, mainly of the lung, are a major cause of treatment related mortality in allogeneic transplantation. Within a prospective phase I/II study caspofungin 50 mg was given iv daily from start of conditioning until stable engraftment to patients with prior fungal infection. No other systemic antifungal was given, but 6 patients received granulocyte transfusions. Patients & Methods: 26 patients were included in this study so far, all of whom had acute leukemias and a history of probable (n=24) or proven (n=2) fungal infections in the past (according to modified EORTC criteria), and consequently a high risk of invasive or systemic fungal infection during transplant: 7 AML, 4 with 2nd AML, 10 AML relapse, 2 Ph+ ALL, 2 ALL relapse, and 1 CML in blast crisis. Prior fungal infection was diagnosed by CT scan of the lungs (n=25) and liver or spleen (n=8) at a median of 2.5 months before transplant; at time of transplantation 6 patients were in CR, 12 had residuals and 8 florid infections. Conditioning was performed with standard myeloablative regimens. Transplants were unrelated (n=17) and related (n=9) with 6 mismatched donors. In unrelated donors and mismatched family donors ATG was added to minimize the risk of GvHD. Results: The incidence of clinically manifest fungal infection under caspofungin prophylaxis was low: In 4 out of of 26 patients (= 15%: 1 CR, 2 residual and 1 florid state prior to transplant) antimycotic treatment was changed to voriconazole, amphotericin B or Ambisome iv. 3 out of 4 patients who failed on caspofungin had delayed engraftment or primary graft failure with 29, 38, and 40 days of neutropenia, and only 1 died of progressive fungal infection. Importantly, in 7 out of 8 cases a florid fungal infection at time of transplant responded to caspofungin alone with 4 PR and 3 CR (one patient with PR died of fungal disease with Scedosporium prolificans). In not a single patient was caspofungin discontinued due to side effects. Toxicity according to Bearman was comparable to historic controls and overall mortality at day +100 was not increased: 6 out of 26 patients (=23%). Conclusion: Prophylactic use of caspofungin is safe and very effective in high risk patients with a history of systemic or invasive fungal infections when undergoing allogeneic transplant.

S. Giebel, R. Maccario, M.A. Avanzini, M. Marconi, G. Rossi, R. Gentile, F. Locatelli, Silesian Medical Academy, IRCCS Policlinico San Matteo (Katowice, PL; Pavia, I) In immunocompetent individuals Cytomegalovirus (CMV) infection is associated with marked decrease of T cell proliferative response to mitogens. This immunosuppressive effect is mediated by IL-10 homologue encoded by the virus. The goal of this study was to examine the effect of CMV infection on T cells of patients receiving allogeneic hematopoietic stem cell transplantation (alloHSCT) and in whom CMV therapy is based on pre-emptive criteria. Forty-nine patients (median age 9.5 years, range 0.5-20.5 y.) given transplant from an HLA-identical sibling (n=15), haploidentical relative (n=12) or matched unrelated donor (n=22) entered the study. CMV monitoring was based on pp65antigenaemia assay and performed twice a week until day 100 post-transplant. Diagnosis of CMV infection was established and the therapy with gancyclovir initiated when >1/2x10 5 cells in a single examination or 1/2x10 5 cells in two following tests were found pp65-positive. T cell proliferative activity was assessed on days +30, +45, +60, and +90 after alloHSCT and additionally in case of persistence of CMV antigenaemia, and was based on 3HTdR-incoropration assay after stimulation with anti-CD3, concanavalin A (ConA), and phytohemaglutinin (PHA). Results were expressed as counts per minute (cpm). At the same time, quantitive reconstitution of T cells subpopulations was examined with the use of flow cytometry. CMV infection was diagnosed in 19/49 (39%) patients at the median time of 37 (12-65) days post transplant. Median initial and maximum number of pp65-positive cells was 2 (1-16)/2x10 5 and 5 (1-76)/2x10 5 , respectively. None of the patients developed CMV disease. Proliferative response to mitogens was significantly decreased on days when CMV antigenaemia was detected as compared to days without antigenaemia (anti-CD3: 20238 (201-64355) vs. 6829 (223-51462) cpm, p=0.01; ConA: 5799 (80-44016) vs. 10668 (75-58166) cpm, p=0.01; PHA: 6609 (89-49798) vs. 10701 (110-92263) cpm, p=0.07). Both sets of tested samples did not differ in terms of median day post-transplant. Furthermore, number of pp65-positive cells negatively correlated with the proliferative response (anti-CD3: R Spearman= -0.45, p=0.02; ConA R= -0.51, p=0.007; PHA R= -0.54, p=0.004). Comparison of patients who did or did not experience CMV infection revealed that significant decrease of proliferative activity was observed on days +30 and +45, when antigenaemia was most frequently diagnosed, whereas no difference was found on days +60 and +90. CMV infection had no impact on the quantitative reconstitution of lymphocyte subpopulations in the early post-transplant period. We conclude that even asymptomatic CMV antigenaemia has negative impact on T-cell proliferative activity after alloHSCT. However, this immunosuppressive effect is transient and restricted to the duration of infection.

A. Dominietto, E. Tedone, M. Soracco, S. Bregante, C. di Grazia, V. Galbusera, F. Gualandi, T. Lamparelli, A.M. Raiola, M.T. van Lint, F. Frassoni, A. Bacigalupo, Ospedale San Martino (Genoa, I) Background. Epstein-Barr virus (EBV) reactivation is a frequent event in patients undergoing allogeneic hematopoietic stem cell transplant (HSCT) and some of these patients progress to develop a lymphoproliferative disease (LPD). The latter can be predicted by early detection of increasing EBV load by quantitative polymerase chain reaction (Q-PCR) (van Esser et al, Blood 2001; 98,972) , and it is particularly high in alternative donor transplants. Objectives. To monitor EBV reactivation by Q-PCR in patients undergoing an allogeneic HSCT from an alternative donor. To test the predictive effect of positive Q-PCR assay on LPD. Methods. Seventy seven patients were prepared for an alternative donor HSCT with cyclophosphamide (CY) and total body irradiation (TBI) or CY and thiotepa. The conditioning regimen included rabbit anti-thymocyte globulin (ATG; sangstat) 7.5 mg/kg. GvHD prophylaxis was cyclosporin A and methotrexate. Stem cell source was unmanipulated bone marrow. The donors were unrelated in 64 (83%) and family 1-2 antigen mismatched in 13 (17%). All patients received foscarnet for CMV prophylaxis. EBV monitoring was performed every week until day+100, and was expressed as DNA copies x 10^5 cells. Median follow-up of patients was 273 days (range 15-1187). Results. Fifty-five patients (71%) showed EBV reactivation with a median number of EBV copies of 375 (107-38336). Median time of reactivation from HSCT was 45 days (range 5-220). We could identify 3 groups of patients: (a) 22 patients (29%) were EBV negative and their transplant related mortality (TRM) was 18%. (b) 40 (52%) patients had EBV reactivation (100-1000 copies) and their TRM was 23%, and (c) 15 (19%) patients had EBV reactivation (>1000 copies) and their TRM was 40% (p=0.2). The total number of LPD was 6/77 (8%): it was 0 (0%), 1 (2.5%) and 5 (33%) in these 3 groups respectively (p= 0.0002). Mabthera was administered to 4 patients with clinically overt LPD and was successful in 2 (50%). Mabthera was given to 11 patients with >1000 EBV copies without clinical signs of LPD , and was successful in 10/11 (90%).

Conclusions. (1) EBV reactivation is a frequent event (63%) in patients undergoing alternative donor HSCT with ATG in the conditioning regimen; (2) the viral load is a significant predictor of LPD and (3) early treatment with rituximab is highly successful and is indicated for patients reactivating with a high EBV load (more than 1000 copies).

Delayed recovery of B-lymphocytes following high-dose sequential chemotherapy plus rituximab (R-HDS) does not increase the incidence of infective events in non-Hodgkin lymphoma patients A. Assanelli, M. Di Nicola, M. Magni, L. Devizzi, P. Matteucci, A. Guidetti, M. Milanesi, C. Carlo Stella, A.M. Gianni, National Cancer Institute of Milan (Milan, I) High-dose sequential chemotherapy associated to immunotherapy with Rituximab (R-HDS) has been proven highly efficient as in-vivo purging procedure obtaining PCR-negative leukapheresis in the majority of the patients with indolent non-Hodgkin lymphoma (NHL). The aim of present study was to evaluate whether R-HDS induces a delay of short and long-term immune reconstitution with an increased incidence of infective complications. To address this issue, from April 2002 to November 2003 we evaluated prospectively immunological reconstitution of 20 NHL patients treated with R-HDS. R-HDS regimen included high-dose (HD) cyclophosphamide (7g/mq) plus Rituximab (375mg/mq), HD-cytarabine (24g/mq) plus Rituximab, HD-etoposide (2.4g/mq) plus cis-platinum (100mg/mq), HD-melphalan (L-PAM, 180mg/mq) and HD-mitoxantrone (60mg/mq) plus HD-LPAM (180mg/mq) associated to reinfusion of peripheral blood stem cells (PBSC). In addition, two further injections of Rituximab were delivered one month after transplant. Before and at +3, +6 and +12 months from transplant, we have evaluated the following parameters: i) percentage and absolute numbers of B and T lymphocyte subsets (CD19+/20+, CD3+/CD8+, CD3+/CD4+, CD4+/45RO+, CD4+/45RA+, CD8+/45RO+, CD8+/CD45RA+, CD16+/CD56+ cells); ii) immunoglobulin levels (IgG, IgM, IgA). As already described, complete CD4+ lymphocyte reconstitution (430+50/µL) was not achieved in the 12 months post-transplant leading to a suppressed CD4/CD8 ratio (0.5, range 0.1-2.1). In addition, in our patients we observed a reduction of naïve CD45RA+ cells after transplant, since most circulating T cells were activated memory CD45RO+. We observed the absence of circulating CD19+/CD20+ B cells during one year after transplant in all evaluated patients. This is associated with median low levels of immunoglobulins, especially of IgM subclass (IgG:352.8+100mg/dL; IgA:35.2+9mg/dL; IgM:37.1+15mg/dL), without recovering after one year. Bacterial and fungal infections or reactivation of cytomegalovirus (CMV), herpes simplex virus, and varicella zoster virus have been monitored in all cases. We documented only two pneumonias (10%) without identification of pathogen agents and one p65 CMV reactivation, successfully treated with Gancyclovir. In conclusion, despite a delayed B-lymphocyte reconstitution, R-HDS regimen is not associated to a significant incidence of infective complications related to transplant. Larger series of patients are useful to confirm this evidence.

Pre-emptive treatment with rituximab efficiently prevents EBV lymphoproliferative disease (EBV-LPD) in paediatric allogeneic stem cell recipients J. Kalpoe, M. van Tol, R. Bredius, D. van Baarle, N. Annels, E Claas, A Kroes, A. Lankester, Leiden university medical center, CLB/Sanguin (Leiden, Amsterdam, NL) Introduction: EBV reactivation is frequently observed following allogeneic stem cell transplantation (SCT). EBV reactivation may proceed to EBV-LPD which is difficult to treat and often fatal. This complication is almost exclusively seen when a graft from an other than a HLA-identical family donor is used, and is a consequence of T cell depletion of the graft and the use immunosuppressive agents. We have previously reported that quantification of EBV-DNA load in plasma is a reliable tool to recognize EBV reactivation in still asymptomatic patients. Using this approach a window-phase is created in which pre-emptive intervention may be more effective compared to therapeutic intervention in a situation of established clinical disease. Methods: EBV reactivation was prospectively studied in a cohort of 25 pediatric alloSCT recipients by RQ-PCR on weekly plasma samples during 4-6 months postSCT. Based on our previous retrospective study, intervention with a single infusion of CD20 antibodies (rituximab; 375 mg/m2) was started if EBV-DNA loads > 10e3 cp/ml in two consecutive samples. A second infusion was given in non-responders. Cellular immune reconstitution was analysed by measuring lymphocyte subsets and tetrameric HLA-EBV-peptide complexes during the EBV reactivation episode. Results: Pre-emptive treatment with rituximab was administered in 8/25 patients. None of these patients developed EBV-LPD. A rapid decrease in EBV-DNA load was seen in 6/8 patients. In 2/8 patients EBV-DNA load declined after a second infusion of rituximab. In 5/8 patients an increase in CD8+ T cells was observed during the EBV reactivation. Data on the EBVspecificity of these CD8+ T cells will be presented. Rituximab treatment resulted in a B lymphopenia that persisted 4-6 months. All patients received IVIG substitution. No treatment-related toxicity was observed. Conclusion: EBV-LPD in alloSCT recipients can be effectively prevented by EBV-DNA load guided pre-emptive therapy with rituximab. The concurrent recovery of CD8+ T cells in some of these patients suggests that this immune respons per se may be sufficient to control EBV reactivation. The combined analysis of EBV-DNA load and (EBV-specific) T cell reconstitution will more accurately define patients at high risk for EBV-LPD. This strategy will now be used to further optimise identification of patients for pre-emptive therapy.

S. Sohn, D. Kim, J. Kim, W. Sung, K. Lee, K. Lee, Kyungpook National University Hospital (Daegu, KOR) An increased incidence of late cytomegalovirus (CMV) infection has been reported during the last decade since the introduction of ganciclovir (GCV) prophylaxis or GCV pre-emptive therapy. Given that a donor lymphocyte infusion (DLI) can induce more severe graft-versus-host disease (GVHD) and delay immune reconstitution, this may predispose a patient to late CMV infection. From 9/98 to 10/02, 64 patients (median age 36, M/F 38/26) underwent allogeneic stem cell transplantation (SCT) using a matched sibling donor with BM (n=9) or PBSC (n=55) as the stem cell source. The overall incidence of CMV infection, early (EI), and late (LI) CMV infection was 46.9, 42.2, and 16.4%, respectively. Early CMV infection was treated with GCV preemptive therapy that produced a 92.6% success rate. Among the 20 patients that received 35 DLIs with a median of 1.31x10 8 /Kg CD3+ cells, late CMV infection developed in 7 out of 19 evaluable cases (36.8%) with the median onset at 127 days post-transplant. The risk factors for late CMV infection in a multivariate analysis included DLIs (p=0.003, HR 40.548, CI [3.382, 486 .094]) and a previous history of early CMV infection (p=0.003, HR 40.548, CI [3.382, 486 .094]). No association was found between the incidence of late CMV infection and the severity of post-DLI GVHD.

In conclusion, late CMV infection was strongly associated with DLIs and a previous history of early CMV infection. Accordingly, extended surveillance of CMV antigenemia is recommended for patients receiving DLIs or who have a previous history of CMV infection.

Invasive aspergillosis in allogeneic stem cell transplant recipients from alternative donor: incidence, risk factors and outcome A.M. Raiola, C. Viscoli, V. Galbusera, A. Dominietto, F. Gualandi, C. Di Grazia, S. Bregante, D. Occhini, T. Lamparelli, F. Frassoni, M. Machetti, M.T. van Lint, A. Bacigalupo, Ospedale San Martino, IST (Genoa, I) Invasive Aspergillosis (IA) remains an important complication with high morbidity and mortality In patients undergoing allogeneic bone marrow transplantation(BMT. We determined, with retrospective analysis, the incidence, risk factors for IA and outcome in 131 patients who received BMT from unrelated(MUD) or family mismatched donor between January 1999 and November 2002 in our Unit. The diagnosis of IA was documented as proven, probable according to the 2002 European Organisation for Research and treatment of cancer (EORTC)/National Institute of Allergy and Infectious Diseases (NIAID) international consensus. We have also considered proven IA the presence of fungal invasion on autopsy. Incidence. The overall incidence of proven or probable IA was 14% (19/131). In the years 1999, 2000, 2001, 2002 the incidence was 25%, 15%, 13%, 7% respectively (p=0.2). These patients did not have previous history of IA. The median time of diagnosis of IA was +80 days from BMT (range10 -449): 57% (11) were seen in the first 100 days. Median time of first radiological sign of lung infection, also not fulfilling major criterion, was on day + 48 post BMT. Median first positive determination of Aspergillus antigenemia (serum Elisa test for galactomannan) was 55 days from BMT. Median follow up was 311 days (range 3 -1576). Risk factors. We studied as risk factors for IA: age, disease, phase of disease at BMT, type of donor (MUD versus related mismatched), conditioning regimen, prophylaxis of GVHD, acute GVHD, CMV. In univariate analysis only acute GVHD grade II -IV was associated with IA (p=0.0009): IA developed in 73% and 33% of patients with or without aGVHD II -IV grade and the diagnosis of GVHD preceded the diagnosis of IA by days 35. 14/19 patients (74%) had at least two positive Antigenemia test. The sensitivity and specificity of galactomannan test were 73% and 98% respectively. Outcome: Of the 19 patients with diagnosis of IA, 15 died for transplanted related mortality (TRM) (79%) versus 44/108 (40%) patients in the group without IA (p=0.002). At death all patients meet criteria of IA diagnosis. Of the 15 deaths in the IA group 6 were autopsied and all were positive for Aspergillosis: 4 were identified as IA only after autopsy. Conclusions: 1-IA is a significant problem after alternative donor allogeneic BMT. 2-Antigenemia is a helpful indicator of IA, although the the diagnosis is not strait forward. 3-There is a significant association of IA with aGVHD. Patients undergoing HSCT are at high risk of developing bacteremias. These bacteremias may be preceded by colonization detectable by surveillance cultures that also provide bacterial susceptibility testing. We undertook a retrospective study in HSCT recipients (1) to determine the incidence, timing and etiology of bacteremias; (2) to examine the ability of routine surveillance cultures to predict bacteremic episodes. We studied 622 (256 allografts and 366 autografts) consecutive patients transplanted between 1982 and 2001. Routine surveillance cultures (stool, urine, sputum, nose, tongue, throat, skin, vagina or penis, anus and blood) were obtained twice weekly. For those occurring before day 60, we examined whether the responsible agent had been detected in routine surveillance cultures obtained before the first day of infection. A total of 404 episodes occurred in 248 patients, due to coagulase-negative staphylococci (n=171, 42%), gram-negative bacteria (n=129, 32%), streptococci (n=48, 12%), other gram-positive bacteria (n=33, 8%), anaerobes (n=9, 2%) and fungi (n=14, 3%). Gram+ outnumbered Gram-bacteria by a margin of at least 2:1 throughout the study period except in the last 2 years. Grambacteremia was 0.28, 0.16 and 0.26 episode/patient in 1982-1992, 1993-1999 and 2000-2001, respectively (p=0.0053) . Bacteremias occurred later and were more frequent in allogeneic compared to autologous transplants (0.96 vs 0.44 episode/patient, p<0.0001). Among them, 212 (53%) occurred before hospital discharge and 192 (47%) thereafter. In 50% of the cases (coag-neg Staph > fungi > Gram-> Gram+ > anaerobes, p<0.0001), the agent responsible for the bacteremic episode was present in routine surveillance cultures beforehand. The predictive value of such cultures is in fact quite low for staphylococci, because 77% of the patients are colonized. For Gram-bacteria, even if we take the conservative figure of 30% resistance to our standard empiric antibiotic regimen, only a very small number of patients (30% of 28 episodes = 8 episodes over 19 years) would be better managed with the help of surveillance cultures. In conclusion, bacteremias remain a frequent complication, particularly in allogeneic transplantation. Routine surveillance cultures can predict bacteremias in 50% of the cases but the practical impact of this observation is limited in view of the costs. Objectives: For the purpose of a prospective study on the benefit of prophylactic immunoglobulins in allogeneic HLA-identical sibling SCT (1), we elaborated a scoring system to assess the severity of the infections occurring after according to the expected rate of mortality in the literature. The aim of the present study is to assess the relationship between the severity of each infection, and the mortality process. Patients and Methods: 440 infectious events occurring within 6 months after transplant were prospectively collected and graded in 190 consecutive patients. 77% patients were leukemic. The median age was 40 y. All received myelo-ablative transplants. The infections were graded as follows: Infections of grade 1 were those usually treated at home (including asymptomatic CMV infection) or febrile neutropenia of unknown origin treated with broad-spectrum antibiotics. Infections of grade 3 were those with an expected mortality rate 60% (i.e., bacteremia or fungemia with severe sepsis, aspergillosis, CMV disease, pneumonia with an initial PaO2 < 65mmHg). Infections of grade 2 were all the other ones. We used multivariable Cox models with time-dependent covariates to assess the relationship between the grade of each infectious episode and the mortality (Splus 2000 (Math Soft, Inc, Seattle, WA) software). Results: The infections were graded as score 1 (n= 274, 62.3%), score 2 (n= 128; 29.1%) or score 3 (n= 38; 8.6%). The occurrence of an infectious episode scored 3 by the severity scoring system significantly increased the risk of death (p< 0.0001), while no additional influence on the death risk was observed after any score 1 (p= 0.53) or score 2 (p= 0.74) infections, whatever the model of analysis. This was independent from GvHD and number of previous infections. This was illustrated by the 11.3% mortality rate observed among the 71 patients who experienced at most an infectious episode scored 1 by the severity score, and 12.7% in the 71 patients who experienced at most a scored 2 infection, compared to 61.3% in those who experienced at most a scored 3 infection Conclusion: Our grading system allows to focus on the most medically important, life-threatening infections and should help in the prospective evaluation of new strategies that aim to reduce the risk of severe infections after allogeneic SCT. The grade 3 infectious events should be reported in priority in prospective trials. 

F. Ciceri, M. Lupo Stanghellini, J. Peccatori, P. Servida, A. Pescarollo, M. Bernardi, D. Giudici, S. Colombo, D. Salaris, G. Torri, M. Bregni, Istituto Scientifico H.S. Raffaele (Milan, I) Non-invasive ventilation (NIV) is considered as first-line treatment in the early phases of acute hypoxemic respiratory failure (ARF). In immunosuppressed patients with ARF, NIV allows assisted mechanical ventilation without the need for an invasive artificial airway and is considered a valid alternative to intubation. In haematologic patients, notably haematopoietic stem cell transplant (HSCT) recipients, avoidance of intubation could be an important objective in order to reduce the risk of secondary infections. Since 1999, we have treated with NIV (continuous positive airway pressure C-PAP and bi-level positive airway pressure BiPAP) 24 patients with ARF in Haematology Unit. The median age of patients was 44,5 years (range 19-62 years), 10 were more than 50-yr old. All of these patients had malignant haematological disease (12 primary and secondary acute myeloid leukaemia, 4 acute lymphoblastic leukaemia, 4 non Hodgkin's lymphoma, 3 Hodgkin's lymphoma, 1 granulocytic sarcoma), 19 patients previously received HSCT. Twelve patients developed ARF during hospitalisation for HSCT, 6 post transplant, 5 after chemotherapy, and 1 for primary TTP. 8/24 patients had, at the moment of ARF, ANC < 0.5 x10 9 /L and 17/24 had platelets <25 x10 9 /L. The aetiology of ARF was known in 11/24 cases: 2 Aspergillus, 2 Candida, 2 CMV, 1 Pneumocystis carinii infection; in 4 patients Pseudomonas aeruginosa was isolated from blood cultures. Before NIV, the median value of O2 arterial pressure (PaO2) in blood samples were 62 mmHg; the median time from clinical onset of ARF to start NIV was 24 hour. Five patients were treated with BiPAP, 19 with CPAP. The median FiO2 was 70%, the median PEEP was 7,5 cmH2O. The median value of PaO2 during NIV was 80 mmHg. The modality of NIV was continuous in 12 patients, cyclic in the others; the median time of application of NIV was 36 hours (range 6-240). Organ failure and shock was concomitant in 13 patients and treated with vasoactive amine infusion. Five patients was referred to ICU for invasive mechanical ventilation without success. At day +30 the survival rate was 25%. Our experience shows that NIV can be easily performed in haematologic department by the local nursing staff. NIV can help in the treatment of ARF in severely neutropenic and transplanted patients.

Candidaemia in allogeneic stem cell transplant recipients: low risk without fluconazole prophylaxis E. Jantunen, A. Nihtinen, L. Volin, E. Juvonen, T. Parkkali, T. Ruutu, V. Anttila, Kuopio University Hospital, Helsinki University Hospital (Helsinki, Kuopio, FIN) Invasive fungal infections (IFI) are common in allogeneic SCT recipients. We have reviewed our experience on IFI with special reference to Candida septicaemia in a cohort of 685 adult patients transplanted in 1983-2002. The donor was a matched sibling in 505 patients and an unrelated donor in 180 patients. BM graft was used in 561 patients and PB in 124 patients. The most common diagnosis were AML (n=217), CML (n=180), ALL (n=112), MM (n=54) and MDS (n=52). Fluconazole prophylaxis was not used during the study period. Definitive or probable IFI was observed in 58 patients (8.5%) with a predominance of Aspergillus infections (45 patients, incidence 6.6%). Candida septicaemia was found in only eight patients (1.2%). The most common causative agents were C. albicans (n=7), C. crusei (n=2) and C. glabrata (n=1); in two patients more than one causative agents were found. The median time to the diagnosis was 93 d (6-249) posttransplant. Six patients were neutropaenic at the time of diagnosis; two patients had not yet engrafted. Six patients had experienced acute GVHD. All patients received antifungal therapy. The median survival was 26 d (6-280). Only one patient was cured. Candida septicaemia seems to be a rare event in allogeneic SCT recipients. Thus systematic antifungal prophylaxis is perhaps not indicated. The prognosis of established Candida infections in this patient population is still poor due to severe co-morbid conditions especially GVHD.

Infectious complications in multiple myeloma patients undergoing autologous stem cell transplantation after high dose melphalan: a single centre retrospective analysis towards outpatient strategy G. Irrera, M. Martino, G. Messina, G. Console, F. Morabito, M. Kropp, F. Iuliano, D. Mamone, M. Brugiatelli, C. Stelitano, V. Callea, C. Musolino, S. Molica, P. Iacopino, Azienda Ospedaliera "BMM", Messina University (Reggio Calabria, Catanzaro, Messina, I) The principal complication in patients undergoing ASCT is infection, which represents an impediment for outpatient basedtransplant. We performed 133 ASCT in patients with MM, following either a total inpatient model (TIM, 56 cases) or outpatient model (OUT, 77 cases). Induction chemotherapy mainly consisted of DAV +/-high-dose CTX. All patients were conditioned with HDM (200 mg/mq). Cases were equally distributes for age, sex and disease status at transplantation. No significant differences between the two groups of patients with respect to the number of stem cells infused (5.9 ± 5.5 x 10 6 /kg in TIM vs. 5.2 ± 2.0 x 10 6 /kg in OUT), the time to granulocyte recovery (9.0 ± 0.8 days in OUT vs. 9.0 ± 0.9 in TIM) and the time to platelet engraftment (12.9 ± 4.4 days in TIM vs. 12.7 ± 2.1 in OUT). The main characteristics of infectious complications and mucositis score are detailed in Table: Overall, these data suggest that: 1) infectious complications are likely to occur in outpatient group; 2) FUO is more frequent in outpatient group; 3) there is no difference between the two groups in mucositis score. Of note, one death occurred in inpatient group because of complications. In conclusion, although some of data could be explained by biased selection, our experience confirms the feasibility of outpatient program. Ambulatory care, when compared with inpatient care, appears to have a low risk of infections and, probably, a better quality of life. Partially supported by Regione Calabria and AIL

Clinical relevance of screening sinus CT scans and results of endoscopic sinus surgery in bone marrow transplant patients I. Yorulmaz, E. Soydan, M. Kizil, B. Ceydilek, M. Arat, Ankara University Faculty of Medicine (Ankara, TR) Bacterial or fungul sinusitis may lead to serious complications and even mortality, in patients who have undergone BMT. Anatomic variations narrowing the sinus ostia or silent chronic sinusitis existing prior to BMT, may increase the risk for life threatening complications of sinusitis. Screening Ear-Nose-Throat (ENT) examination and imaging of the paranasal sinuses has been felt to be useful in determining which patients need elimination of risks for post-BMT sinusitis. This study reviews the pre-BMT screening sinus data and post-BMT outcome, in 82 patients who received allogenic BMT, between 1998 and 2002. Patients were analyzed in two groups. The first group consisted of fifteen patients who passed a standart ENT physical examination and plain sinus X-rays. One patient (7%) were lost due to invasive fungal sinusitis and another five patients (33%) were found to be in need of surgical treatment for their chronic sinusitis detected in the post-BMT period, although none had developed a serious complication of sinusitis in the neutropenic state of BMT. The second group consisted of 68 patients who were evaluated with careful nasal endoscopy and coronal CT scan of the paranasal sinuses. Sixty percent of the patients had normal findings, 22% of the patients had minor anatomic variations that did not effect the sinuses and 18% (12/68) of the patients had either chronic sinusitis or chronic sinusitis plus leading anatomic variations that necessitated surgical treatment.. Ten out of 12 patients with chronic sinusitis were operated endoscopically before BMT, the remaining two patients did not accept surgery. None of the patients experienced a serious sinus problem in the neutropenic state of BMT and none showed recurrence of chronic sinusitis requiring revision surgery after the initial surgical procedure. One patient who had normal screening findings initially developed chronic sinusitis after the neutropenic state of BMT and was treated surgically. The results of this study show that diagnostic nasal endoscopy and screening CT scan of the paranasal sinuses reveal sinusitis requesting surgical therapy approximately in one fifth of the patients. Standart ENT examination and plain sinus X-rays may fail to detect minor chronic sinus disease that may endanger the neutropenic patient. Minimally invasive endoscopic sinus surgery is a safe method and reduces the risk for life-threatening complications of sinusitis, in patients undergoing BMT.

Human herpesvirus-6 antigenaemia: an association to acute GvHD?

L. Volin, I. Lautenschlager, E. Juvonen, A. Nihtinen, V.-J. Anttila, T. Ruutu, Helsinki University Central Hospital (Helsinki, FIN) Human herpesvirus-6 (HHV-6) has a tendency to remain latent in the body after primary infection and then reactivate in an immunocompromised host. After stem cell transplantation (SCT) the incidence of HHV-6 viremia studied by PCR has been reported to be 28-75%. However, qualitative PCR often cannot distinguish between latent and active infection, and more recently quantitative PCR has been explored to better correlate the virus load to clinical symptoms. The clinical significance of HHV-6 viremia after SCT is, however, not clear. Immunoperoxidase staining provides a method to demonstrate HHV-6 spesific antigens in peripheral blood mononuclear cells. We have prospectively monitored HHV-6 by an antigenemia assay in 58 consecutive adult allogeneic SCT recipients who were transplanted for a malignant haematological disease (CML 15, AML 15, ALL 10, NHL 8, MM 6, CLL 2, MF 1, and HES 1) . The conditioning was myeloablative in 44 and non-myeloablative in 14 cases. If the donor was unrelated, antilymphocyte globulin (Thymoglobuline) 6-12 mg/kg was given. 36 of the patients had a sibling and 22 a matched unrelated donor. 34 patients received a bone marrow and 24 a peripheral blood stem cell graft. In myeloablative SCT GVHD-prophylaxis consisted of cyclosporine, methotrexate, and in sibling SCT of methylprednisolone (MP) in addition, and in non-myeloablative SCT of cyclosporine and mycophenolate mofetil. All patients received acyclovir as herpes simplex virus prophylaxis for 35 days after SCT. Blood samples for the detection of HHV-6 (two variants HHV-6A and HHV-6B) antigenemia were obtained weekly during the first three months after SCT. In this study we observed that 47/58 (81%) of the patients demonstrated HHV-6B antigenemia on day 4 as a median (range 4-25). The cumulative incidence of acute GVHD was 26%. The onset of acute GVHD occurred significantly (p = 0.034) earlier in the 9 HHV-6B antigenemia positive patients compared to the 6 HHV-6B negative patients, on the median day of 24 (range 7-42) and 43(range 17-72), respectively. The overall incidence and grade of acute GVHD did not significantly differ in this small material. In this study HHV-6 antigenemia was detected in a large proportion of patients after SCT and our finding suggests that HHV-6B antigenemia may be associated to an early onset of acute GVHD.

The use of dendritic cells transfected with in vitro transcribed mRNA for the analysis of CMVpp65-specific Tcell reconstitution after allogeneic stem cell transplantation R.G. Meyer, C.M. Britten, A. Gstoettner, C. Huber, T. Woelfel, W. Herr, Johannes Gutenberg University (Mainz, D) CMV-specific T-cell reconstitution after allogeneic stem-cell transplantation has been investigated by various methods. Current assays based on HLA/peptide multimer staining or cytokine secretion (e.g. ELISPOT) frequently use defined peptide epitopes derived from CMV proteins (mainly pp65) to examine the frequency of CMV-specific CD8+ T cells among peripheral blood mononuclear cells (PBMC). The use of peptides, however, is limited to known epitopes and to the presence of their restricting HLA-alleles. Herein we applied CMVpp65 mRNA as an antigenic format to detect among PBMC of two allografted patients CMV-specific CD8+ T cells in IFN-gamma ELISPOT assays. Dendritic cells (DC) generated from the patients´ posttransplant PBMC served as antigen-presenting cells (APC). DC were transfected with mRNA using an electroporation protocol. Assays were performed on CD8+ lymphocytes positively isolated from PBMC samples obtained prior to, and at several time-points after transplantation. The patients and their corresponding donors were CMV-seropositive. Patient 1 had CML in chronic phase and underwent transplantation with a HLA-identical sibling stem-cell allograft. He received immunosuppressive medication for severe acute GvHD starting at day +30. Among the patient´s PBMC collected before transplantation, 1/460 CD8+ lymphocytes recognized autologous DC transfected with pp65-mRNA. After SCT, pp65-specific CD8positive T cells were detected as early as on day +22 (1/210), increased to a peak on day +86 (1/170), and were seen at slightly reduced levels on days +148 (1/294) and +267 (1/200) . Patient 2 had ALL and underwent transplantation with an unrelated donor allograft mismatched for HLA-DRB1. In this patient, the pre-transplantation frequency of CMVpp65-specific CD8+ T cells was 1/370. After SCT, anti-CMVpp65 reactivity was not detectable on day +22. However, on day +63 the frequency of pp65-specific CD8+ T cells was 1/116 and remained stable through day +105.

Our data indicate the feasibility of using mRNA-transfected DC as APC in IFN-gamma ELISPOT assays for the sensitive and HLA-independent monitoring of CMV-specific CD8+ T-cell reconstitution. The approach might be also applicable to other pathogens, in particular when immunodominant epitopes and their HLA-restriction are poorly defined.

T-cell depletion rather than persistent graft-failure determines the risk of viral but not fungal deaths in children undergoing second attempt HSCT for graft dysfunction L.M. Ball, M.S. Page, A.C. Lankester, R.G.M. Bredius, M.J.D. van Tol, R.M. Egeler, Leiden University Hospital (Leiden, NL) Primary failure of engraftment or early rejection is known to be associated with a high mortality despite further attempts at retransplantation. Overwhelming infection is commonly associated with a fatal outcome. Factors influencing the type of infectious agent could, if known, lead to more specific intervention strategies with the aim of improving outcome. We reviewed the results of children undergoing second hemopoietic stem cell transplantation (HSCT) for graft dysfunction in a single center. We aimed to determine factors associated with deaths occurring within 100 days post second HSCT attributable to infection. Data from children with more than one documented attempt at HSCT from 1990 HSCT from -2003 were included in the study. Deaths were analyzed in relation to transplant parameters and proven infections. Ten percent of children (n=33) transplanted in our unit required a second or subsequent attempt HSCT for graft dysfunction, ten of whom survived. Infection accounted for 15/23 deaths (65%). Non-survivors could be grouped into those who had persistent graft failure despite subsequent attempts (n=9), of whom four died as a result of infection. In contrast, 11 (78.5%) of the successfully engrafted non-survivors (n=14) died from infection. Engraftment in these children was independent of T-cell depletion. In non-survivors, T-cell depletion of the graft (n=13) was associated with death due mainly to viral infections (6 disseminated adenovirus, 3 gram negative sepsis, 1 C. albicans), whereas, in non-T-cell depleted grafts (n=9), 5 (55%) died mainly from fungal infection (4 Aspergillus fumigatus, 1 CMV). Viral but not fungal infection was associated with subsequent rejection episodes. Our study shows that infection is the most common cause of death in children undergoing second HSCT, occurring despite successful engraftment and neutrophil regeneration. In vitro Tcell depletion and early repeated HSCT defined children at high risk for disseminated adenoviral infection. Non T-cell depleted grafts were associated with Aspergillus fatalities.This suggests that within the immediate post-transplant period following 2nd attempt grafts, host T cell immunity may be functionally more important in the control of viral than fungal infections. Successful engraftment can be achieved with the use of additional immune suppression but predisposes to death from infection in this highrisk population.

A. Zambelli, D. Lilleri, P. Pedrazzoli, J. Peccatori, F. Baldanti, V. Fregoni, F. Ciceri, M. Renga, E.-P. Alessandrino, G. Gerna, G.-A. Da Prada, S. Siena, M. Bregni, Fondazione S. Maugeri, IRCCS Policlinico San Matteo, Ospedale Niguarda Ca Granda, IRCCS HS Raffaele, IRCCS Fondazione S. Maugeri (Pavia, Milan, I) Human cytomegalovirus (HCMV) infection is the most frequent infectious complication after conventional allogeneic stem cell transplantation (alloSCT). From December 1998 to December 2002, we prospectively monitored HCMV reactivation in 59 patients affected by solid tumors and undergoing nonmyeloablative allogeneic stem cell transplantation (NST). Patients were allografted from HLA-identical sibling donors after Fludarabine/Cyclofosphamide-based conditioning regimens. Seventeen out of 59 patients (28.8%) presented HCMV antigenemia, and 14 of them received ganciclovir with successful HCMV clearance in all cases. No patient developed HCMV viremia/disease. The median time to HCMV reactivation was 54 days (range 16-245) after NST. These patients were compared with a cohort of hematological patients, who were treated by conventional myeloablative alloSCT. Matching criteria included HCMV risk group, SCT source, donor type and age. In the myeloablative group the HCMV active infection was observed in 47/55 patients (85.4%) at a median time of 30 days (range 13-64) . This HCMV incidence rate is higher (p<0.001) and the appearance of infection was earlier (p=0.001) than that observed in NST patients.

In conclusion, patients affected by solid tumors undergoing NST showed a reduced and delayed incidence of HCMV active infection. HCMV infection does not seem to represent a major clinical problem in this setting.

A. Rovelli, P. Corti, A. Balduzzi, D. Longoni, M. Dassi, P. Perseghin, G. Gaipa, R. Parini, G. Uziel, A. Biondi, C 

GvHD has been correlated with a poorer neuropsychological outcome in patients with inherited metabolic storage diseases (IMSD), therefore we include T-cell depletion by MACS technology (TCD) in the procedure for these patients. Due to the residual B-cell amount after two-step negative selection (1 HSCT in this series) and the increased number of B-cells in the final product after the T-cell addback following positive selection (the other 11 HSCTs) (see BMT 2003; 31, 857-860) , since 1999 a rituximab single dose on day + 3 was included in the treatment plan to carry out a B-cell depletion in vivo to prevent EBV-PTLD. Ten consecutive patients with different IMSD who underwent 12 HSCTs (11 unrelated, 6 of them with some degree of mismatching, and 1 related donor; age at HSCT 1.2 to 32.4 years) were scheduled to receive the anti-CD20 monoclonal antibody. Rituximab was administered following 11 HSCTs to 9 pts. and not to 1 for lacking of parents'consent. Conditioning regimen included BuCy (7) or FluBuCy (4) or TTCy (1) + ATG. All patients were monitored weekly for EBV reactivation by quantitative PCR. Engraftment occurred in all cases from day +10 to +15; 2 pts. who rejected, at +257 and +343 days, respectively, underwent successfully to a 2nd HSCT from a different unrelated donor. The median number of cells infused (x10 6 /kg) was: CD34+ 5.6, CD3+ 0.35 and 1.0 (at 1st and 2nd S198 HSCT, respectively). Only 1 pt. developed grade I acute skin GvHD and 2 pts. limited chronic GvHD. One pt. died; at a median follow-up of 2.7 years (0.6 -4.0) 9/10 pts. are alive with complete (7 pts.) or partial (2 pts.) chimerism (70% and 85% donor, respectively); 7/9 with improved neuropsychological function and 2 with stable disease but some disabilities developed after HSCT as a result of the primary damage. None of the pts. developed EBV reactivation, with the surprising exception of the only pt. who did not receive rituximab as prophylaxis and who was at that point (+71 days) successfully treated with the anti-CD20 antibody (2 infusions). Only 2 pts.

have not yet recovered CD19+ cell count: the one who developed EBV reactivation and one who is only 7 months after HSCT. We used rituximab empirically thinking it reasonable; due to the rarity of IMSD and the limited number of such a procedure/manipulation at our center, a specific study was not feasible. Our observation is intriguing and prompts for randomized studies in settings where a higher number of pts. can be recruited.

A. Picardi, G. Gentile, P. de Fabritiis, A. Capobianchi, L. Cudillo, T. Dentamaro, A. Tendas, L. Cupelli, M. Ciotti, A. Volpi, S. Amadori, P. Martino, University Tor Vergata, University La Sapienza (Rome, I) The effective anti-CMV treatment strategy must include a sensitive and reliable diagnostic assay to rapidly evaluate active CMV infection. Pp65 antigenemia (pp65Ag) is considered the standard assay to detect CMV infection; however, whether PCR assay for detection of CMV DNA in plasma may anticipate pp65Ag is still controversial. The aim of the study was to investigate the usefulness of a quantitative plasma PCR test and compare it with the pp65Ag for detection of CMV infection following allogeneic stem cell transplantation (SCT). Between December 2002 and October 2003, 21 patients underwent an allogeneic SCT from either an HLA-identical sibling (19) or an unrelated donor (2) . All patients were weekly monitored for CMV infections by both quantitative CMV-PCR in plasma (COBAS AMPLICOR CMV MONITOR Test with lower limit detection of 400 copies/ml plasma) and pp65 Ag during the first 100 days after SCT. As CMV prophylaxis, Acyclovir was given either at standard or high dose in related or unrelated transplants, respectively. No patients received specific Ig, Gancyclovir, Foscarnet or Cidofovir as CMV prophylaxis. Pre-emptive therapy with Gancyclovir or Foscarnet was started at the first detection of antigenemia ( >= 1 positive cell). Plasma CMV DNA was not considered for clinical decision making. Overall, 12/21 patients (57.1%) had CMV infection within 100 days from SCT: in 8 patients, CMV was first detected by pp65 alone; in 3, by both methods and in 1, by PCR alone. Pp65Ag positivity after SCT was earlier (mean 37 days ±12.1, range 14-56) than plasma PCR assay (mean 45 days ±34.2, range 7-100). A total of 261 blood samples were analyzed. CMV was detected in 27 samples (10.3%) by a single or both methods. PCR detected a median of 1000 copies/ml (range 770-10000) while pp65Ag showed a median of 5 positive/150000 total PMN examined (range 1-80). Overall, 7 samples were found to be positive by PCR and pp65antigenemia, 12 samples were pp65Ag positive but PCR CMV negative, 1 was PCR positive/pp65Ag negative, and 234 were negative by both assays. Five patients (23%) died after a median of 49 days (28-84) from SCT for progressive disease (3) or acute GVHD (2) in the absence of CMV infection. Only 1 patient developed intestinal CMV disease during the study period despite negative pp65Ag and PCR assays, responding to antiviral therapy. In conclusion, pp65Ag detected CMV infection earlier than plasma PCR in our allogeneic SCT recipients.

Lamivudine for treatment and pre-emptive therapy of Hepatitis B virus reactivation following haematopoietic stem cell transplantation A. Locasciulli, L. Forte, L. Pescador, M. Gabriel Arana, M.R. Capobianchi, I. Majolino, Ospedale S. Camillo-Forlanini, IRCCS L.Spallanzani (Rome, I) HBV reactivation after HSCT in patients HbsAg positive before transplant has been reported extensively. 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-up. We report the results obtained with Lamivudine treatment in 9 HSCT patients (3 viraemic before transplant and 6 developing HBV reactivation during follow-up). Among 106 patients undergoing autologous or allogeneic HSCT in our Unit between 2001 and 2003, 9 (8.5%, 1 female and 8 male) showed HBV active infection either before (n.3) or after transplant (n.6). The underlying disease was Acute leukemia in 4, Chronic Leukemia in 2, Multiple Myeloma in.1 and Aplastic anaemia in. 2. One received autologous and 8 allogeneic HSCT ( from HLA identical sibling donor in 7 and from mismatched parent in 1). Lamivudine protocol wa as follows: treatment (100 mg/day), started concomitant to conditioning regimen in HBV carriers and preemptive therapy patients showing viraemia and HBV antigenemiaduring 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 month-treatment. HBV Genotype and sequences analysis was carried out in 5/9 cases. Results: HBV infection = 3 patients (1 carrier and two with postHSCT reactivation) cleared the infection and seroconverted to anti-HBs within three months from the beginning of treatment. Seroconversion from HbeAg to anti-Hbe occurred in 2 /5 cases with ongoing infection , but evidence of viraemia still persists after 160-200 days from treatment. HBV-related liver disease: no patient developed fulminant hepatitis or severe flare-up; 1/9 patients showed overt hepatitis concomitant to HBV reactivation, but transaminases returned to normal after 30 days of treatment, while 7 had either a transient and mild increase in ALT values or normal liver function. Toxicity: we did not observe marrow or other relevant organ toxicity.

In conclusion, the impact of Lamivudine treatment and preemptive therapy on HBV -related liver disease seems remarkable and efficacy on HBV infection is promising.

Comparable risk of cytomegalovirus infection after allogeneic haematopoietic stem cell transplantation following non-myeloablative or myeloablative preparative regimen in patients affected by haematologic malignancies D. Caldera, D. Lilleri, P. Bernasconi, A. Colombo, F. Montanari, F. Ardizzone, M. Parea, M. Lazzarino, G. Gerna, E. Alessandrino, IRCCS Policlinico San Matteo (Pavia, I) Objective: The aim of this study was to compare incidence and outcome of human cytomegalovirus (HCMV) infection in two groups of patients with hematologic malignancies who received hematopoietic stem cell transplant (HSCT) from an HLA-identical related donor treated by two different preparative regimens. Patients and Methods: Group A included 27 patients receiving a reduced intensity conditioning regimen with Thiotepa and Fludarabine, whereas group B included 31 patients treated with a standard myeloablative conditioning regimen (BUCY2). Patients from the two groups were homogeneous for age, gender, and donor/recipient serostatus for HCMV. Virological monitoring was conducted by biweekly determination of pp65-antigenemia and viremia. Patients were preemptively treated with ganciclovir 5 mg/kg every 12 hrs. at first appearance of virus in blood or after confirmed positivity if a single pp65-positive leukocyte was detected. No patient received anti-HCMV prophylaxis. Results: In group A 19/27 patients had active HCMV infection, as well as 24/31 group B patients. The actuarial probability of developing HCMV infection within day +100 was similar between the two groups (77% in group A vs 80% in group B) as well as median time to HCMV appearance in blood: 39 days (range: 24-79) vs 42.5 days (range:13-64 ). Antiviral therapy was successful in clearing HCMV infection in both groups (no patient developed symptomatic HCMV disease). A trend toward a shorter median duration of treatment in group A (18 days, range 6-42) vs group B (24 days, range 6-49) was found. Conclusion: No difference was found in incidence, time to appearance and outcome of HCMV infection in patients receiving non-myeloablative or myeloablative conditioning regimen.

H. G. Heuft, L. Goudeva, N. Pulver, L. Grigull, B. Hertenstein, R. Blasczyk, Hannover Medical School (Hannover, D) Objectives: To investigate the therapeutic effect of granulocyte transfusions (GTX) in critically ill neutropenic patients with septic infectious complications during treatment for acute leukemia or allogeneic peripheral blood stem cell transplantation. Study design and methods: We evaluated 54 patients with 271 GTX (33 children, aged 8 [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] per GTX corresponding to 1.46x10 9 granulocytes/kg in children and 0.58x10 9 granulocytes/kg in adults. Results: The WBC counts increased from baseline values of 0.2 (0-0.5x10 12 ) G/L for both pediatric and adult patients to peak values of 3.8 (0.4-18.2)x10 12 G/L (children) and 0.9 (0.3-9.4)x10 12 G/L (adults) at one hour after GTX and to 1.4 (1.1-13.6)x10 12 G/L (children) and 0.4 (0.3-8.6)x10 12 G/L (adults) at 24 hours after GTX. In 42 out of 54 patients (80%), the CRP levels significantly declined (60 (11-91)%; p<0.001) during the granulocyte transfusion period; in almost all cases (39/42; 90%) after the initial or 2nd transfusion. Thirty-eight patients (71%) were alive at day +30 after termination of neutropenia and GTX. Patients without CRP response to GTX (6/9, 66%) and patients with severe viral infections 6/7 (86%) were not among the day +30 survivors. Conclusions: GTX are helpful to overcome systemic bacterial or fungal infections even in severely immunocompromised adult neutropenic patients but are ineffective in the treatment of viral infections. Declining CRP levels are an early predictive marker of a successful GTX treatment.

Secondary prophylaxis with voriconazole for invasive aspergillosis in stem cell transplantation D. Mattei, N. Mordini, C. Lo Nigro, M. Musso, D. Rapezzi, A. Gallamini, Az. Ospedaliera S. Croce e Carle (Cuneo, I) Patients with a history of IA during chemotherapy appear to be at high risk of recurrence when undergoing further chemotherapy. However, if treatment and secondary prophylaxis with effective antifungal drugs is promptly started, these patients can complete the scheduled chemotherapy and undergo SCT. We report our experience with secondary prophylaxis with voriconazole (VOR) in 6 SCT patients with a history of IA. The patients characteristics are reported in table 1. Despite antifungal prophylaxis with itraconazole 200 mg twice daily, all the patients developed IA during induction chemotherapy-related neutropenia. According to the EORTC/MSG classification, there was 2 proven (1 AML+1 MM) and 4 probable cases (4 AML), respectively. Three patients failed previous amphotericin B deoxycholate (D-AMB) and liposomal amphotericin B (L-AMB) treatment, in 1 case D-AMB was given before VOR, VOR was the front-line treatment in 2 cases. At the time of the first VOR infusion, chest CT scan revealed lung lesions and ELISA assay for Aspergillus spp galactomannan (GAL) turned out to be positive (>1 ng/ml) in all the patients. VOR schedule was 6 mg/kg iv twice daily on day 1 and then 4 mg/kg iv twice daily,then 200 or 150 mg orally twice daily. All patients completed the scheduled chemotherapy program during VOR treatment and then underwent SCT during secondary prophylaxis. Source of SC and conditioning regimens are described in table 2. VOR was administered for a median time of 136 days (range 104 to 270),time to GAL reduction <0.7 ng/ml was 7 days. At the time of VOR discontinuation, chest CT scan showed lung lesion healing and negativity of GAL ELISA assay in all the patients. Side effects included WHO grade 2 liver abnormalities in 4 patients and visual disturbances in 2 cases, and required only a brief discontinuation and/or dose reduction in 2 patients. AlloSCT recipients required CyA dosing reduction during VOR treatment. No IA recurrence occurred at any time, and at a median follow up of 262 days (range 54 to 1017) after SCT 5 out of 6 patients are alive without clinical, radiological or laboratory signs of IA. An AML patients died of refractory leukemia after a second alloPBSCT. We conclude that VOR secondary prophylaxis is effective and well tolerated in patients with a previous IA history undergoing auto/alloSCT. Moderate increase in liver enzymes is frequent but not harmful; the dosing of CyA must be reduced to avoid toxicity P759 Delayed reactivation of Epstein-Barr virus in recipients of reduced-intensity conditioned allogeneic stem cell transplantation with campath-1H may impact on surveillance and pre-emptive strategies Q. Hill, T. Collyns, A. Hale, T. Fawcett, A. Hancox, G. Cook, St. James´ University Hospital, Health Protection Agency Laboratory (Leeds, UK) Elevated EBV DNA copy numbers have been shown to be predictive for EBV-induced lymphomas (EBV-LPD). Increased EBV-DNA levels following RIC-HSCT protocols containing ATG, occurring 1-3 months with EBV-LPD developing <6 months post-transplant. Campath-1H in RIC-HSCT protocols has been associated with delayed lymphocyte recovery, it has been suggested that B-cell depletion can reduce the levels of EBV infected B-cells (donor & recipient) resulting in reduced reactivation. We conducted a study to examine the incidence & timing of EBV reactivation in RIC-HSCT. 18 patients undergoing RIC-HSCT for haematological malignancy (AML n=6; ALL n=1; CML n=3; CLL n=1; MDS n=1; PNH n=1; FCL/MCL n=3; HD n=2) with a median age of 42 years (range 20-57) received in vivo T-cell depletion with Campath-1H (20mgs on days -8 to -4); 17 patients received fludarabine (150mg/m 2 ) & melphalan (140 mg/m 2 ) and 1 patient received cyclophosphamide (120mg/kg) & TBI (200cCy). 11 patients received HLA-matched sibling, 6 received MUD & 1 received a mismatched MUD grafts (PBSC n=15, BM n=3). Post-graft cyclosporin A alone was used as GvHD prophylaxis. Pre-transplant recipient EBV serology (IgG) was positive in 17 cases & negative in one. EBV serology was positive in 14 donors (one IgM +ve), unavailable for testing in 4 & negative in one (EBV +ve recipient). Median time to engraftment (ANC> 0.5x10 9 /L) was 16 days (range 12 -29) . EBV DNA copy numbers in recipient patients' stored plasma, taken 1-19 months post transplant were determined by a real-time polymerase chain reaction (Q-PCR) assay. Lower limit of detection was 200 EBV DNA copies/mL plasma). EBV reactivation was detected in 6/18 patients (33%) with EBV copy numbers ranging from 3x10 2 to >3x10 5 /ml plasma. No reactivation has been detected before 8 months post transplant. Therefore, in patients surviving >8 months post-transplant, 6/10 (60%) had EBV reactivation. No EBV-LPDs were diagnosed. The recipient of the EBV IgM positive stem cells did not reactivate. 10 (56%) patients were treated for CMV reactivation (as determined by Q-PCR), which occurred within the first 2 months in each case. The delay in EBV reactivation compared to published studies in ATG-containing protocols may relate to the depletion of the B-cell pool. However, the risk of late reactivation is significant and thus surveillance strategies should be implemented & consideration given to preventative therapy including monoclonal antibody immunotherapy.

Invasive aspergillosis in immunocompromised patients: clinical findings and therapeutic options C. Peters, S. Matthes-Martin, A. Lawitschka, M. Minkov, M. Dworzak, T. Lion, P. Höcker, H. Gadner, St. Anna Kinderspital, Children's Cancer Research Institute (Vienna, A) Despite new antifungal drugs the development of invasive aspergillosis (IA) is a life threatening event in patients with impaired immunity. We investigated 68 cases of aspergillus infection seen in our hospital since 1995 to evaluate the disease's clinical characteristics, to ascertain the factors influencing the outcome and whether early diagnosis and multimodal therapy (chemotherapy, granulocyte transfusions, surgery) could improve the response. The patients suffered from various haematological malignancies, solid tumours or congenital haematological or metabolic disorders. Antifungal prophylaxis consisted of amphotericin B (AmB) or liposomal AmB in patients at high risk for fungal infections. Diagnostic procedures included X-ray, CT-scans, galactomannan-test, broncho-alveolar lavage and biopsy of suspicious infection sites. The most common sites of IA were the lungs, brain and skin. The multivariate analysis revealed as independent risk factors for development of IA severe long lasting neutropenia (>20 days), diagnosis of AML, chemotherapy-resistant leukaemia and immunosuppression with corticosteroids for treatment of graft vs. host disease. Preemptive antifungal therapy was initiated in all neutropenic patients with fever not responding to appropriate antibiotic therapy. In patients with proven or possible IA combination therapy (e.g. AmB and intraconazole, AmB and caspofungin, AmB and voriconazole) was given. Patients with severe neutropenia additionally received granulocyte transfusions from prednisolone-or G-CSF stimulated donors. After consolidation of the clinical situation surgical resection of the aspergillus lesions was initiated. Antifungal therapy was given at least for 6 weeks. In patients who underwent allogeneic stem cell transplantation after IA antifungals were given until immunological recovery. Two patients developed severe liver toxicity after treatment with voriconazole in combination with 6-mercaptopurin for maintenance treatment of acute leukaemia. 14 patients were non-responders to the given treatment and died due to pulmonary failure, multi organ failure or bleeding. Autopsy revealed disseminated IA in 10 cases. In conclusion, prophylactic use of antifungal drugs has not been shown to consistently reduce IA in patients at high risk for fungal infections. Thus careful clinical and radiological examinations are necessary to initiate appropriate therapeutic steps. However, disseminated IA remains a largely incurable disease.

Sirolimus -itraconazole drug interaction after allogeneic stem cell transplantation X. Schiel, J. Tischer, C. Rieger, A. Haas, M. Vogeser, U. Spoehrer, H.J. Kolb, H. Ostermann, University of Munich -Grosshadern (Munich, D) Background: Graft versus Host Disease (GvHD) and invasive fungal infections contribute significantly to morbidity and mortality after allogeneic stem-cell-transplantation. Sirolimus is a new drug for GvHD prophylaxis and therapy. Azoles like Itraconazole and Voriconazole pertain to the antifungal armamentarium. Sirolimus as well as the Azoles are metabolized via P-450 cytochrome. No data exist about clinically relevant interactions between Itraconazole and Sirolimus. Methods: We retrospectively analyzed serum concentrations of Itraconazole and Sirolimus coadministered in patients after allogeneic stem cell transplantation. Results: 14 patients (4 female) were included. Median age was 38 years (20-60). 4 HLA-identical siblings, 7 unrelated and 3 haploidentical sibling transplantations were carried out. The underlying diseases were 4 ALL, 4 CML, 2 AML and one each NHL, Severe Aplastic Anemia, Paroxysmal Nocturnal Hemoglobinuria and Multiple Myeloma. 674 treatment days were analyzed. Mean duration of combined treatment was 18 days (5-46) . Sirolimus was applied on 28 days with a cumulative dose of 3.22 (0,24-14,91 ) mg/kg body weight (BW). Itraconazole was given on 48 days (30-90) with a cumulative dose of 95 (47-193) mg/kg BW. 12 patients were treated with Sirolimus as second or thirdline GvHD therapy, 2 for GvHD prophylaxis. Sirolimus therapy was started between day +34 and +139. Itraconazole was administered in most patients (10) for fungal prophylaxis. Patients with combination therapy received significantly (p< 0.0001) less Sirolimus (0.10 mg/kg BW ±0.07) compared to patients without Itraconazole therapy (0.15 mg/kg BW ±0.11). However Sirolimus serum levels were significantly (p< 0.002) higher in patients receiving both drugs (14.5 ng/ml ±15.3 versus 8.7 ng/ml ±6.9). We could also detect a trend to higher Itraconazole levels in patients, who were treated with both drugs 682 ng/ml ±750 with Sirolimus (p = 0.07) versus 488 ng/ml ± 378 without Sirolimus. In 6 patients GvHD improved with Sirolimus therapy. In 8 patients Sirolimus treatment was discontinued because of gastrointestinal bleeding (2), infections (2) , severe mucositis (1), progress of GvHD (1) or death (2) . Conclusions: We could demonstrate a significant interaction between Sirolimus and Itraconazole resulting in increased levels of Sirolimus when coadministered with Itraconazole. Close monitoring of Sirolimus serum levels is necessary in patients receiving Itraconazole as comedication.

G. Bogdanovic, P. Priftakis, G. Giraud, M. Kuzniar, P. Kokhaei, H. Mellstedt, M. Remberger, P. Ljungman, J. Winiarski, T. Dalianis, Karolinska Institutet, Huddinge University Hospital (Stockholm, Huddinge, S) The aim of this study was to identify BMT children and grownups at risk for developing hemorrhagic cystis (HC) in order to be able to treat or prevent the disease. Background: BK virus (BKV) is excreted in the urine of most BMT patients, leading to HC in around 30% of the cases. HC is rarely lethal, but painful and the need of blood transfusions prolongs hospitalisation, hence the importance of early identification, treatment and prevention. Mutated and increased BKV excretion in the urine occur at HC, but the time point when they occur and if children and adults show a similar profile is unknown. Patients, materials and methods: During 2002-2003, 170 urine samples were collected (1-360 days) after BMT from 31 patients (18 children and 13 adults) and tested for if presence and increase in BKV excretion leads to HC. All urine samples were tested for the presence of BK virus by a nested PCR and BKV was quantified by a quantitative (Taqman) PCR in BKV positive samples and samples from HC patients Results: BKV DNA was detected in the urine samples of 16 (52%) out of 31 BMT patients by nested PCR, more specifically 10/18 (56%) children and 6/13 (43%) adults excreted BKV and BK viruria could be observed from one week before up to 8 months after BMT. However, there was no clear association between the patients' preparative regimen, or time of engraftment and frequency of BK viruria. Patients with BK viruria had a tendency to experience a somewhat more severe GVHD. HC was observed in 6 (19%) out of 31 patients, more specifically in 3/18 (17%) children had HC and 3/13 (23%) adults. In 5 of these 6 patients BKV DNA was detected by nested PCR in the urine samples before the onset of HC, and BK viruria was also confirmed during the episode of HC. For one patient there was no sample available from the period before and during HC, but BKV DNA was detected in the sample taken after HC. BKV DNA could be quantified by Taqman PCR in 69/86 (80%) of BKV positive samples by PCR and could be detected in most samples (59/69) when urine samples were diluted 1/10. The viral load was not correlated to the intensity of HC symptoms and in some cases the viral load was highest before the outbreak of HC. Conclusion: BKV can be detected by nested PCR and by a quantitative Taqman PCR in BMT patients before, during and after the outbreak of HC, but the viral load is not always correlated to the severity of the symptoms and cannot alone be used to predict the development of HC.

M. Musso, F. Porretto, A. Crescimanno, V. Polizzi, R. Scalone, Ospedale La Maddalena (Palermo, I) Cytomegalovirus infection in allogeneic stem cells transplantation recipients is a major cause of morbidity and mortality and is associated with more death than any other infections agents. It was reported that the incidence of CMV infection depends on the intensity of the conditioning regimen used. We report here on the impact of prophylactic therapy in patients underwent sibling allogeneic stem cell trasplantation with Cidofovir 3 mg/kg every 14 days. A total of 29 patients: 18 Female 11 Male, median age: 48y (18-67y) received reduced intensiy Fludarabine based conditioning consisting in association with Busulphan (3) with L-PAM (4) or Cyclophospamide (22). 17 patients suffered with haematological malignancies (AML: 4; CML: 1; HD: 3; CLL: 1; IM: 4; MM: 3; ALL:1), 12 patients had solid tumour (BC:9; Renal Ca.: 2; Melanoma:1) In 13 patients either the ANC or the PLT count never fell under 0.5x109/L or 20x109/L respectively. In the other 16 patients ANC and PLT engraftment occurred after a median of 13 days (range 8-19 days) and 14days (12-20) respectively. Acute Graft versus Host disease grade II-IV was observed in 5 patients (median onset day +34) Either all but one recipients and all donors were CMV seropositive. An initial episode of viremia occurred in 5/29 patients (17%) at a median day of 43 (26-156). aGVHD was documented in 3/5 patients. Infected patients were treated with Cidofovir 5 mg/kg every 7 days. No second viremia episode occurred. Only one patient developed disease (pneumonia) and died. Neither haematological or extrahaematological toxicity related to the schedule proposed was noted. The schedule proposed was extremely well tolerated and induced a low incidence of CMV viremia.

Candida colonisation of patients undergoing haematopoetic stem cell transplantation and receiving fluconazole prophylaxis J. Sinkó, N. Radka, P. Reményi, S. Lueff, A. Bátai, G. Kriván, M. Réti, T. Masszi, St. László Hospital (Budapest, HUN) Study objectives: 1.To evaluate patterns of candida colonization in patients undergoing hematopoetic stem cell transplantation (HSCT) who received fluconazole prophylaxis. 2.To assess the risk of invasive candidosis in colonized individuals. Methods: A retrospective analysis of records of patients undergoing HSCT between January 2000 and December 2002 at St. László Hospital, Budapest. While neutropenic, patients received 200 mg fluconazole daily (oral/infusion) for antifungal prophylaxis. As a part of the routine protocol to test colonization, fecal and urine samples as well as oral swabs were cultured twice weekly. Results: Data from a total of 188 (46 allografted and 142 autografted) patients were evaluated. Candida colonization could be detected in 139 cases (73,9%), fecal samples accounted for the majority of positive cultures (111 patients). 22% of patients were colonized by C. albicans, 58% by a nonalbicans species, whereas samples of the remaining 20% grew both. From all positive samples C. albicans could be isolated in 31%, C. glabrata and C. krusei were seen in 26% and 33%, respectively. From Day-14 to Day+14 the proportion of C. albicans colonization decreased from 68,4% to 16,6%, while colonization by C. krusei increased from 0 to 50%. Throughout the three years' observation period, only two cases of invasive candidosis occurred, both of them during the late post-transplant period with chronic graft-versus-host disease (after Day+200). Conclusion: Candida colonization is common in the studied population. With the use of prophylactic fluconazole, proportion of nonalbicans strains (especially C. krusei) tends to increase over time. In spite of widespread colonization, invasive candidosis remains an infrequent complication usually occurring late after transplantation.

J.S. Kalpoe, R.M.Y. Barge, R. Willemze, N. Vaessen, A.C.M. Kroes, Leiden University Medical Center (Leiden, NL) We recently described an in-vitro T-cell depleted alloSCT protocol following non-myeloablative conditioning with fludarabine, ATG, busulphan and Campath-in-the-bag as a suitable platform for subsequent cellular immunotherapy (Exp. Hematol. 2003; 31: 865-872) . In this study we evaluated whether the incidence and severity of CMV-viraemia was enhanced in this non-myeloablative group (n = 17) compared to patients receiving in-vitro T-cell depleted grafts using Campath incubation after myeloablative conditioning (n = 16). Patients were monitored weekly from transplantation for CMV-DNA load using real-time quantitive PCR. Data analysis included the calculation of the area under the curve over time (AUC), which is an informative marker for the level and duration of the viraemic episode. A pre-emptive treatment protocol was used in all patients. During the first 180 days after alloSCT, CMV viraemia was diagnosed in 10/17 (59%) patients in the non-myeloablative group, compared to 8/16 (50%) in the myeloablative group. One patient in the non-myeloablative group developed CMV disease. The median time to onset of CMV viraemia after alloSCT in the nonmyeloablative group and the myeloablative group were 31 days (range: 8 -54) and 23 days (range: 14 -31) respectively. The median CMV DNA peak loads were comparable in both groups: 1x10 5 copies/ml (range: 3.2x10 3 -4.0x10 5 ) and 2x10 5 copies/ml (range: 1.3x10 4 -2x10 6 ) in the nonmyeloablative and the myeloablative groups respectively. The median duration of CMV viraemia appeared to be higher in the non-myeloablative group (41 days; range: 11 -112) than in the myeloablative group (35 days, range 15 -56). Analyses of the AUC of both groups showed a higher AUC-value in the non-myeloablative (70; range: 14 -200) versus myeloablative group (56; range: 4 -120). The differences between the two groups in this study were not statistically significant, which is most likely secondary to the small sample size. Furthermore, successful pre-emptive treatment strategy might have masked lager differences between the two groups. Therefore we are currently analyzing the number of weeks of anti-CMV treatment in both groups. We conclude that the overall incidence of CMV viraemia is comparable in both alloSCT groups. However, AUC analyses revealed that the severity of the CMV viraemia is moderately increased in the non-myeloablative group due to a prolonged duration of the viraemia episodes in this group.

Secretory IgA of the oral saliva normalises earlier than IgA in serum after haematopoietic stem cell transplantation in children R. Haefer, M. Steinbrenner, B. Gruhn, A. Mueller, D. Fuchs, F. Zintl, University of Jena (Jena, D) Hematopoietic stem cell transplantation (HSCT) has become a well recognized treatment modality in patients with leukemia, lymphoma, solid tumors, and autoimmune diseases. After transplantation there is a time period with a strong immunosuppression. The oral cavity appears to be a special locus for opportunistic infections and the local immune defense may be contribute to patients' outcome. Because of the different reference ranges reported in the literature about secretory IgA of the oral saliva (sIgA) we have first performed a study for determination of normal sIgA values in 77 healthy volunteers. In children at the age of 7-14 years the range of sIgA was 143 mg/L -6204 mg/L and in those at the age of 14-25 years the range was 243 mg/L -8160 mg/L (percentile of 2.5 and 97.5, respectively). Saliva was collected by means of Salivette (Sarstedt, Germany). Serum IgA and sIgA were determined by radial immunodiffusion (The Binding Site, UK). 35 consecutive patients with ALL (n=8), AML (n=5), CML (n=4), MDS (n=3), NHL (n=3), and solid tumors (n=12) were monitored monthly until 18 months after transplantation and 24 months after transplantation in a single center. 15 patients received autologous HSCT and 20 patients underwent allogeneic HSCT. 18 patients received bone marrow transplantation and 17 peripheral blood stem cell transplantation. The levels of serum IgA and sIgA concentrations decreased continuously after transplantation to a nadir (mean: 290 mg/L and 110 mg/L, respectively) and increased afterwards. But the nadir of sIgA level was reached already at 3 months after transplantation whereas the nadir of serum IgA levels was observed at 6 months after transplantation. The sIgA levels normalized already at 12 months after transplantation whereas the normalization of the serum IgA levels was observed at 24 months after transplantation. Therefore, we conclude that the recovery of sIgA is faster than that of serum IgA and that sIgA recovery may be independent of the immune reconstitution in the peripheral blood.

A retrospective study examining the United Kingdom Cancer Care Study Group's fungal management strategy in practice R. Nolan, J. McDerra, S. Peters, J. Cornish, Bristol Royal Hospital for Children (Bristol, UK) Paediatric SCT has seen an increase in the number of high-risk patients (pts) as a result of more intensive leukaemia protocols, transplantation of pts in higher remission states (e.g. CR3), higher intensity preparative regimens and the use of increasingly mismatched donors (e.g. haploidentical). The profound neutropenia and immunosuppression seen in these pts result in as many as 40% of pts developing an invasive fungal infection (IFI), a major cause of morbidity and mortality. The UKCCSG fungal management strategy was written in an attempt to improve fungal therapy. This study ia an evaluation of this approach. Strategy: Pts are stratified to a risk group and receive either prophylaxis or pre-emptive therapy. Empiric therapy is commenced at 96 hours of unresponsive PUO. The therapy to be given in each category, including treatment of IFI, is specified by the strategy. Patients and methods: 79 paediatric pts (aged 10mths to 19 yrs) undergoing SCT at Bristol BMT unit over a 2.5 yr period. Data were collected retrospectively. All details of antifungal (AF) therapy, along with any additional treatments of IFI, namely use of GM-CSF or granulocyte transfusions, were noted. Other complications, e.g. GvHD, CMV and ADV were recorded. Results: 48 (62%) pts were treated in accordance with the strategy. However 49 (62%) had been assigned an incorrect risk status. Prophylaxis: 77 (97%) pts received itraconazole liquid (itra) as prophylaxis. Of these, 29 pts could not take the itra liquid, with another 10 stopping itra during mucositis. This led to 39 (49%) receiving AmBisome (AmB) for prophylaxis solely due to problems with itra administration. Itra was also stopped in 10 (13%) pts due to elevated LFT's. Empiric Therapy: 23 (29%) pts received AmB 1mg/kg and 3 (3.8%) received AmB 3mg/kg. AmB therapy was altered in 3 (4.9%) pts due to renal impairment. Treatment: 17 (21%) pts were treated for suspected or proven FI, of these, 16 (94%) received AmB 3mg/kg, 3 (18%) had this dose increased to 5mg/kg. After failing sole treatment with AmB, 3(18%) received caspofungin and 4(23%) received voriconazole. Six pts (35%) had combination AF therapy. Outcome: 3 (3.9%) died of probable IFI, 1 (1%) died of proven IFI. At Day +100 14 pts (18%) had died: relapse 4 (9%), FI 4(5%), ADV 3(4%) Conclusions: Itra intolerance was surprisingly high; could NG administration alter this? A positive finding was that AmBisome had much lower rates of toxicity than previously reported.

S. Cantero, G. Sanz, F. Moscardó, J. Sanz, M. Remigia, I. Lorenzo, G. Martín, I. Jarque, C. Jiménez, J. Martínez, J. De la Rubia, M. Sanz, Hospital Universitario La Fe (Valencia, E) Objectives: To analyse the influence of CD34 positive selection in the incidence of CMV infection after allogeneic peripheral blood stem cell transplantation (allo-PBSCT) Patients and methods: Seventy adult patients (38 males and 32 females) undergoing a first allo-PBSCT between January 1996 and April 2002 were analysed. Median age was 34 years (range: 16-60). Fifty patients (72%) had acute leukemia. Most patients (83%) were conditioned with busulfan and cyclophosphamidecontaining regimens. Fifty-one patients (73%) received a manipulated graft with CD34 positive selection performed with an immunomagnetic technique (CliniMACS) and a fixed CD3 cell dose of 0.3x106/Kg. All CMV-seropositive recipients and those with CMV-seropositive donors received prophylaxis with acyclovir followed by ganciclovir until day +100. CMV infection was defined as the presence any degree of pp65 antigenaemia. Acute graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine and prednisone in 90% of the patients. Results: Twenty-four patients (34%) developed CMV infection at a median time of 39 days (range: 11-176) . Actuarial probability of CMV infection at one year was 41%, and was greater in CMVseropositive recipients (53% vs. 0%; p=0.006) and in patients with acute GVHD equal or greater grade 2 (61% vs. 35%). No difference was found between patients receiving CD34+ selected grafts ( 42%) or unmanipulated grafts ( 45%). Five patients (7%) developed CMV disease, 3 (6%) with CD34+ selected grafts and 2 (11%) with unmanipulated grafts. Conclusions: CD34 positive selection did not influence the risk of CMV infection after allo-PBSCT. Serologic status of the recipient and the development of acute GVHD were the most important risk factors for CMV infection.

Infections after reduced-intensity conditioning for allogeneic blood stem cell transplantation in metastatic solid tumours L. Barkholt, M. Blomkvist, P. Hentschke, L. Klingspor, I. Lewensohn-Fuchs, J. Mattsson, R. Milosavljevic, P. Pisa, M. Remberger, P. Svenberg, A. Thörne, P. Wersäll, O. Ringdén, Karolinska Institutet (Stockholm, S) Objective: What is the incidence of infections in patients with metastatic solid tumors during the first year after reduced intensity conditioning (RIC) and allogeneic stem cell transplantation (SCT)? Methods and Patients: In total 20 patients, 8 with metastatic colorectal, 12 with renal carcinoma, and metastases in all, underwent allogeneic SCT. RIC included Flu 30 mg/m 2 for 3 days, using sibling donors and 5 days using unrelated donors followed by 2 Gy TBI or Cy (60mg/kg for 2 days). Antithymoglobuline 4 mg/kg was administrated to patients with unrelated donors (n=6). Peripheral blood stem cells were collected after G-CSF stimulation of all donors. The CD34+ cell dose was median 7.5x10 6 /kg (1.7-21.8) . Immunosuppression was by CyA, combined with MMF (n=16) or MTX. Results: Bacterial septicemia was found in 6 patients within 6 months after SCT, associated with central venous line (Staph. epidermidis 4 cases), urosepticemia (Enterococcus faecalis 1 case) or acute GVHD of the bowel (Pseudomonas aeruginosa, E.coli 1 case each). Fungal infection was diagnosed by PCR in 11 patients (Candida albicans in 8 or Aspergillus fumigatus in 9 patients), but only one patient suffered from fungal septicemia. CMV infection and syndrome developed in 13 and 3 patients, respectively. In 6 patients, CMV reactivated after the initial preemptive antiviral therapy. No organ invasive fungal or CMV disease developed. No infections were seen after 6 months. The cumulative incidence of grades II-IV acute GVHD was 57%. CMV disease was associated with aGVHD (p=.05). Thirteen patients died =<1year: 4 transplant-related complications, 1 head trauma and 8 progressive disease. Three patients (1 CC, 2RCC) are alive 30-38 months. Conclusion: Following RIC and allogeneic SCT, acute GVHD developed in 57% of the patients, and was associated with risk for CMV disease. Bacterial septicemia, CMV disease and invasive fungal infection were uncommon. However, positive PCR for CMV and fungi occurred in 65% and 55% of all patients, respectively.

Autologous stem cell transplantation in HIV-infected patients affected by lymphoma: virological and immunological parameters during high-dose chemotherapy M. Michieli, C. Simonelli, M. Rupolo, M. T. Bartolin, M. Mazzucato, S. Zanussi, M. Spina, L. Abbruzzese, P. De Paoli, L. De Marco, V. Gattei, M. Berretta, U. Tirelli, National Cancer Institute (Avione, I) Given the refractory nature of HIV-Lymphomas, novel therapeutic approaches are needed. In our Institution refractory and/or relapsed non-Hodgkin Lymphomas (NHL) and Hodgkin's Lymphoma (HD)in HIV-pts are treated with High Dose Chemotherapy (HDCT) followed by Autologous Stem Cells Transplantation (ASCT). So far we enrolled 6 patients (pts). 3 pts had a relapsed NHL, 1 refractory NHL and 2 had relapsed HD. 5 pts were chemosensitive to the debulking therapy, and underwent to whole ASCT program. We report the virological and immunological parameters of 4 pts who completed the therapeutic program. At the time of the second line CT, the median CD4+ count was 198/uL (range: 172-460) . Pts were all on HAART and HIV-viraemia was <50 cp/ml in all. CD4+ count reached the nadir (median value 99/uL; range 8-268) soon after aplastic period (+14 days from ASCT). The median value of CD4+ remained low (103/uL; range 93-275) 3 months later. 3 pts continued HAART during the whole conditioning treatment (BEAM) and their HIV-viraemia was <50 cp/uL. 1 pt, just before HDCT, discontinued HAART, because G3 liver toxicity; HIV viraemia was >500000 cp/ml (after 14 days from ASCT), then restarted the same HAART and HIV viraemia decreased to undetectable level within 3 months. Serology for EBV was positive in all pts, but EBV-DNA was detectable in 2 pts; an increase of EBV-DNA was observed only in 1 pt. Two pts has a positive serology for HCV. HCV-RNA was detectable only in 1 pt, but the HCV-RNA cp number remained stable during the whole treatment. Serology for HHV-8 was performed in all 4 pts and HHV8 viraemia will be performed in the positive ones. HIV, HBV, HCV and HHV8 dynamics during treatment will be discussed at the meeting.

P. Topcuoglu, M. Arat, E. Soydan, K. Dalva, A. Ciftcioglu, H. Akan, O. Ilhan, M. Beksac, Ankara University Faculty of Medicine (Ankara, TR) CMV infection(CMVi) is a major cause of mortality but the role of CMV viremia(CMVv) has not been analyzed extensively. Previously, we reported a negative effect of CMVv on overall survival (OS) in 62 patients (EBMT,2002, 96% vs 78%, p=0.03 ).

Here we report the results of a prospective study. Patients and Methods: Between March 2000 and January 2003, 101 consecutive patients [M/F: 55/46; 31 (16¨C58) year] with CML/AML/ALL/other (n=41/30/14/16), received allogeneic stem cell transplantation (AHSCT) from bone marrow (BM, n=33) or peripheral blood (PB, n=68) of an HLA identical sibling donor or three patients (one paternal, one maternal and one matched unrelated donor), following an ablative (MA, n=76) or a reduced intensity conditioning regimen (RIC, n=25). All pairs were CMV seropositive and received leukofiltered/irradiated blood products. CMV-DNA was monitored twice weekly by Hybrid Capture assay (Digene,USA). Absolute CD3, CD4, CD8 counts were performed by FACS [n=86(24.4% CMVi(+) , 59.4%CMVv(+)]. Results: The incidence of CMVv(>= 3pg/ml) was 72.2% and occurred at a median of 37 days (0-788 d). 83% these were earlier than day 100. CMVi suggesting findings were present in 33% of the CMVv(+). Borderline viral load (3-14pg/ml) was detected in 32 patients who also did not present clinical findings. >= 15pg/ml was observed in 41 patients (15-203pg/ml). The incidence or the time of CMVv was not significantly different between PBSCT and BMT [PB: 67.6%, , BM: 81.9%, p=0.136 and p=0.847 ]. In the PB group, conditioning regimen, MA versus RIC did not cause a difference (64.3%, 31days, 0-533d vs 76%, 44days, 0-408d, p=0.317 and p=0.475) . Ganciclovir was given either as therapeutic dose (n=24) or as preemptive therapy (n=6) (>= 15pg/ml). The therapeutic dose was required more often following BMT compared to PBSCT (p=0.039). Pretransplant lymphopenia (<1.5x10 9 /L) was observed in 51%. Neither the absolute nor ratio value of CD4, CD8 had an effect on the incidence or time of CMVi or CMVv. OS (3y) was 31% and 58.4% among patients with or without CMVv (p=0.16) or 23.4% and 72.1% in patients with or without CMVi (p=0.037). Both acute and chronic graft versus host disease (GVHD) was associated with more frequent CMVv at any time (79% to 91%). In conclusion, both CMVi and with a less significance CMVv shortened OS. Pretransplant CD4/CD8 counts were not influential. Although not statistically significant, PBSCT was associated with less frequent CMVv.

A. Georgala, K. Mboumi, P. Martiat, J. Debruyne, V. Duchateau, M. Aoun, Institut Jules Bordet (Brussels, B) Background: CNS toxoplasmosis is a rare but often life threatening infection occurring in BMT-recipients. It usually occurs after reactivation of a latent infection . Clinical signs and symptoms are unspecific and the diagnosis might be extremely difficult. Thus, the correct clinical, radiological and microbiological assessment of BMT patients may be the key in the successful diagnosis and treatment of CNS toxoplasmosis in these patients. Methods: We examined, retrospectively, all the cases of CNS toxoplasmosis that occurred in our hematologic unit during a 5year period (1998) (1999) (2000) (2001) (2002) (2003) . The incidence of CNS toxoplasmosis is 2,18% among our BMT patients. Patients characteristics, clinical features, radiological and microbiological documentation and outcome were assessed in order to define the criteria that could help the clinician to diagnose and treat efficiently the CNS toxoplasmosis. Results: Seven cases of CNS toxoplasmosis occurred in BMT recipients. There were 2(28.6%) female patients for 5(71.4%) male patients. Most of them had acute leukaemia (4/7-57.14%). Six (85.71%) received an allograft, either bone marrow or peripheral blood stem cells (PBSC) and only one had an autograft (PBSC). All the allograft recipients received immunosuppressive therapy in the 30 previous days. All patients presented with fever. Other clinical signs include headache, confusion, tremor and encephalopathy. Cerebral magnetic resonnance imaging (MRI) was relevant in all of the cases. Microbiological documentation including blood-polymerase chain reaction (PCR), blood cultures, cerebrospinal fluid (CSF)-PCR, CSF cultures and serology was positive in 2 patients. All 6 patients who received pyrimethamine associated with IV clindamycin, responded to therapy. Only one patient died of autopsy-proven CNS toxoplasmosis, because of non compliance with oral therapy (pyrimethamine + sulfadiazine). Conclusions: CNS toxoplasmosis is an opportunistic infection occurring in highly immunosuppressed patients such as BMT recipients. Rapid diagnosis using clinical, radiological and microbiological documentation and therapy with pyrimethamine associated with clindamycine resulted in a better survival than reported in the literature.

Prospective surveillance of nosocomial infection among allogeneic haematological stem cell transplant recipients stratified by grade of neutropenia at a teaching hospital R. Fagnani, L.G.O. Cardoso, S.R.P.E. Dantas, M.L. Leichsenring, M.C.S. Carvalho, P. Trabasso, Univ. Est. de Campinas/TMO/UNICAMP (Campinas, BR) Introduction: Allogeneic Haematological Stem Cell Transplant (allo-HSCT) recipients are at high risk for acquiring nosocomial infections (NI), and neutropenia is the most frequent risk factor (RF). The most common method used to assess the risk of acquiring infection is dividing the number of infection by the total amount of days in which the RF is present. Although accepted for non-neutropenic, this method was not yet validated for neutropenic patients. Objective: To assess the density incidence of NI stratified by grade of neutropenia among allo-HSCT recipients at a Brazilian Teaching Hospital. Methods: All allo-HSCT recipients cared for at the Hospital of Universidade Estadual de Campinas from Jun/02 to Oct/03 were followed from admission to discharge. Demographic data and information of grade and duration of neutropenia and utilisation of central venous catheter (CVC) were collected. CVC were stratified in long-term semi-implantable (LTSI), long-term totally implantable (LTTI), and short-term (ST). Epidemiological data were total number of patients, admissions and discharges, and total number of patients in each level of neutropenia. Patientsday, neutropenia-day and device-day rates were calculated and the incidence of infection were reported as number of infection/1000 patients-day, number of infection/1000 neutropenia-day and number of device-related infection/1000 device-day. Results: A total of 89 patients were followed, meaning 1935 patients-day. Fifty-six (63%) patients used LTSI-CVC, 17 (19%) ST-CVC, 9 (10%) LTTI and 7 (8%) other types of CVC. A total of 65 infections were diagnosed, being Blood Stream (BSI) N=33 (50.8%), Fever of Unknown Origin (FUO) N=15 (23.1%) and Pneumonia (PN) N=7 (10.8%) the most frequent. The number of infection and the infection rates/1000 patients-day stratified by level of neutropenia were as shown in Table 1 .

Conclusion: Assessment of NI using incidence of infection/1000 neutropenia-day showed higher rates rather than usual methodology, strengthening the necessity of the validation of specific rates for severely immunosuppressed individuals.

L. Clement, A. Salmon, L. Mansuy, S. Paczesny, P. Bordigoni, Hopital d'enfants CHU Nancy (Vandoeuvre les Nancy, F) Invasive fungal infections after HSC transplantation are one of the main causes of morbidity and mortality. Fluconazole prophylaxis(Px) is effective for prevention of major invasive yeast infections. Itraconazole and voriconazole are antifungal agents with activity against yeasts and molds, including Candida and Aspergillus. Their use as antifungal Px agents is interesting after HSC transplantation, but perhaps encouraged emergence of other fungal pathogens as zygomycosis (zm). Zm was never diagnosed in our unit transplant until the next 6 months, when 4 cases were identified. We reviewed them in details. Results: patient's median age was 26 yrs. Primary disease diagnosis was haematological malignancies for all. Pts 2 and 4 received HLA-identical sibling HSC transplant, pt 3 was transplanted from unrelated donor and pt 1 underwent a mismatched sibling transplantation. All pts were treated for chronic graft versus host disease. Diagnosis was made within a median of 6 months after transplant. Pt 1 received oral voriconazole as antifungal Px. Itraconazole was prescribed to pt 2. Pt 3 received itraconazole before hospitalisation and then voriconazole intravenous (iv). Pt 4 received iv voriconazole as antifungal Px and caspofungin in addition when lung infiltrates appeared. Voriconazole plus caspofungin were given as empiric therapy for all pts. Pt 1 developed 2 hepatic lesions, 1 lesion in each kidney and then cerebral and pulmonary lesions. Biopsy of hepatic lesion demonstrated the presence of many hyphae. Stool, nasal and buccal cultures yielded Absidia sp. Pt 2 presented sinusal pain with facial swelling, necrotic nasal mucosal and ankles pain (infarction of femoral heads). Rhizopus sp was isolated from sinusal puncture. The 2 others pts were hospitalized in intensive care unit for respectively pulmonary aspergillosis (pt3) and adenoviruses infection (pt4). Rhizopus was identified histologically for pt 3. Mucor was isolated from a BAL specimen made because of an increasing progressive pulmonary failure in pt 4. Three of 4 pts died. Pt 2 is alive, with 9 months of therapy with caspofungin and liposomal amphotericin B. Conclusion: our analysis of these 4 cases suggests an increased incidence of zm as the result of our change in antifungal prophylaxis or empiric therapy. Questions are: first, is emergence of zm due to the use of voriconazole for Px? Second, could we prescribe voriconazole in first intention as empiric therapy in the context of HSC transplantation?

A.M.T. van der Velden, A.M.E. Claessen, H. van Velzen-Blad, D.H. Biesma, G.T. Rijkers, Sint Antonius Hospital, University Children's Hospital/UMCU (Nieuwegein, Utrecht, NL) Objectives: After autologous stem cell transplantation (ASCT), patients are prone to infections with encapsulated organisms such as Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae. Although vaccination against these pathogens is recommended, no standard protocols exist. We are conducting a prospective follow-up study to determine the immune response to vaccination with Hib and pneumococcal (conjugate) vaccines and to establish a vaccination schedule for patients who underwent ASCT. Methods: A total of 25 patients will be included in the protocol. Currently, 22 patients have entered the study of which 6 have completed the full vaccination schedule. Of these first 6 patients, 4 had multiple myeloma and 2 non-Hodgkin's lymphoma as underlying disease for ASCT. At 6, 8 and 14 months after transplantation, patients were vaccinated with a tetanus toxoid conjugated Haemophilus influenzae type b vaccine (PRP-T). At 6 and 8 months after transplantation, a heptavalent pneumococcal polysaccharide-protein conjugate vaccine was given. At 14 months after transplantation, patients were vaccinated with a 23valent polysaccharide vaccine. Antibody responses were measured by ELISA in serum samples obtained before and 21 days after each vaccination. Results: For Hib, adequate IgG antibody responses (> 50 U/ml) were found in one, four and five patients after one, two and three vaccinations, respectively. Geometric mean titre after 2 doses PRP-T was 31 U IgG/ml, after 3 doses 137 U IgG/ml. The response to 2 doses pneumococcal conjugate vaccine was poor as judged by the increase in IgG antibody titers against vaccine serotypes 6B, 9V and 19F. Furthermore, pneumococcal conjugate vaccination did not prime for a booster response to polysaccharide vaccination at 14 months. No major adverse effects were noted. Conclusions: vaccination with a conjugated Haemophilus influenzae type b vaccine at 6, 8 and 14 months after ASCT in patients with multiple myeloma and non-Hodgkin's lymphoma seems safe and effective. Vaccination with pneumococcal vaccine seems less effective at these timepoints. These preliminary conclusions will be substantiated with an increased number of patients analysed.

A.M. Raiola, C. Viscoli, V. Galbusera, A. Dominietto, F. Gualandi, C. Di Grazia, S. Bregante, D. Occhini, T. Lamparelli, F. Frassoni, M. Machetti, M.T. van Lint, A. Bacigalupo, Ospedale San Martino, IST (Genoa, I) Background: The risk of reactivation of fungal infection in patients with haematological malignancies is very high during neutropenia post chemotherapy o post stem cells transplantation (SCT). Voriconazole is a broad -spectrum triazole that is active against Aspergillus species. Patients: We report eight cases of patients who developed invasive Aspergillosis (IA) during induction and consolidation therapy and then were undergo stem cells transplantation (SCT) with secondary voriconazole prophylaxis. The diagnosis of IA was documented as proven (2 patient), probable (2 pts) or possible (4 pts, 3 of them showed at CT scan typical cavitating lesions but there were not microbiological evidence of moulds) according to the 2002 European Organisation for Research and treatment of cancer (EORTC) international consensus. The diagnosis were 5 AML (1 relapse ), 2 ALL (resistant), 1 SAA. The donor was matched unrelated for 3 patients and HLA identical sibling for 5 patients. The median age at SCT was 41 years (range 21 -51). Conditioning regimen and GVHD prophylaxis was standard for all patients. At SCT all patient showed radiological evidence of IA, but the clinical signs and symptoms were absent. Voriconazole was started on day -7 i.v.: 6mg/Kg of body weight twice a day the first day, 4mg/kg twice a day for two weeks and then orally: 400mg twicwe a day. The median time of voriconazole prophylaxis was 80 days (range 48 -230). The patients were monitored twice weekly with Aspergillus antigenemia (serum Elisa test for galactomannan), clinically, radiologically and microbiologically. The median follow up is 5 months (range 2 -10). Results: The median time for neutrophils engraftment was + 22 days from BMT (range 17 -41). With voriconazole prophylaxis patients did not developed any major infectious complications, aspergillus antigenemia remained negative and the CT scan performed at discharged from the Transplant Unit did not show new lesions. Two patients had to discontinue Voriconazole (after two months of therapy) for suspected side effects. Conclusions: 1-This experience suggests that antifungal secondary prophylaxis with voriconazole is safe and can be administrated throughout the peri-transplant period. 2-None of the seven patients developed clinical signs of IA. 3-A previous history of IA is not a contraindication to further stem cells transplantation. 3-It remains to be determined how long must be prophylaxis secondary post SCT.

Invasive aspergillosis and haematopoietic stem cell transplantation in paediatric patients N. Crassard, M.-A. Piens, C. Galambrun, T. Basset, J.-C. Berthier, C. Pondarré, N. Philippe, G. Souillet, Y. Bertrand, Hôpital Debrousse (Lyon, F) Aim: to investigate incidence and outcome of Invasive Aspergillosis (IA) in HSCT pediatrics receivers. Patients and methods: 379 allograft were rewieved from 1986 to 2000. EORTC criteria of IA were used to define proven and probable infections. All patients received immunosuppressive conditioning regimen and were nursed in LAF room with HEPA filters.

Results. -Ten patients presented proven (2) or probable (8) IA after HSCT. Median age was 9.5 years (8 months-17 years). Incidence of IA was 2.67%. Five patients received allogeneic marrow graft from unrelated donors, 3 patients from siblings, 1 patient received peripheral blood stem cell from his father and 1 patient received cord blood from an HLA identical sibling. Prevention of GVHD was CSA for 10 patients plus MTX for 7. Underlying disease was familial lymphohistiocytosis (1), Hurler disease (1), Fanconi anemia (2), JMML (1), AML (1), ALL (2) and SCID (2) . All but one had pulmonary involvement: isolated for 6 patients, associated with pericardium involvement for 1, brain (1), peritonitis during peritoneal dialysis and meningitis (1). One patient had an isolated middle ear involvement. Median onset was 64 days : for 7 patients, complications occurred (GVHD, VOD, infections) and the median onset of IA was 177 days. For the 3 non complicated HSCT, it was 15 days. Nine patients died, including 8 from or with IA. Median survival was 45 days.

-Five patients underwent BMT (4 from unrelated donors, 1 from HLA identical siblings) after proven IA. Underlying disease was ALL (2), AML (2) and idiopathic aplastic anemia (1). Median age was 7 years. Four patients presented with pulmonary involvement and one with mastoiditis and meningeal involvement. The median time from IA to BMT was 110 days. All patients underwent antifungal therapy and surgery. Secondary antifungal prophylaxis consisted of Amphotericin B (1), Amphotericin B plus itraconazole (1), itraconazole (3) . IA relapsed in 2 patients: 1 died from IA, 1 survived. Three patients died: 2 from other co morbidities and 1 from IA. Two patients are alive and well with a follow-up of 36 and 54 months.

S. Fürst, Q.H. Le, A. Michallet, F. Nicolini, X. Thomas, Y. Chelghoum, J. Troncy, A. Thiébaut, F. Persat, S. Picot, M. Piens, M. Michallet, Hopital E. Herriot, Faculté de Médecine, Hôpital E. Herriot (Lyon, F) The majority of opportunistic fungal pathogens are represented by Aspergillus and Candida species in hematological malignancies. Amphotericin B (Ampho-B) has been the goldstandard treatment during decades. In a prospective randomized trial, Voriconazole compared to Ampho-B, as initial therapy in patients with SFI, has shown an improved outcome and a better survival. Nevertheless, SFI remained a pejorative prognostic factor for survival in patients with hematological malignancies. This monocentric non randomized retrospective study concerned 95 patients [37 males, 48 females with a median age of 52 y (21-82)] harbouring an hematological malignancy, who underwent either intensive chemotherapy [first induction (1I) (n=51), induction for relapse (2I) (n=15), consolidation (n=12)] or allogeneic (n=14) or autologous transplantations (n=3). We stratified patients in 2 groups: group 1 received Ampho-B as first line therapy (n=40) before Voriconazole was commercialized in France and group 2 received Voriconazole either in first line (group 2-1: n=20) or in second or more line therapy (group 2-2: n=35) for SFI [proven, probable, possible (EORTC criterias)] or >or=2 increasing titers of aspergillus antigenemias. We performed a descriptive (Table  1 ) and a multivariate analysis ( Table 2 ) using a PH cox model regression on age, sex, diagnosis, therapeutical strategy, EORTC criterias and SFI treatment. In group 1, 16 out of 40 patients died (40%) [6 from SFI (37.5%) and 10 from disease progression (DP)], in group 2-1, 6 out of 20 died (30%) [3 from SFI (50%) and 3 from DP] and in group 2-2, 18 out of 35 died (51.5%) [7 from SFI (39%) and 11 from DP]. The 6 month probability of survival was 65% (95%CI 48.5-81.4) for group 1, for group 2-1 and 15.6% (95% CI:0-40) for group 2-2. The results of multivariate analysis showed the significant impact on survival of age, SFI diagnosis and SFI treatment.This retrospective study did not show any survival benefit of Voriconazole compared to Ampho-B as first line therapy for patients with hematological malignancies presenting SFI and receiving intensive chemotherapy, followed or not by autologous or allogeneic transplantations. Nevertheless, patients receiving Voriconazole presented more proven and probable SFI than patients treated by Ampho-B (75% vs 25%) but these 2 types of SFI have no significant impact in the multivariate analysis.

Single-certre experience S. Guidi, C. Nozzoli, M. Bonolis, A. Orsi, L. Lombardini, R. Saccardi, A. Vannucchi, A. Bosi, Ospedale di Careggi (Florence, I) Epstein Barr virus (EBV) is important cause of morbidity and mortality after allogeneic hematopoietic stem cells transplantation (HSCT). EBV reactivation may evolve in lymphoproliferative disease (PLTD). Pre-emptive EBV treatment seems to reduce the PLTD incidence in HSCT recipients. A highly sensitive EBV screening has been available since one year by quantitative polymerase chain reaction (Q-PCR). We evaluated the incidence, risk factors, and treatment in 27 consecutive recipients of HSCT observed by us during last year. One T depleted autologous and 26 consecutive allogeneic HSCT, 11 id. sibling recipients and 15 alternative donors (13 matched unrelated and two aploidentical) transplant were studied. Diagnosis were: AML 10, ALL 7, NHL 4, CLL 1, SAA 1, LH 1, MMM 1, multiple myeloma 1, 15 male/11 female, 18 pts conventional conditioning and 8 reduced intensity. 14 pt received ATG as part of conditioning regimen and 13 no. EBV DNA Q-PCR was done weekly. All patients received prophylaxis with gancyclovir pre transplant and then foscarnet untill take. Only patients with Q-PCR EBV plasma copies > 2.000 or with clinical symptoms were treated with cidofovir and rituximab. With a mean follow up of 84 days (range 20-420), 22/27 patients were found EBV DNA positive: 12 qualitative PCR only and they were not treated, 10 patients resulted Q-PCR positive (range 1680-800.000 blood viral copies). 5/10 patients experienced fever and were treated, one of them developed a PLTD with CNS involvment. Those pt were treated with cidofovir 5mg/kg i.v., and Rituximab 375 mg/mq weekly for 4 doses or till complete response achieved . One pt died because EBV and GvHD the other pts recovered. The CNS-PLTD pt added intrathecal MTX plus methilprednisolon and stopped cyclosporine . Q-PCR EBV DNA reactivation was more frequent in ATG pts (12/14) instead of no ATG cohort in which 1/13 was positive. All but one patients who received EBV treatment had experienced GvHD requiring at least 2mg/kg methilprednisolon. EBV reactivation is more frequent than previously known especially in patients undergoing highly immunosuppressive transplant as MUD or mismatched ones or in autologous T deplete transplant. In our experience ATG and steroid treatment as part of prophylaxis or therapy of GvHD, represent the most important risk factor for EBV reactivation and we strongly suggest a weekly determination of EBV DNA by Q-PCR. Harsdorf, M. Bommer, T. Zenz, M. Ringhoffer, R. Schlenk, T. Schmid, W. Hampl, S. Stilgenbauer, H. Döhner, T. Mertens, D. Bunjes, Medizinische Klinik und Poliklinik, Ulm University (Ulm, D) CMV -disease remains a major complication of allogeneic stem cell transplantation, especially if T cell depletion is used as GvHD -prophylaxis. We have compared 4 CMV -prophylaxis regimens for their effect on CMV reactivation and disease in 132 seropositve patients (pts) who received a myeloablative allogeneic stem cell transplant between 1996 and 2002. The median age of the pts was 43y (range 17 -63y). The diagnoses were CML n=43, AML n=39, ALL n=13, multiple myeloma n=9. The stem cell source was bone marrow in 19 pts and peripheral blood stem cells in 113 pts. The donors were HLA -id. sibs in 65 pts, compatible unrelated donors in 49 pts and mismatched family donors in 17 pts. GvHD -prophylaxis consisted of CD34+ selection in 68, Campath (CP) in the bag in 27 and CP i.v. in 9 pts. The CMV prophylaxis regimens evaluated were: i.v. CMVhyperimmuneglobulin (IVIG) alone 20g every 14d till d+120 in 40 pts, Gancyclovir (GCV) 5mg / kg / d + IVIG in 19 pts, Foscarnet (PFA) 2 x 60mg/kg /d till d+14 in 19 pts and Cidofovir (CDV) 5mg / kg loading dose followed by 3mg/kg weekly till d+14 in 54 pts. CMV -antigenaemia was monitored.weekly. CMVantigenaemia was documented in 99 pts (75%), CMV -disease was observed in 11 pts (11%). 10 pts developed CMV -IP and 7 died. The incidence of grade II -IV acute GvHD was 21%, chronic GvHD developed in 33% of pts. The reactivation rate in the IVIG alone group was 77%, in the PFA + IVIG the incidence was 58%, in the GCV group 79% and in the CDV group 78%. Therefore CMV -prophylaxis with antiviral drugs was of no benefit in this group of pts.

Tuberculosis post stem cell transplantation C. Burton, E. Thomson, E. Kanfer, T. Rogers, J. Goldman, J. Apperley, Hammersmith Hospital (London, UK) Mycobacterial infection is an infrequent complication after stem cell transplantation. We reviewed the data for 354 allogeneic and autologous stem cell transplants performed during the last 3 years at the Hammersmith Hospital. Mycobacterial species were isolated from three patients: mycobacterium tuberculosis (TB) was isolated from two patients by bronchoalveolar lavage; from a third patient Mycobacterium avium intracellulare and Mycobacterium chelonae were both isolated on blood cultures. Thus one can extrapolate to an incidence of mycobacterial infection of 850/100,000 (0.85%) in this patient population. The background incidence of TB in London is 40/100,000 (0.04%); the incidence of TB in patients in the post stem cell transplant setting is therefore about twenty times the background incidence. The incidence in this patient population is therefore comparable to the incidence of mycobacterial infection reported in other immunosuppressed patient groups; for example, renal dialysis patients have an incidence of 1187/100,000 (1.19%) . In three Asian countries where tuberculosis is to a greater or lesser extent endemic, the incidence of tuberculosis after stem cell transplantation has been reported as 0.62-1.38%; these figures are similar to our own. We conclude that tuberculosis post stem cell transplantation is a more common complication than previously acknowledged and that a relatively high index of suspicion is warranted. The use of antituberculous treatment should be considered promptly in the context of pyrexia of unknown origin post transplant when resolution with conventional antimicrobials is not achieved. The need for prophylaxis for mycobacterial infection should also be considered, especially in 'at risk' patient subgroups. Background: Cytomegalovirus (CMV), Epstein-Barr virus (EBV) and Human Herpes virus 6 (HHV-6) infections are significant causes of disease and mortality among immunocompromised patients e.g. AIDS patients, cancer patients and transplant recipients. Anti-viral regimens are available and needs to be started at an early timepoint in order to achieve the best effect. Continuos monitoring of these viruses in patients at risk of developing disease, using sensitive diagnostic tests, is therefore important. Today, quantitative PCR is used in several laboratories for the monitoring of viral loads. However, truly standardised procedures are not available for all three viruses. The affigene(R) OPI (Opportunistic Infections) panel for Patient Disease Management (PDM) is comprised of three assays for efficient monitoring of CMV, EBV and HHV-6 viral load in serum/plasma specimens. Methods: The assays were performed according to method manuals and evaluated utilizing QCMD reference panels, clinical specimens and artificial specimens comprised of serum/plasma spiked with each specific virus. The three assays share a common sample preparation, which enables the user to analyse all three viruses from one single specimen preparation. Results: All three assays can reliably measure decrease and increase in viral loads over time in clinical samples and good correlation was determined compared to expected viral loads e.g. reference panels, electron microscopy and in-house validated assays. Limit of detection (LOD), defined as more than or equal to 95 % positivity rate, is 500, 500 and 1000 copies/mL for affigene(R) EBV VL, affigene(R) HHV-6 VL and affigene(R) CMV VL, respectively. Conclusions: The affigene(R) OPI panel facilitates the monitoring of active viral infection in patients at risk of opportunistic infections. The performance of the assays make them suitable for pre-emptive monitoring.

In children undergoing HSCT, multiple and simultaneous non-albicans yeasts commonly occur but do not signify a risk of early dissemination or anti-fungal therapy resistance L.M. Ball, M.A. Bes, E.J. Kuijper, T. Boekhout, B. Theelan, R.M. Egeler, Leiden University Hospital, CBS, Centre for Fungal Diversity (Leiden, Utrecht, NL) Candida albicans is the most frequently isolated yeast in children undergoing hemopoietic stem cell transplantation (HSCT) but nonalbicans Candida (NAC) species are increasing. NAC's exhibit different sensitivities to antifungal treatments and variable mortality rates compared to C. albicans. Amplified fragment length polymorphism (AFLP) analysis is a PCR based method, which can reliably and reproducibly identify medically important Candida sp. We investigated the use of AFLP analysis of routine, serial surveillance cultures to determine the frequency of NAC's and whether there was, as a consequence, an increased risk of dissemination or antifungal resistance. Five consecutive non-selected children undergoing allogeneic HSCT (4 MUD; 1 syngeneic) were included in the study. All received total gut decontamination with non-absorbable polyenes and piperacillin/tazobactim. Three children also received oral fluconazole due to fecal overgrowth of Candida. All isolates (n=54), identified as Candida albicans by rapid MUREX analysis, were retrospectively re-analyzed using a standardized AFLP protocol, with investigators blinded to sample origin and patient identity. All children had, in the study period (+100days), positive Candida isolates. One child became rapidly colonized with C. albicans, this strain being present throughout his admission. Another child had persistent colonization with C. albicans that developed marked variation in DNA "fingerprinting" over time, consistent with microevolution. C. dubliniensis, C. lusitaniae and Saccharomyces cerevisiae were identified in the three remaining patients, two of whom had multiple, simultaneous and transient colonization with different species. Species identities were later confirmed by D1/D2 domain sequencing. No correlation with any transplant parameter or therapy administered was associated with the multiple / NAC yeast colonization. The presence of NAC's did not lead to dissemination or an increase in azole resistance (only evident in one C. albicans isolate). Prophylactic antifungals did not affect colonization but may have contributed to lack of dissemination. The presence of NAC's or of multiple simultaneous yeasts had not been previously appreciated. Lack of dissemination or therapy resistance illustrates the complex interaction between host and colonizing yeasts. Our study demonstrates that AFLP is a potent and reliable tool for future research into the dynamics of yeasts occurring in HSCT patients.

Blood transfusion requirements and febrile complications after autologous PBSCT: cause or consequence?

C. Scheid, C. Fritzsche, U. Holtick, M. Fuchs, D. Waldschmidt, K. Hübel, V. Diehl, D. Söhngen, University of Cologne (Cologne, D) Blood transfusions are thought to be exert an immunosuppressive effect. To investigate whether the amount of blood transfusions might interfere with the recovering immune system after autologous stem cell transplantation we studied the duration of fever in 172 autologous peripheral stem cell transplants in 138 patients in a retrospective analysis. The transplants were grouped according to the duration of fever (0 days, 1-3 days, 4-6 days or > 6 days). Fever was defined as a temperature > 38,0 °C on at least two occasions during the same day. Fe found a significant correlation between the number of transfused red cells (p<0.001) and platelets (p<0.001) and duration of fever. With the exception of the group with > 6 days of fever there was no difference in the number of transplanted CD34+ cells or in the time to recover a neutrophil count > 500/ul between the groups. The data are summarised in table 1(values are given as median (range)). To explain the correlation between fever duration and amount of blood transfusions one could argue that patients with a prolonged hematopoietic recovery (and therefor requiring more transfusions) may have a greater risk to develop infections and fever during neutropenia. In fact we have previously shown that a lower CD34+ cell dose is associated with an increased incidence and duration of fever (Scheid et al. Bone Marrow Transplant 1999 23:1177 . However with the exception of the group with >6 days of fever all other groups had a similar CD34+ cell dose and time to neutrophil recovery. We therfor favor the hypothesis that blood transfusions are rather the cause than the consequence of fever in this setting, either by inducing febrile transfusion reactions or by exerting an immunosuppressive effect and thereby facilitating oportunistic infections. As fever during neutropenia always requires antibiotic and sometimes antimycotic coverage, any strategy that reduces febrile complications after PBSCT should reduce resource utilisation. In conclusion our data support an even more restricitve use of blood transfusions after PBSCT, a finding which is in line with results in intensive care patients (Hebert et al. N Engl J Med 1999 340:409-17) .

Anti-fungal primary and secondary prophylaxis with liposomal amphotericin B (AmBisome) in patients undergoing family haploidentical allogeneic haematopoietic cell transplantation for high-risk haematologic malignancies F. Ciceri, M. Tassara, J. Peccatori, F. Lunghi, P. Servida, E. Guggiari, M. Bernardi, A. Pescarollo, M. Bregni, Istituto Scientifico H.S. Raffaele (Milan, I) Haploidentical stem cell transplantation (haplo-SCT) is a therapeutic option for patients with haematologic malignancies lacking an HLA matched donor. The delayed immune recovery secondary to T cell depletion results in prolonged risk of posttransplant infections and high mortality. The complexity and high cost of therapy and most of all the high case fatality rate of systemic fungal infections are reasons for a prophylactic approach. Therefore, we investigated the introduction of primary and secondary prophylaxis with liposomal amphotericin B (AmBisome) in reducing the occurence of proven, probable, and possible invasive fungal infection. We treated with AmBisome 23 patients with a median age 44 (range 21-60), affected by AML (17), ALL (1), NHL (2), HD (2), MM (1) . Five patients experienced a previous invasive fungal infection (IFI) before allogeneic HSCT; sites of documented infections were lungs (2 patients) and sinusal (3) . For 18 patients, primary prophylaxis was applied at transplant. All patients received AmBisome at standard dose of 1 mg/kg daily from day -10 to day + 30 after allogeneic HSCT from haploidentical family donor. Patients were monitored weekly for Aspergillus antigenemia in peripheral blood. CT HR scan of lungs was performed in case of fever of unknown origin (FUO) lasting more than 5 days under standard antibiotic multitreatment. Failure to AmBisome primary and secondary prophylaxis was defined as the occurrence of FUO requiring antifungal treatment and/or the documentation of IFI according to EBMT definitions. In case of failure, AmBisome 3 mg/kg/daily treatment or voriconazole was prescribed. Overall, we collected a total of 10 failures (43%) of AmBisome prophylaxis; 4 out of these 10 failures were registered in the secondary prophylaxis group of patients (80%); conversely, 6 out of 18 patients (30%) of the primary prophylaxis group experienced a failure. Possible, probable and proven IFI was detected in 3, 2, 2 patients respectively. Death occurred in 1, 1, 2 patients respectively. In 3 patients the failure of prophylaxis was represented by a FUO responding to anti-fungal treatment doses of AmBisome. No significant toxicity secondary to AmBisome prophylaxis and treatment was registered in this series. Our data support the use of primary prophylaxis in the setting of T-cell depleted allogeneic HSCT from haploidentical family donors.

Retrospective analysis of the use of valganciclovir in the prevention of cytomegalovirus following stem cell transplantation J. Subedi, J. Chappell, W. Ingram, M. Zuckerman, S. Devereux, A. Ho, G. Mufti, A. Pagliuca, King's College Hospital (London, UK) Cytomegalovirus (CMV) reactivation is a significant cause of morbidity following Stem Cell Transplantation (SCT) requiring IV therapy usually as an inpatient. Oral valganciclovir has equivalent bioavailability to IV ganciclovir. We performed a retrospective analysis of valganciclovir in 20 patients (14M/6F) post SCT. Median age was 52years(17-67) with a variety of myeloid (12) and lymphoid (8) disorders. 18 received Campath (17 reduced intensity SCT, 1 standard), 2 non-Campath standard SCTs. CMV status pre SCT for recipient/donor was -/-3/20, +/+9/20, -/+1/20, +/-7/20. 16/20(80%) patients had evidence of reactivation by CMV DNA PCR (CMV copy number >1000copies/ml) 4/20 patients (20%) had no evidence of reactivation by PCR but were commenced on treatment as they were deemed to be at high risk of reactivating. The median duration to reactivation was . 19/20 patients with 1st reactivation were admitted and treated with IV ganciclovir +/-foscarnet. Of those treated with valganciclovir 10/20(50%) had evidence of 1 previous CMV reactivation, 3/20(15%) had 2, 2/20(10%) had 3 and 5/20(25%) had no previous CMV reactivations. Valganciclovir was commenced for secondary prophylaxis in patients with previous CMV reactivation at high risk of further reactivations (3/20); evidence of low but positive CMV copy numbers >1000 on more than 1 occasion (4/20); continuation of therapy post ganciclovir or foscarnet in high risk patients (9/20) or patients considered to be at high risk of reactivation due to confounding medical disease states e.g. graft versus host disease, but had no evidence of CMV reactivation on PCR (4/20). Median duration of treatment dose valganciclovir was 24days (6-120). 7/20 patients continued on prophylactic dose, median of 14 days (5-60). 2/20 patients had a further reactivation after stopping valganciclovir. 1 patient required conversion to IV ganciclovir due to rising CMV copy numbers. 1 patient discontinued therapy due to neutropenia, no adverse effects were otherwise reported. The median follow up post SCT was 287days (124-826).We have demonstrated the need for trials of the use of valganciclovir in the post SCT setting. The indications, optimal dose and duration of treatment need to be determined.

Anti-fungal combination therapy for the treatment of invasive aspergillosis in patients undergoing allogeneic stem cell transplantation J. Tischer, X. Schiel, C. Rieger, G. Ledderose, H. Ostermann, H.-J. Kolb, LMU-Munich, Klinikum Grosshadern (Munich, D) Objectives: Invasive aspergillosis is a a well known major cause of morbidity and mortality in stem cell transplant recipients. The lack of effective treatment of invasive aspergillosis (IA) leads to the concept of combination therapy. Liposomal amphotericin B targets the ergosterol components of the fungal cell membrane. Caspofungin is an echinocandin inhibiting fungal cell wall growth by interfereing with 1,3-beta-glucansynthase. Combining these two compounds could result in a potentiation of antifungal efficacy by additive or synergistic effects. Patients and Methods: From December 2001 to March 2003 fifteen patients (pts.) with hematological malignancies undergoing allogeneic stem cell transplantation and possible, probable or proven IA (according to the EORTC/MSG definitions) received antifungal combination treatment with caspofungin (70 mg i.v. on day 1 followed by 50 mg i.v. once daily) and liposomal amphotericin B (1-3 mg/kg i.v.) for primary or salvage therapy or as secondary prophylaxis. Renal and hepatic toxicity was evaluated by clinical and laboratory parameters. Results: Fifteen pts. (median age: 51, five females) received the combination treatment for primary (2 pts.) or salvage (11 pts.) therapy or as secondary prophylaxis (2 pts.) for possible (6 pts.), probable (8 pts.) and proven (1 pt.) IA. The combination therapy was administered for 7 to 131 days (median 17 days). 14 patients had evidence of acute GVHD Grade II-IV requireing high dose corticosteroids and further immunosuppressive therapy in 13 pts. At the end of combination treatment 12/15 pts. were alive, 2/15 pts. had died due to progressive IA. 8/15 pts. (53,3 %) showed a clinical, radiological and/or microbiological improvement. Success of treatment was documented in 7/11 pts. with salvage therapy, 3/8 pts. with probable and 5/6 pts. with possible IA. 2/15 pts. had to be withdrawn from combination therapy due to renal impairment. 7 pts. showed increased liver parameters, probably related to aGVHD in 6/7 pts.. Conclusions: Antifungal combination therapy with caspofungin and liposomal amphotericin B in the treatment of IA in patients undergoing allogeneic stem cell transplantion is feasible. The overall response rate was 53,3 %. However, progressive IA is still associated with poor prognosis. Renal and hepatic toxicity was acceptable. Infectious complications, particularly bacterial and fungal, are the most significant causes of death and morbidity in patients with SAA. We performed a retrospective evaluation of incidence, type and outcome of infections in SAA children followed in our institution between 1991-2002. Patients: 17 children (5 females and 12 males; mean age 100.5 months) treated with WPSAA-GITMO immunosuppression (IS) schedules (ALG+CSA with or without G-CSF). Patients undergoing allogeneic BMT were excluded from the study at time of transplant. All patients but one received G-CSF and all had antimycotic (nistatin/fluconazole and/or B-amphotericin), anti-Pneumocystis (trimethoprim-sulfamethoxazole or pentamidine) prophylaxis, and gut decontamination (colimycin) during severe neutropenia. Follow up: 15 alive patients (88 %): 14 in complete remission (3 after unrelated BMT) and 1 transfusion-dependent; 2 patients died for infections. Infctions: 8/17 (47%) presented at least one episode with a total of 17 episodes (3 major, requiring long term hospitalization, 9 minor and 5 CVL-related). No severe viral infections was observed. Major episodes (bilateral pneumonia by Staphylococcus aureus plus Aspergillus fumigatus, bilateral maxillary-sinus Aspergillosis with septicaemia, Streptococcal septicaemia) occurred early within a mean of 31,6 days from diagnosis and was associated with very severe and persistent neutropenia. Two out of 3 recovered, though persistent neutropenia (one after leukocytes transfusion from the father). Half of minor episodes and all CVL-related Is (1Enterobacter endocarditis and 4 Staphylococcal subcutaneous tunnel infections, incidence of 0.8/1000 days CVL) occurred later (4-42 months) while assuming full dose CSA. Conclusions: the high response to IS plus G-CSF and improvements in supportive management reduced infection related mortality in children with SAA. Major infections occurred exclusively during severe persistent neutropenia preceding response to IS; nevertheless also responsive patients during prolonged CSA administration are at risk of developing infections, and CVL removal must be considered as soon as possible.

V. Milovic, S. Brioschi, J. Altclas, G. Jaimovich, A. Requejo, V. Listello, G. Drelichman, M. Garate, L. Feldman, ICTEM (Buenos Aires, AR) Liver disease is a major complication after stem cell transplant , with mortality rates ranging from 5 to 10%. Patients (pts)who are positive for HBV/HCV are at risk for the development of hepatitis reactivation and hepatic failure. It has also been suggested a relationship between HBV/HCV and veno-oclusive disease(VOD) of the liver. From October 1993 to January 2003, 325 pts underwent stem cell transplant in our BMT unit, 160 autologous and 165 allogeneic. Twentyfour (7.4%) pts and 2/165 (1.5%) donors were HBV/HCV positive before transplant. For their retrospective analysis, 4 groups were considered, according to their serological status: 1-patients HbsAg positive : 5 pts (1.5%) Two of 5 developed hepatitis reactivation with positive viremia (PCR). Both were treated with lamivudine, with progression and death due to hepatic failure in one of them. 2-patients HbsAg negative and anticore positive: 11 pts ( 3.5%) Two showed hepatitis reactivation , with spontaneous complete remission in one, and following lamivudine treatment in the other. 3-HCV positive patients: 8 pts (2.4%) In 6 ,viremia was performed and was negative. One pt died of severe VOD. 4-Donors HBV/HCV positive : 2 .None of the recipients who received their graft from a HBV/HCV positive donor, developed hepatic disease during their initial 2 years follow up . Conclusions : Four out of 24 ( 16.6 %) HBV/HCV positive patients showed viral hepatitis reactivation , with progression to hepatic failure in one (4.1%). Viral reactivation was observed exclusively in allogeneic transplant pts , and unexpectedly , biochemical hepatitis occurred during immunosupression in most cases. Based on our experience ,we conclude that a close follow up, and the early use of antiviral drugs, allow HBV/HCV positive patients and/or donors to be considered for bone marrow transplantation. Background: Patients undergoing autologous hematopoietic stem cell transplantation (AHSCT) are at standard risk for serious opportunistic infections since the expected duration of neutropenia is <10 days. Monotherapy may be sufficient as firstline empirical treatment in this setting. Inclusion of a glycopeptide in the initial regimen is controversial. To assess the role of initial glycopeptide we report the results of a study based on the use of imipenem/cilastatin (I/C) for empirical therapy in AHSCT recipients. Patients and Methods: An observational, prospective, multicenter study was carried out in two consecutive cohorts of AHSCT recipients with febrile neutropenia. From June 2001 to July 2002 I/C was given alone (500 mg/6 hours), and from July 2002 to October 2003 it was given in combination with a glycopeptide (I/C+G), either vancomycin (1 g/12 hours) or teicoplanin (400 mg/day). Results: A total of 386 patients (I/C, 181; I/C+G, 205) were assessable for response. There were 221 men and 165 women aged between 18 and 74 years (median, 52). The most prevalent underlying diseases were multiple myeloma (42%) and lymphoma (39%). Baseline patient characteristics were comparable in both groups. Oral antibacterial prophylaxis was administered in 62% (fluorquinolone in 44%) of cases. Peripheral blood was the source of stem cells in 96% of patients, and granulocyte colony-stimulating factor was given in 84%. Infection was microbiologically documented in 30% of cases (25% with bacteremia) and clinically documented in 28% of patients. Fever of unknown origin (FUO) occurred more frequently in patients treated with I/C+G (50% vs. 31%, P=0.0004). Successful response without modification of the initial regimen was consistently higher in the I/C+G study, especially in patients with microbiologically documented infection by gram-positive bacteria (see Table) . One patient died within 10 days of empirical antibiotic therapy (from idiopathic pneumonia) in the I/C+G group. Median time to defervescence, superinfection rate and length of hospital stay were not different in the two treatment groups. Conclusion: In AHSCT recipients inclusion of a glycopeptide in the initial empirical antibiotic regimen for febrile neutropenia leads to higher success rates without the need to modify the regimen. However, this benefit has no clinical impact since patients with gram-positive bacteremia can be appropriately treated later without an increase in morbidity or mortality. Background: The purpose of this study was to evaluate the role of partial growth hormone (GH) deficiency in metabolic syndrome observed as a late effect several years after stem cell transplantation (SCT) performed in childhood. Methods: The patients were 31 SCT survivors, with median age 15 yrs (range 7-34), transplanted for leukemia (n=26) or nonmalignant hematologic disease (n=5) with grafts from unrelated donors (13) and siblings (18). The median posttransplant followup was 6.0 yrs (range 1.2-20.5). Three had been primed without TBI. None was on steroids or GH therapy. Oral glucose tolerance test (OGTT) with measurement of blood glucose and serum insulin levels, as well as assessment of serum level of triglycerides and cholesterol were performed after an overnight fast. Patients with both hyperinsulinemia (serum insulin >20 mU/L after fast or >150 mU/L during OGTT ) and hypertriglyceridemia (serum triglycerides > 1.7 mmol/L ) were considered to have the core signs of the metabolic syndrome. GH secretion was evaluated either by provocative test with GH releasing hormone and arginine, clonidine or insulin-arginine-ACTH. A GH peak level of <20 mU/L was considered deficient. Results: Twelve patients (39%) had metabolic syndrome (hypertriglyceridemia and hyperinsulinemia combined). The median peak GH level in provocative testing was 12.3 mU/L (range 7.0-39.9) in the patients with metabolic syndrome and 31.3 mU/L (range 2.1-203.0) in those without (p=0.03). Nine of the 12 patients (75%) with metabolic syndrome had partial GH deficiency. In a multiple regression model, including age at SCT, use of TBI, donor, time interval postSCT, chronic GVHD, hypogonadism, hypothyreoidism and partial growth hormone deficiency as dependent factors, metabolic syndrome was independently associated with partial GH deficiency (R=0.35, p=0.04). Conclusion : The disturbances in insulin and lipid metabolism were evident in younger patients and at an earlier stage posttransplant than has been previously shown (1) . Partial growth hormone deficiency should be considered as a risk factor for metabolic late effects in children advancing to adulthood after allogeneic SCT. References 1. Taskinen M, Saarinen-Pihkala UM, Hovi L, Lipsanen-Nyman M Impaired glucose tolerance and dyslipidemia as late effects after bone marrow transplantation in childhood. Lancet 2000; 356: 993-997.

J. Gaziev, P. Polchi, C. Giardini, P. Sodani, G. Lucarelli, G. Visani, E. Angelucci, D. Baronciani, G. Leopardi, F. D'Adamo, B. Guiducci, M. Mele, Pesaro Hospital (Pesaro, I) There is a concern that high doses of cytotoxic drugs and radiation used for pretransplant conditioning may increase the risk of malignancies in long-term survivors. We studied 986 recipients of allogeneic bone marrow transplantation (BMT) for thalassemia and sickle cell disorders who received transplants between 1981 and 2002 to determine the incidence of malignancies. There were 8 malignancies (0.8%): 3 -early post transplant non Hodgkin lymphomas, 1-late non-Hodgkin lymphoma and 4 solid tumors (spinocellular cancer, colon cancer, melanoma and Kaposi's sarcoma respectively). We compared these results with the incidence of secondary malignancies in a cohort of 407 patients who received allogeneic BMT for acute myeloid leukemia (n=117), acute lymphoid leukemia (142), chronic myeloid leukemia (n=120), myelodisplastic syndromes (n=18), non Hodgkin lymphoma (n=8) and Hodgkin lymphoma (n=2) at the same time at our Center. In these patients we observed 6 secondary malignancies (1,5%): 1 Hodgkin lymphoma, and 5 solid cancers (melanoma, kidney cancer, breast cancer and 2 squamos cell cancer respectively). Although the overall incidence of tumors after BMT in patients with thalassemia was low than patients with malignancies this difference was not statistically significant. The incidence of post transplant solid cancer was low in thalassemic patients (0,4%) as compared with patients who received BMT for malignant diseases (1,2%; p=0.08, Fisher exact test). These data indicate a low incidence of solid cancer in patients undergoing BMT for thalassemia.

Immune reconstitution following allogeneic haematopoietic stem cell transplantation: 8 year, single-centre experience from more than 300 patients and nearly 1000 lymphocyte subset determinations A. Spyridonidis, D. Behringer, E. Bernhardt, C. Bachl, M. Egger, H. Bertz, J. Finke, University of Freiburg, Augusta Hospital (Freiburg, Bochum, D) Analysis of immune reconstitution after allogeneic HSCT has been a subject of a lot of studies, however results hampered by the small numbers of pts studied. We routinely evaluate lymphocyte subsets in allogeneic HSCT recipients since 1996 by two-color flow cytometric immunophenotyping and analyses with an internal standardized analysis programm (LYREG). We collected all LYREG-data generated between 01/1996 and 07/2003 in order to perform a retrospective analysis of immune reconstitution in allogeneic transplants. We generated two databases: The first includes data from 356 patients and 936 LYREG determinations done at 1m (16%), 3m (17%), 6m (25%), 9m (16%) and >=12m (24%). In order to more precise analyse the evolution of immune recovery in each patient and avoid influence of unknown parameters (like availability of the patient) we generated another database from 156 pts from which LYREG data existed from 1m, (3-6)m and (9-12)m. CD3+ T-lymphocyte numbers normalized after 6m in 49% of the pts and after 9m in 46% of the pts. 35% of the pts continued to be lymphopenic by 1 year post-transplantation. T-lymphocyte regeneration was mainly due to CD8+ T cell recovery which reached normal values in 67% pts by 6m and 82% pts by 1y. In contrast, CD4+ Tlymphocytes reached normal values by 12m only in 27% of the pts. 1 year after transplantation, 33% of the pts still had CD4 numbers <200/µl and 9% pts <100/µl. In our preliminary univariate analysis within distinctive, homogenous subgroups showed that a strong factor positively influencing CD4 recovery was the sibling donor compared to the unrelated donor. Naive CD4+CD45RA+ T cells were characteristically low and reached normal values by 1 year only in 20% of the pts. CD19+ B lymphocytes were strongly reduced in nearly all pts at 1m (median 5 cells/µl) and reached normal values in 25% pts at 6m and 75% pts at 1 year. Our results from a big database clearly demonstrate that recipients of allogeneic HSCT experience a prolonged period of profound impairement of lymphocyte numbers. We collected in our 2 large databases all variables that have been reported in the literature to influence lymphocyte numbers and currently complex multiple tests which take in account the interactions between different categories are in process.

Endocrine complications after paediatric haematopoietic cell transplantation G. Carreras, I. Badell, N. Pardo, M. Torrent, C. Lopez, J. Cubells, Hospital de la Santa Creu i Sant Pau (Barcelona, E) Aim: To evaluate the type and prevalence of endocrine complications in the children submitted to HCT in our unit. Patients and methods: Between October 1988 and January 1998, 265 children underwent 271 HCT. There were 88(33%)disease-freee survivors. Clinical records of the 69 survivors followed up at our centre(54% boys, 93% with malignant disease) were reviewed. Allogeneic transplantation made up 26% of HCT. Conditioning treatment included total body irradiation (TBI) in 57% and cyclophosphamide(Cy)in 65%. Mean age was 8.2+/-4.2 years (range 0-16)and mean follow-up 7.2 +/-2.6 years. Five patients had reached final height before HCT, and 83% of patients were Tanner stage I. Thyroid hormones were determined serially in 50 patients, and gonadotropines were determined in 37/55 patients who reached puberty age. Weight and height were evaluated each visit.

Bone metabolism and risk-adjusted ibandronate drug therapy after allogeneic blood stem cell transplantation B. Steiner, D. Wolff, M. Freund, University of Rostock (Rostock, D) Background:

Bone-related events including aseptic osteonecrosis and steroid-induced osteoporosis complicate the long-term outcome of allogeneic blood stem cell transplantation (BSCT) patients in up to 60%. In addition, there are no established treatment guidelines. Therefore, we investigated bone metabolism in BSCT patients and evaluated the effects of a prophylactic and risk-adjusted bisphosphonate drug therapy. Methods: Bisphosphonate drug treatment was administered to 47 patients after BSCT for 6-60 months (mean 26.8). Treatment consisted of 2 or 4 mg of ibandronate (Bondronat) every 3 months for one year. Risk adjustement was considered in that patients selected for either clinical bone events or steroid therapy or pathological quantitative CT scans were offered long-term therapy with 2 or 4 mg ibandronate every 4 weeks. In addition, calcium and vitamin D were supplemented before and throughout the study. Results: Supplementation with calcium and vitamin D was requested to normalise their blood concentrations before and throughout ibandronate therapy. During this one-year trial, 29 patients (62%) underwent dose adjustement. From these, 20 patients (69%) demonstrated less signs and symptoms related to skeletal complications, improved bone mineral density as verified by quantitative CT scans, and biochemical evidence of improved bone metabolism. The remaining 9 patients (31%) demonstrated no subjective or objective changes in bone status. Conclusions: In the present feasibility study, the effects of ibandronate drug treatment were demonstrated as related to both, bone mineral density and clinical improvement. These preliminary results form the rationale of an ongoing randomised trial directed to assess the clinical efficacy of ibandronate after BSCT.

Quality of life as an important outcome of high-dose therapy+autologous stem cell transplantation (HDCT+ASCT) in multiple sclerosis patients Y. Shevchenko, A. Novik, T. Ionova, I. Lesukov, A. Kulagin, O. Malysheva, V. Melnichenko, G. Bisaga, L. Chelombit, A. Kishtovich, National Pirogov's Medical Surgical Center, Russian Cooperative Group for Cellular Therapy, Multinational Center of Quality of Life Research (Moscow, Novosibirsk, St. Petersburg, RUS) Background: Quality of life (QoL) is increasingly used as a treatment outcome along with traditional clinical outcomes in multiple sclerosis (MS) patients. It is of importance if new interventions are being used. HDCT+ASCT is a new treatment strategy for patients with MS. The aim of the study was to provide MRI, neurological and QoL monitoring in MS patients before and at different time-points after HDCT+ASCT. Methods and Patients: Ten patients with MS were included in the study (EBMT protocols). QoL was assessed by FACT-BMT and FAMS. Integral QoL index was assessed by integral profiles method. QoL and EDSS evaluation was provided at baseline, at discharge, at 3, 6, 8,12 months and later every half a year. Median EDSS at base-line was 5.0 (range 1.5-7.5). The median follow-up duration was 32 months (range 9-54 months). The comparison of EDSS and QoL parameters median with the corresponding values at base-line was performed by Wilcoxon signed-rank test. Results: Clinical examination and MRI revealed disease stabilization (DS) in 8 patients and progression (DP) in 2 patients (one new lesion appeared in 1,5 years after APSCT). Comparison of EDSS at base-line and follow-up revealed its decrease in 6 patients; in 2 patients it remained the same. EDSS increased in the patients with DP (from 6.5 to 7.0 and from 6.0 to 6.5). Distinct QoL improvement was observed in all the patients with DS at the end of follow-up. Integral QoL index increased dramatically as compared to base-line: from 46.5 to 87.0 (FACT-BMT) -4.5 years follow-up; from 95.5 to 158.0 (FAMS)-3 years follow-up; from 62.0 to 167.0 (FAMS)-2.5 years follow-up. In patients with long-term follow-up no significant change in EDSS was observed whereas significant improvement of QoL index was found out. Conclusion: QoL is a measurable outcome of HDCT+ASCT in patients with MS. HDCT+ASCT results in distinct improvement of QoL parameters whereas there is only a slight decrease of EDSS level. QoL appears to be a more sensitive outcome than EDSS in MS patients after HDCT+ ASCT.

Quality of life assessment in long-term survivors post allogeneic haematopoietic cell transplantation. Are quantitative methods accurate enough?

A. Hotidou, P. Kaloyannidis, C. Kartsios, I. Sakellari, A. Anagnostopoulos, A. Fassas, University of Rennes, The George Papanicolaou General Hospital (Rennes, F; Thessalonica, GR) Aim-methods: The purpose of our study was to estimate QoL and adjustment of long-term survivors after Allo-HCT using the EORTC QLC-30 version 3.0 questionnaire and semi-structured interviews. Thirty-four patients (pts) aged 27(20-52) years were evaluated 2-12 years (median, 4 years) after Allo-HCT for leukemia (27), NHL (5) and MM (2); 17 pts were under medication for complications, mainly GvHD. Questionnaire: Pts main physical disruptions included fatigue (59%), dyspnoea (47%), insomnia (29%) and chronic pain (26%). Global health status was good to excellent (score>67, median: 83) in 65% of the responders. Physical, role, emotional, cognitive and social functioning were satisfactory (score>67) in 82%, 56%, 59%, 79% and 62% respectively, 29% reported financial difficulties. Interview: Survivors participated in a semi-structured interview regarding their adjustment in their familial, professional and social life. They designed specific factors influencing their QoL. Interestingly, 63% of pts with high global health status score revealed serious social and/or family problems. Content analysis identified consistently emerging themes such as: uncertainty, fear of disease relapse, physical and emotional limitations, appearance changes, energy loss, difficulty in future planning, work-related concerns and social isolation. Support and family relationships determined the degree of physical and emotional recovery. Discordance between pre-Allo-HCT expectations and current functional status resulted in psychological distress. Conclusions: QoL is influenced by a combination of biopsychosocial variables that affect the post-Allo-HCT recovery process. Social role retention is associated with a positive perception of QoL. Many pts tried to describe a better self-status through the questionnaire. On the other hand, interview revealed data closer to pts perspective of QoL. Integration of quantitative and qualitative methods could provide more accuracy and sensitivity in QoL assessment. Interventions based on knowledge of the influencing factors might facilitate patient's adjustment. Background Autoimmune thyroid disease (AITD) may occur in patients after stem cell transplantation (SCT). We investigated the actuarial incidence and association of AITD in adult Southern Chinese patients after SCT, and reviewed all reported cases. Material and methods 452 Chinese adult SCT cases from 1991 to 2002 with over one year survival were studied. Thyroid function was tested in recipients and donors before SCT, and at annual follow up. Anti-thyroglobulin (Tg) and microsomal (Tm) antibodies (Ab) were assayed by a particle agglutination test to confirm AITD. Relative risk (RR) associated with HLA alleles and donor / recipient gender was analysed. Results: Ten cases of AITD were diagnosed (6 hyperthyroid, 2 hypothyroid, 1 unique case of hypothyroid followed by hyperthyroid). The median age was 32 (range 19-42) and time from SCT was 36 months (range 12-94), which was not different from 14 reported cases. Combined with 14 reported AITD cases, there were equal number of male and female recipients, but 20 allogeneic donors were exclusively females. The 5-year actuarial incidence of AITD was 5% after allografts and 2% after autografts. There was strong association with the HLA alleles B46 (RR:3.7, p<0.0001) and DR9 (RR: 3.1, p=0.001) and with the haplotype A2B46DR9 (RR:2.9, p=0.0215), present in 13% of Chinese. The presence of AITD in donors and in patients before SCT do not predict for subsequent thyroid dysfunction. Conclusions: We conclude that female donor lymphocytes with predisposing HLA alleles have a high propensity to cause AITD in SCT recipients, and may warrant close monitoring. Post SCT AITD is common in Chinese due to high incidence of the susceptible HLA combination.

Nephrotic syndrome as late complication of chronic graftversus-host disease after allogeneic haemopoietic stem cell transplantation A.A. Colombo, C. Rusconi, P. Bernasconi, D. Caldera, P. Bertazzoni, C. Esposito, M. Lazzarino, E.P. Alessandrino, IRCCS Policlinico San Matteo (Pavia, I) Nephrotic syndrome (NS) is a rare complication of allogeneic transplantation; aim of this study was to evaluate the frequency of such complication in 301 consecutive patients (pts) with haematological malignancies who underwent allogeneic transplant from HLA identical related or unrelated donor. NS was observed in 6 out 221 evaluable pts (2.7%). All patients were male with a median age of 50 years (range 32-57). Five of them received HLA matched sibling peripheral stem cells, one case was grafted with an HLA well matched unrelated marrow. As preparative regimen 4 pts had a standard myeloablative treatment, 2 patients a reduced intensity conditioning. The median time of NS appearance was 23 months (range 13-43) from transplant. At the NS onset all patients were with active extensive chronic-Graft Versus Host Disease (c-GVHD) and on treatment with: Cyclosporine-A (CSA) and 6-Methylprednisolone (6-MP) given as maintenance therapy (4 pts), 6-MP alone (1pt), 6-MP and Methotrexate (1pt). The most common clinical signs referred were fatigue and edema of the lower extremities; laboratory tests revealed urine proteins median level of 6.5 g/24h (range 3-12); serum albumin was 2.4g/dL (range 2.3-3.4) and serum creatinine 0.9 mg/dL (range 0.7-1.07). In 4 of 6 patients renal biopsy was performed and the pathological diagnosis was: Membranous Glomerulonephritis (2 pts) and Minimal Change Disease (1 pt), in one pt biopsy specimen was not diagnostic. The treatment for NS was Prednisone 1 mg/Kg/day associated with a low dose of CSA in 5 patients. One pt was treated with Ciclophosphamide 200 mg /day for 5 days and Prednisone 1mg/Kg/day for two weeks. Response was achieved in a median time of three months (range: 2-6) months. At last follow up five pts are alive and well with a 80% decrease of proteinuria. One unresponsive patient died within 6 weeks from NS diagnosis of acute respiratory distress. Patients with c-GVHD may be considered at risk of NS: a careful monitoring of renal function is advisable particularly in patients receiving allogeneic peripheral stem cell.

E. Morello, R. Gaiardoni, M. Sorio, N. Pescosta, M. Casini, F. Benedetti, Azienda Sanitaria di Bolzano, University of Verona (Bolzano, Verona, I) Introduction: Adrenal gland insufficiency after allogeneic bone marrow transplantation (BMT) is mainly due to the prolonged steroid based immunosoppressive treatment for Graft-versus-Host Disease (GVHD). The physiologic role of DHEA (Dehydroepiandrosterone) and DHEAs (Dehydroepiandrosterone sulfate) is under investigation. Many authors report low blood levels of DHEA after BMT. Replacement therapy with DHEA in women with adrenal gland insufficiency is reported to be safe and to improve physical, psychological and sexual wellness (Arlt, nejm 1999) . Design of the study: We performed a pilot study in the BMT Units of Verona and Bolzano in order to evaluate the possible impact of DHEA replacement on the health status in a setting of hematopoietic cell transplanted patients. We proposed to 16 patients (12 in Verona and 4 in Bolzano) with latent adrenal gland insufficiency the daily dose of 50 mg of a galenic formulation of DHEA made by the hospital pharmacies, until complete recovery of DHEA's blood levels. Inclusive criteria were allogeneic bone marrow or peripheral blood precursor transplantation, low blood levels of DHEA, withdraw of steroid-based immunosuppression. After 3 to 6 months the patients were evaluated for DHEA blood levels, and in case of complete recovery the replacement was stopped. Each patient received a health-status questionnaire (SF12) at the entry in the study, and after 1, 3 and 6 months. At each physical examination Karnofsky Performance Status (KPS) was calculated. The results of SF12 questionnaire were evaluated according to the authors' guidelines. Results: Fourteen patients (87.5%) reached the end of the study, with good blood levels of DHEA and two patients are three months after enrollment. None developed side effects after DHEA assumption. All the patients showed an improvement of KPS (median improvement 10%, range 10-40%) and felt better than at enrollment in the study. The SF12 questionnaire shows also an improvement between the situation before replacement therapy and after 1, 3 and 6 months, but these results are not statistically significant.

Discussion: This pilot study shows the feasibility of a DHEA replacement in a group of hematopoietic cell transplanted patients with latent adrenal gland insufficiency. This treatment was well tolerated and seems to improve the health status of these patients, but a (multicenter) controlled randomized trial is needed to confirm these data.

Osteoporosis after autologous bone marrow transplantation: a short-term compliance R. Ria, A.M. Scarponi, F. Falzetti, S. Ballanti, M. Di Ianni, M. Cimminiello, C. Gasbarrino, B. Pallone, F. Russo, T. Cirulli, G. Di Pietro, A. Vacca, F. Dammacco, E. Mannarino, M.F. Martelli, A. Tabilio, University of Bari Medical School, University of Perugia Medical School (Bari, Perugia, I) Patients who underwent to autologous stem cell transplantation (ASCT) are prone to decreased bone mineral density (BMD). The post-transplantation bone damage is probably attributable to the direct impairment of osteoblasts by chemotherapyc drugs and total body irradiation (TBI), and the difficulty of stromal stem cell compartment to regenerate a normal number of osteoblasts. Mechanisms of this type may account for the late recovery of bone mass after ASCT. Therefore, we measured BMD in 180 patients (98 M and 82 F) who underwent ASCT for hematologic malignancies. Patients were evaluated with a median of 6.2 years (range: 24-192 months) after ASCT. Median age was 46 (range: 25-68 ) years. 20 patients (12 M and 8 F) who received only chemotherapy (median age 52, range 34-72 years) were evaluated for BMD as controls. The BMD was assessed at either the L1-L4 vertebrae (124 [68%] patients) or at the wrist (56 [32%] patients). At the L1-L4 vertebrae 51 (41%) patients had osteopenia and 32 (26%) patients had osteoporosis. At the wrist, 21 (37%) patients had osteopenia and 12 (21%) patients had osteoporosis. On the logistic regression analysis, patients with decreased BMD were older than those with normal BMD (p=0.03). Gender, oncotype, debulking therapy, response to treatment and type of conditioning regimen, with or without TBI, were not associated with decreased BMD. The loss of bone mass was greater during the first year after ASCT, since majority of patients showed recovery of BMD during the following years. We also evaluated the incidence of secondary hyperparatyroidism, osteoblast activity (as osteocalcin serum levels) and bone resorption markers (as pyridinium urinary escretion). During the first year after ASCT 148 (82%) patients gave increased PTH serum levels (median 81.5 ng/mL, range 60.6-177.8; p<0.01), 155 (86%) lower osteocalcin serum levels (median 16.5 microg/mL, range 11.1-55.0; p<0.05), and 132 (73%) higher pyridinium urinary escretion (median 39.0 mg/24h, range 19.0-89.0; p<0.05). All patients showed normalization of these bone turnover markers in the following years. To sum up, after ABMT, over half of the patients show osteopenia or osteoporosis independent of the sex. According to other groups our results emphasize the potential usefulness of antiresorptive agents to prevent or treat post-ASCT osteopenia or osteoporosis, and the importance of the measurement of BMD as an integral component to the follow-up of ASCT. The objective of the present study is to assess the clinical and ethiological features of neurological complications (NCs) of allogeneic bone marrow transplantation (BMT). We have reviewed the clinical records of 183 patients who underwent allogeneic BMT during the years 1997-2001 at the Seràgnoli Institute, Bologna. NCs were found in 36% of patients (66/183), mostly occurring within 6 months after BMT. Indeed, 64% of patients developed NCs between 1 month before and 6 months after BMT, 11% between 1 week and 1 month after BMT, 8% during the first week after BMT and only 17% after 6 months. The most frequent disturbances, observed in 52 patients (80%), involved the central nervous system (CNS) with tremor (39%) and seizures (21%). Twelve patients experienced NCs involving the Peripheral Nervous System (PNS) with sensorial polyneuropathy (25%, 3 patients), toxic myopathy (25%) and Guillame-Barrè syndrome (18%, 2 patients). In 5% of patients we found both CNS and PNS complications. Cerebrovascular events were relatively rare (2 patients). The ethiology was multifactorial. The main cause of CNS symptoms was CsA toxicity (62%, 31 patients); other co-factors were infections, relapse of hematologic malignacy, thrombocytopenia, methabolic alterations, toxicity of the conditioning regimen and type of transplant (VUD vs sibling). PNS complications were attributed to steroids (25%, 3 patients), CsA (17%, 2 patients), relapse (2 patients) and GvHD (in form of Guillame-Barrè Syndrome, 2 patients). Thirty-three (50%) patients with NCs died within 1 year; the high mortality rate was mainly due to CNS infections. In univariate analysis the type of the transplant (VUD vs sibling) and Busulfan-based regimen resulted in a significant association with NCs within 6 months from BMT, whereas in multivariate analysis only the association between VUD transplants and NCs resulted statistically significant.

Severe hypogammaglobulinaemia in patients with lymphoid neoplasia after allogeneic HSCTwith rituximab during preceding therapy A. Buser, C. Bucher, D. Heim, G. Petitjean, T. Hauser, A. Rolink, A. Tichelli, J. Passweg, A. Gratwohl, Kantonsspital Basel, Institute for Immunology (Basel, CH) Hypogammaglobulinemia and lymphopenia are well known complications of HSCT, but severe Immunoglobuline deficiency outside the context of severe GvHD is rare. We observed in a patient a very severe hypogammaglobulinemia and absolute Bcell lymphopenia at 3 months after HSCT. The patient was part of a pilot protocol treating 10 patients with refractory lymphoid neoplasia with BEAM followed by allogeneic RIC 28 days later. We compared the levels of Immunoglobulins and lymphocytes of these patients with 10 consecutive patients with standard HSCT at day 90 after HSCT. 5/7 alive of the BEAM group had had Rituximab within preceding treatment. All of these showed severe hypogammaglobulinemia but no lymphopenia at day 90 posttransplant compared to the ten patients without Rituximab. Rituximab prior to HSCT even long before, might impair B-cell reconstitution of donor B-cells post allogeneic HSCT.

Salivary gland function following stem cell transplantation: a prolonged study comparing non-myeloablative versus myeloablative conditioning R. Nagler, L. Barness-Hadar, S. Slavin, A. Nagler, Rambam Medical Center, Hadassah University Hospital, Chaim Sheba Medical Center (Haifa, Jerusalem, Tel-Hashomer, IL) SCT related salivary gland injury and dysfunction results in local and systemic manifestation, which may last very long and are associated with high rate of morbidity and even mortality due to increased risk of infections. Non myeloablative and low intensity conditioning (LIC) regiments were shown to be associated with decreased rate of transplant related organ toxicities. We, therefore, assessed salivary gland function up to 12 months post SCT in thirty patients who underwent SCT post LIC (study group, n=16) in comparison to myeloablative (control group, n=14) conditioning. Eighteen were male and 12 female with a median age of 36 (range 7-58) years. Disease categories included acute leukemia -13, malignant lymphoma -5, chronic myeloid leukemia -6, multiple myeloma -3, genetic diseases -2, solid tumors -1. Salivary gland function was assessed by sialometic and biochemistry studies which included measuring total protein, albumin, secretory IgA and the antioxidants (peroxidase, TAS and SOD) in the collected saliva. We observed a major decrease of saliva secretion in the first few months post SCT mainly in patients that developed graft versus host disease (from 0.7 ± 0.1 ml/min to 0.2 ± 0.1 ml/min, respectively). A significance difference was observed between patients that received LIC in comparison with myeloablative conditioning. In the LIC group a good recovery of saliva secretion was observed, one year, post SCT, while in the myeloablative group saliva secretion remained significantly low (0.7 ± 0.2 ml/min vs 0.2 ± 0.02 ml/min, respectively) (P< 0.05). No difference was observed between the two groups in saliva biochemistry and antioxidant profile.

We, therefore, conclude that myeloablative conditioning, has an immediate effect on the salivary glands, as well as is a prolonged effect, disabling the recovery of the glands, for at least one year post SCT. In contract, low intensity conditioning is associated with decreased rate of transplant related salivary glands toxicity.

Renal function shortly and 1-year after allogeneic haematopoietic stem cell transplantation M. Ertem, T. Ileri, F. Azik, F. Yalcinkaya, M. Ekim, Ankara University (Ankara, TR) Renal dysfunction has been increasingly recognized as a common adverse effect of allogeneic hematopoietic stem cell transplantation (HSCT). Since the data suggest that compared with adult kidneys, developing kidneys may be more vulnerable to the preparative regimen, children should be studied separately. In this prospective study, we aimed to investigate the effect of an allogeneic HSCT on renal function in children. Renal ultrasonography and DMSA were performed to evaluate the structural renal abnormalities and evaluation of renal function included measurement of the glomerular filtration rate (GFR), creatinine concentration, concentrating capacity of the kidneys, tubular phosphorous reabsorbtion capacity, urine level of betamicroglobulin, and also dipstick urinalysis before, shortly after and 1-year after HSCT. Patient characteristics, conditioning regimen, major complications, and the use of potentially nephrotoxic medications were evaluated. In a 3-year period, 29 children with hematologic disorders (leukemia,13; thalassemia,12; aplastic anemia,3, FHLH,1) underwent allogeneic HSCT. Median age of the patients were 10.0 years (1.9 to 17.0). None of the patients received TBI in their conditioning regimen. Before HSCT, minimal structural renal abnormalities were detected in 5 patients (17.2 %) however, renal functions were found to be normal in all patients. Of these 29 patients, 11 (38 %) had significant acute renal dysfunction (grade 2 toxicity-renal insufficiency-in 8 and grade 3 toxicityrenal failure-in 3 patients) shortly after HSCT. In these patients, the identified causing factors were the use of cyclosporin (n: 5), hepatic veno-occlusive disease (VOD) (n: 5), and sepsis (n: 1). Among the potential risk factors for acute nephrotoxicity, hepatic VOD was found to be the only significant risk factor (p< .05). After allogeneic HSCT a total of 5 patients (17 %) died, 4 from transplant related complications and 1 from relapse of the disease. One year after HSCT, 2 (12.5 %) of 16 evaluable children had chronic renal insufficiency (GFR<70 ml/min/1.73 m 2 ) and no patient had chronic renal failure requiring dialysis. In only 3 patients structural renal abnormalities persisted 1-year after HSCT. In conclusion, acute and chronic renal dysfunction was found to be a frequent complication after allogeneic HSCT in children. Follow-up of the patients with chronic renal insufficiency is needed in order to predict the long term prognosis.

The role of inherited hypercoagulable abnormalities in paediatric haematopoietic stem cell transplantation M. Ertem, F. Azik, T. Ileri, E. Akar, A. Ulu, N. Akar, Ankara University (Ankara, TR) A decrease in the natural anticoagulants(protein C, protein S and antithrombin III), factor VIII elevation or mutations of the factor V Leiden and prothrombin 20210 G-A are known as predisposing factors for thrombophilia. Recent reports from different groups pointed to this predisposing factors in the development of thrombotic processes after hematopoietic stem cell transplantation(HSCT) in adults. In this prospective study, we aimed to investigate the effect of inherited hypercoagulable abnormalities on HSCT outcome in children. The laboratory evaluation of inherited hypercoagulable abnormalities included functional anticoagulant assays for protein C, protein S, antithrombin III(AT III), factor VIII and genetic analysis of the factor V Leiden and prothrombin 20210 G-A mutation were performed before conditioning therapy. Patient characteristics, thrombotic processes(catheter related thrombosis, pulmonary embolism, deep venous thrombosis) and veno-occlusive disease(VOD) were evaluated. In this ongoing study, 33 children(median age:10 years, range:1,1-17 years) who underwent HSCT for leukemia(n:13), hemoglobinopati(n:11), aplastic anemia(n:3), MDS(n:2), JCMML(n:2), CML(n:1) and FHLH(n:1) were included. Laboratory evaluation of inherited hypercoagulable abnormalities indicated a patient had heterozygote protein C deficiency. Protein S and AT III levels were normal in all of the patients. Factor VIII levels were elevated in five(%15,1) patients. Factor V Leiden heterozygosity was seen in 7(%21,2) patients, but none of the patients had prothrombin gene 20210 G-A mutation. Thrombotic complications were diagnosed in 8(%24,2) patients. There were 6 (%18,1) cases of VOD and 2 (%6) cases developed catheter related thrombosis. VOD was diagnosed on the basis of clinical criteria as previously described by Seattle. 2 patients had severe and 4 patients had moderate VOD. 3 of the 6 VOD had factor V Leiden heterozygosity. Catheter related thrombosis was not associated with inherited hypercoagulable abnormalities in our 2 cases. In conclusion hereditary thrombophilia did not increase the risk of thrombosis in pediatric HSCT patients except VOD. We recognise that the number of patients in this study is small to draw a conclusion. However, we believe that factor V leiden mutation may increase the risk of VOD in HSCT patients and early intervention for VOD should be considered for patients with this mutation.

K. Higgins, C. Noon, V. Cartwright, M. Davies, T. Howlett, A. Hunter, Leicester Royal Infirmary (Leicester, UK) The European Group for Blood and Marrow Transplantation highlighted the risk of premature death in survivors of bone marrow transplantation (BMT). Survivors of childhood BMT have an increased risk of features of the metabolic syndrome. Case reports describe early cardiovascular morbidity in this group. Little is known of the metabolic sequelae of adulthood BMT. True cardiovascular morbidity is unknown. We recently set up a Late-Effects clinic for adults treated with BMT. Its aim is to monitor endocrine late effects and screen for features of the metabolic syndrome. We measured: anthropometry, blood pressure (BP), urinalysis, 75g oral glucose tolerance test, biochemical, lipid and hormone profile. Of 16 patients (10men:6women), mean age 38(25-54)yr, mean time from transplant 6(1-12)yr, 93% had total body irradiation prior to transplantation. One received oral steroids in the previous year but this had been stopped for 6months. Two patients had evidence of chronic graft versus host disease (skin). One set of notes was lost and clinical data (anthropometry and BP) is missing. Single biochemical data points are absent in 3 patients (high density lipoprotein (HDL) cholesterol levels twice and albumin:creatinine ratio once). Forty-seven percent of patients (7/15) had central obesity (waist:hip ratio >1.0 men, >0.88 women), 63% (10/16) had hypertriglyceridaemia (>1.7mmol/l), 33% (5/15) had low HDL cholesterol (<1.0mmol/l men, <1.2mmol/l women), 20% (3/15) were hypertensive, 27% (4/15) had an elevated albumin:creatinine ratio (>3.5mg/mmol) and 13% (2/16) had abnormal glucose metabolism. One patient had IGT, the other type II diabetes (according to WHO criteria). One patient had all 6 features of the metabolic syndrome, 1 had 5 features, 1 had 3 features, 7 had 2 features and 3 had 1 feature. Three men had primary hypogonadism, none were on hormone replacement therapy (HRT). All six women had biochemical evidence of early menopause (2/6 were not on HRT). One patient was found to be hypothyroid, one to have borderline hypothyroidism and two to be on thyroxine replacement but inadequately replaced. Two patients have low IGF-1 levels and are awaiting investigation. This observational report shows that features of the metabolic syndrome are common in patients who have had BMT in adulthood. Early identification of these risk factors and prompt intervention may reduce cardiovascular mortality in a group known to be at risk of premature death.

Psychosocial late effects of bone marrow transplant in adolescents G. Peykerli, S. Anak, S. Ozgenc, E. Can, H. Bilgen, E. Tugrul Saribeyoglu, G. Gedikoglu, Istanbul Scool of Medicine, Our children leukemia foundation, Cerrahpasa Blood Bank Unit (Istanbul, TR) Although BMT is a life saving treatment option, patients and families also encounter severe psychosocial problems. These problems not only occur during BMT, but also years after. The aim of this study was to determine the psychosocial problems of BMT survivors and the impact of these problems on their daily lives. To fulfill this aim, only patients, whose BMT was performed 3 -10 years ago, were selected. Rosenberg Self Esteem Scale was applied to 25 adolescents, whose BMT was performed between 1989-2000. Age matched 25 adolescents without any health problems were the control group,. Rosenberg Self Esteem Scale consists of twelve subtests which are self-esteem, vulnarability to criticism, continuity of self-concept, trust on people, depressive mood, imagination, psychosomatic symptom, threat on interpersonal relation, involvement degree on discussion, degree of parental care, relation to father, psychologic isolation. The results were evaluated with chi square test. The median age of all children (patients and control group) was 17 years. Gender distribution in the patient group was 11 female/14 male, control group 13 female/12 male. BMT had been performed for aplastic anemia (3 patients), thalassemia (1 patient), AML (13 patients), CML (2 patients), Hodgkin's disease (2 patients), ALL (3 patients) and MDS (1 patient). Both the patients and control group were from low or middle socioeconomical level. As a result, a statistical difference for self esteem, continuity of self-concept, trust on people, vulnarability to criticism, depressive mood, imagination, phsysicosomatic symptoms, involvement degree on discussion, degree of parental care, relationship to father, psychologic isolation, could not be demonstrated between the study and control groups (p>0.05). Only threat on interpersonal relation was higher in the patient group (p<0.05). These results suggest that, psychosocial status of BMT survivors improves to the level of healthy adolescents in 3 years after BMT, except for threat in interpersonal relations and lack of self trust , suggesting a total mistrust to the outer world. Longterm psychosocial follow-up is a must after BMT to achieve a better quality of life. Immune recovery after haploidentical haematopoietic stem cell transplantation (SCT) is delayed and is of clinical concern. We studied thirty-five children (19 males, 16 females) with a variety of malignant and non-malignant disorders who received SCT from haploidentical family donors. Patients with relapsed acute leukaemia constituted the largest diagnostic group (n=20) followed by myelodysplastic syndrome (n=3), Fanconi's anaemia (n=3), primary haemophagocytic lymphohistiocytosis (n=3), primary immunodeficiencies (n=2), juvenile myelomonocytic leukaemia (n=2), adrenal leukodystrophy (n=1) and osteopetrosis (n=1). The mean age of the patients at the time of SCT was 7.3 years (range 0-16) and the median follow up was 17 months (range 1-55). Patients received G-CSF mobilized, CD34+ positively selected peripheral blood stem cells from 32 parental and 3 sibling donors. The mean CD34+ dose infused was 12.4 X10 6 /kg (range 0.29-34.2 X10 6 /kg). Lymphocyte subset recovery was analysed for 27 patients. The CD56+ subset (NK cells) was the earliest to recover, reaching normal levels at a median of 45 days (range 15-240) followed by CD19+ subset at 90 days (range 45-240). The median time for CD3+ and CD8+ subsets to normalise was 172 days (range 45-600) and 120 days (range 28-420) respectively. The CD4+ lymphocyte population was the last to recover and reached normal levels only after a median of 315 days (range 45-600). Fifteen patients developed opportunistic infections, which included CMV reactivation (n=6), pulmonary aspergillosis (n=3), disseminated varicella zoster (n=2), disseminated HSV (n=1), EBV reactivation (n=1), adenovirus reactivation (n=1), parainfluenza virus type 3 (n=1), RSV (n=1) and Mycobacterium kansasii (n=1). Fourteen patients (40%) died following transplantation of which 8(23%) deaths were due to disease relapse. Four (11%) patients died of opportunistic infections and there were 2(6%) toxic deaths. Conclusion: Lymphocyte subset recovery particularly the CD3+, CD8+ and CD4+ cell subsets is delayed after haploidentical SCT. This increases the risk of opportunistic infections and measures to minimize the infection related morbidity and mortality including, antimicrobial prophylaxis and intravenous immunoglobulin till immune recovery should be undertaken. We will discuss factors, which may influence the delayed recovery and wide variability in the time to lymphocyte subset normalisation in these patients.

Haploidentical transplantation in paediatric patients with ALL: a comparison with unmanipulated grafts P. Lang, J. Greil, P. Bader, R. Handgretinger, T. Klingebiel, P.-G. Schlegel, D. Niethammer, University Children's Hospital (Tuebingen, D) Between 1995 and 2003, 30 pediatric patients with high risk or relapsed acute lymphatic leukemias have been transplanted with CD34+ selected or CD133+ selected stem cells (or a combination of both) from 1-3 loci mismatched related (haploidentical) donors. All patients lacked a matched related or unrelated donor and received a median number of 24x10 6 /kg stem cells purified with magnetic microbeads (Miltenyi Biotec), including several boosts given 14-90 days after initial transplantation. Chimerism state was monitored weekly. To evaluate the clinical impact of this method, the outcome was compared to that of a patient group, who received unmanipulated grafts from matched unrelated donors (n=18). The rate of sustained engraftment after initial conditioning was slightly lower in the haploidentical group (83% vs. 100%). All patients with graft failure were rescued by reconditioning regimens. In contrast, incidence of primary GvHD exceeding grade I was higher in the group with unmanipulated matched grafts, despite the fact, that the patients of the haploidentical group did not receive any posttransplant immunosuppression. (3% haplo vs. 67% unmanipulated). Probability of disease free survival at 2 years for patients with CR1-3 was 44% in the haploidentical group and 39% in the unmanipulated group (p=0.8, median follow up 2 and 4.8 years). Relapse probability was also not significantly different (0.5 vs. 0.3, p=0.13). Causes of death in the haploidentical group and in the unmanipulated group were relapse, viral and fungal infections (17%) and others (7%). However, no lethal viral infection was observed in the last 2 years, probably due to improved diagnostic methods and prophylactic and preemptive antiviral therapy. Conclusions: profound T cell depletion was capable of minimizing GvHD but did not result in significantly increased relapse rates. To overcome the HLA barrier, high stem cell doses and additional stem cell boosts were necessary. Some patients needed intensified conditioning regimens. Surveillance of virus load and chimerism state were important tools. Taken together, this approach resulted in a favorable and stable DFS (44%) in patients with ALL, which is similar to that of patients with unmanipulated MUD grafts.

Unrelated donor marrow transplantation in children withhigh risk haematologic malignancies in a single-centre from 1989 to 2002 C. Galambrun, G. Souillet, V. Dubois, N. Bleyzac, M.-P. Pages, C. Pondarré, Y. Bertrand, Hôpital Debrousse (Lyon, F) We evaluated the outcome of sixty three paediatric patients treated by sixty seven stem cell transplantations from unrelated donors (URD BMT) between May 1989 and September 2002. Four patients received a second stem cell transplantation because of relapse disease. All of them had high-risk haematologic malignancies with acute lymphoblastic leukaemia (n=35), acute myeloid leukaemia (n=17) or myelodysplastic syndrome (n= 15). HLA matching was retrospectively defined using DNA high-resolution typing of both HLA class I and class II loci. Twenty four patient/donor pairs were fully matched (36%). Among the forty three mismatched pairs, twenty two were mismatched at only one HLA (33%) locus and twenty one at two or more locus (31%). Most of the patients received rabbit anti-Tlymphocyte globulin (ATG) as in vivo T depletion before transplantation with a median total dose of 15 mg/kg. Bone marrow was used as the source of haematopoietic stem cell in 64 patients whereas cord blood was used for the three others. The median nucleated cell dose was 4,3 x 108/kg in patients given a transplant of bone marrow stem cells. Overall survival was 50%. Acute GVH disease developed in 46% with 9% having grades III-IV. Disparity for more than one HLA antigen increased the risk of severe GVH from 6,5% to 19%. The probability of GVHD-related mortality equalled 7,4%. The incidence of chronic GVH was 12%. The cumulative incidence of transplantation-related mortality (TRM) was 26%. The estimate for relapse after bone marrow transplantation was 24%. Use of high resolution typing methods in routine matching improve unrelated donor selection and URD BMT should be considered for paediatrics patients with high-risk haematologic malignancies.

Treatment of liver failure due to veno-occlusive disease in a two-year old child after stem cell transplantation with living donor liver transplantation from the same HLA-identical donor K. Mellgren, A. Fasth, J. Abrahamsson, R. Saalman, M. Olausson, Queen Silvia Children's Hospital, Sahlgrenska University Hospital (Gothenburg, S) Objective: We describe a case where liver transplantation from the same donor as used for stem cell transplantation (SCT) was performed as treatment of liver failure due to veno-occlusive disease (VOD). Methods: Two monozygotic twins were diagnosed with acute myeloic leukemia (AML) in March and July 2002, respectively. They were treated according to NOPHO AML -93 protocol, and both went into complete remission (CR) after the induction course. Results: Twin I relapsed 4 months after end of primary AML therapy. With FLAG reinduction CR was achieved and she was transplanted with peripheral blood stem cells (PBSC) from her HLA-identical father in February 2002. Four months after transplantation she relapsed and was given two infusions of donor lymphocytes (DLI). CR3 was achieved after first DLI but she subsequently developed grade IV liver graft-versus-host disease (GvHD). Six weeks after the second DLI she relapsed again and is presently receiving palliative treatment. Twin II started treatment 4 months after her sister. At the first relapse of her twin sister we decided to intensify treatment with SCT in CR1. She was transplanted in January 2003, with PBSC from her HLA-identical father. After transplantation she developed a severe VOD with irreversible fibrous obliterative changes and progressive liver dysfunction and it was decided to proceed to liver transplantation. Segments 2+3 were transplanted from her father. Additional immunosuppression with one week of steroids was given. After 4 weeks, tacrolimus was withdrawn and no rejection has developed. Reoperations due to stenosis of the bile duct anastomosis have been necessary. The patient remains well with satisfactory liver function. She is still in CR1 11 months after SCT. Conclusion: Liver transplantation with living donor is a treatment option for VOD, in this case without need for additional immunosuppression.

Role of echodobutamine in the evaluation of cardiac dysfunction in children more than 10 years after bone marrow transplantation C. Uderzo, E. Cavatorta, G. Trocino, A. Rovelli, D. Longoni, E. Viganò, A. Tagliabue, A. Balduzzi, G. Masera, Ospedale San Gerardo (Monza, I) Introduction: In the past few studies addressed the possible cardiotoxicity as important late effect in children undergoing Bone Marrow Transplantation (BMT). The aim of the current study is to evaluate the cardiac contractility reserve in children more than 10 years (yrs) after BMT and the role of echodobutamine in detection of subclinical cardiac dysfunction. Patients and Methods: Twenty nine patients (pts), 24 males and 5 females, have been followed prospectively by echocardiogram since the beginning of BMT performed in childhood for malignant diseases (median age at BMT 9,2 yrs, range 1, [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] 9) . 20/29 underwent an allogeneic BMT from HLA matched sibling, 1/29 a singeneic BMT, 1/29 an haploidentical BMT, 6/29 an autologous BMT and 1/29 an unrelated BMT. 22/29 pts received preBMT anthracycline (median cumulative dose 300 mg/m2, range 120-610) while 23/29 received fractioned TBI and 29/29 high dose Cyclophosphamide (median dose 135 mg/kg, range 40-330) as a conditioning regimen for BMT. 20/29 pts were affected with a-GVHD and 7/29 with c-GVHD. All the patients were recalled more than 10 yrs after BMT (median follow-up 14,8 yrs, range 9,9-23,3) to perform both a clinical evaluation and an echocardiogram before and after administration of low-dose Dobutamine (10mcg/kg/min). Results: At the time of echodobutamine evaluation the median age of the pts was 20,9 yrs (range 14,2-32). Despite nearly all pts (28/29) were asymptomatic and with normal echo in basal conditions, we were able to demonstrate, through echodobutamine, a group of 7/29 pts with low cardiac contractility reserve (Ejection Fraction -EF-increase after Dobutamine<10%). These seven pts compared with the group with normal reserve (22/29 pts) presented: 1. lower EF increasing after stress (5,8% versus 18,9%) 2. lower increasing of left ventricular end-diastolic volume and diameter 3. lower systolic thickening of left ventricular posterior wall and interventricular septum Conclusion: This study suggests that echodobutamine is an important tool to evaluate late cardiac effects in pts potentially cured by BMT performed during childhood. The fact that 24,2% of our asymptomatic population showed a reduced response at stress, i.e. a systolic and diastolic dysfunction, calls for a continuous surveillance in order to provide more information on the long term quality of life.

E. Gorczynska, D. Turkiewicz, K. Rybka, J. Toporski, K. Kalwak, A. Dyla, A. Chybicka, Wroclaw Medical University, Institute of Immunology and Experimental Therapy (Wroclaw, PL) The aim of the study was to evaluate the frequency and clinical implication of BKV infection in children after alloHCT. (2), SAA (4), immune deficiencies (3), leukodystrophies (2) . Methods: Conditioning regimen was based on TBI (5), Busulfan (59) or other chemotherapy agents (4). Patients transplanted from MUD or PMFD received antibodies (ATG, CAMPATH 1H) in preparative regimen. Patients transplanted from PMFD received additionally OKT3 as rejection prophylaxis. GvHD prevention consisted of CsA +/-MTX (MFD), CsA+MTX (MUD) or ex-vivo Tcell depletion (PMFD). BK virus DNA was detected in plasma and urine with PCR-based assay every second week from admission till discharge and then during each medical check-up. Results: BKV-DNA was detected in 32 patients (46%), in 9 transplanted from MFD (42.9%), 22 from MUD (64.7%) and 1 from PMFD (7.7%). In 6 patients BKV was detected in the urine before HCT. In 26 other patients first positive result was obtained from day +5 to +92 (median +22). BKV DNA was transiently present in peripheral blood of 9 patients together with BKV viruria. BKV viremia occurred exclusively among patients transplanted from alternative donors (MUD -8, PMFD -1). Cumulative incidence of HC in patients with both BKV viruria and viremia was 74% and 33% in patients with viruria only (p=0.05). Only one BKV-patient developed severe HC, in this child ADV etiology was proven. Median age of children with or without BKV viruria was 13.5 and 7.7 years and the difference was significant (p<0,01). Only 7 children (22%) finally eliminated BKV from urine after median time of 181 days. In the remaining patient BKV viruria persisted until the end of follow-up (range 5 to 384, median 60 days). Conclusion: The frequency of BKV infection was high, especially among patients transplanted from MUD. Surprisingly low incidence of BKV viruria among patients transplanted with T-cell depleted graft may be related to younger age of recipients. Strong correlation between BKV positivity and age may indicate mechanism of reactivation, not de novo infection. Detection of serum BKV DNA indicates the highest risk of HC occurrence.

High-dose chemotherapy followed by autologous peripheral blood stem cell transplantation in children with solid tumours: a paediatric hospital experience G. Deb, A. Landolfo, C. Ciscato, I. Ilari, R. Cozza, L. De Sio, P. Fidani, A. Jenkner, A. Castellano, G. M. Milano, F. Zinno, G. Isacchi, A. Donfrancesco, Ospedale Bambino Gesù (Rome, I) Objectives: in the last decade, high-dose chemotherapy with peripheral blood stem cell (PBSC) support has been increasingly used to treat metastatic or high-risk pediatric solid tumors. Patients and methods: From May 1989 to September 2003, 498 leukaphereses were performed in 258 pts (194 pts affected by solid tumors), with an average of 2 collections per pt, employing a Baxter CS-3000 Plus. A total of 143 transplants were done in 126 pts (neuroblastoma 36, CNS tumor 22, Ewing sarcoma 16, Wilms tumor 13, other sarcomas 9, PNET 9, retinoblastoma 4, rhabdoid CNS tumor 4, germ cell tumor 3, osteosarcoma 2, NHL 2, other histologies 6). Pt age ranged from 6 months to 19 years, weight from 6 to 73 kg, there were 70 boys and 56 girls. All pts received high dose chemotherapy. 11 pts underwent 2 transplants and 3 pts had 3 transplants. In 113 pts the treatment regimen at first transplant (ETC) consisted of etoposide (E) 600 mg/m 2 , thiotepa (T) 750 mg/m 2 and cyclophosphamide (C) 120 mg/kg. The remaining pts were treated with ET or CE or TLPam or LPam alone. 124 procedures were supported by peripheral blood stem cells, 12 by bone marrow and 7 by a combination of the two. To reduce the number of PBSC harvests, timing of the collection followed CD 34+ count monitoring after conventional chemotherapy and administration of growth factors. PBSC rescue was given 72 hours after completing -dose chemotherapy.

Results: There were no treatment-related deaths. Presently, 15/36 neuroblastomas, 7/10 sarcomas, 7/13 Wilms tumors, 7/13 Ewing sarcomas, 6/9 PNETs, 7/23 CNS tumors, 10/14 other tumors are in CR (47%) and 3/36 neuroblastomas, 1/2 osteosarcomas, 1/3 germ cell tumors and 5/23 CNS tumors are alive with disease. Conclusions: In our experience high-dose dose chemotherapy shows evidence of feasibility for the treatment of pediatric solid tumors, but we need a larger number of patients to demonstrate a survival advantage with this approach. Alternative strategies, possibly including antiangiogenic agents after PBSC transplant, are required.

A. Prete, R. Rondelli, F. Locatelli, S. Pierinelli, F. Fagioli, C. Messina, A. Garaventa, A. Pession, Unità di Terapia Cellulare (Bologna, Pavia, Turin, Padua, Genoa, I) Objective: A phase II not controlled trial to study rHuIL-2 low doses was performed with the aim to amplify natural immune mechanisms against cancer and control minimal residual disease in NB after autologous BMT. Patient and methods: From 1/92 to 12/98, 82 consecutive pts submitted to autologous BMT because affected by high risk NB were enrolled. 25 and 31 pts received rHuIL-2 (Proleukin, Aldesleuchina) respectively according to a ev (1) and a sc (2) schedule of administration, 26 refused or did not received IL-2 treatment for medical staff decision (3) . Treatment schedule 1 consisted of two cycles of 24-h iv for 5 d (2-4-6-8-8 MU/sqm/d respectively) followed by 11 monthly and 6 bimonthly cycles of rHuIL-2 administered sc for 5 d (2-4-4-4 MU/sqm/d), for a total of 18 cycles/pts. Treatment schedule 2 consisted of rHuIL-2 administered sc for 5 d (4 MU/sqm/d), twice a month for a total of 12 cycles/pts. Before IL-2 treatment, disease status of pts was 8 in CR, 10 in VGPR and 7 in PR (schedule 1); 10 in CR, 7 in VGPR and 14 in PR (schedule 2); 15 in CR, 2 in VGPR and 9 in PR (no treatment). Results: Overall pts received 317 (schedule 1) and 366 (schedule 2) cycles of IL-2. Iperpirexia and trombocytopenia were the only rHuIL-2 dependent toxicity observed during 1 or 2 treatment protocol. Immunological analysis evidenced: 1) increment of NK cells number and activity; 2) increment of activated T limphocyte cells number in both protocol. The 5 years DFS (IC 95%) is 28% (12-46), 41.7% (24-58) and 23.6% (15-43) respectively for the pts enrolled in schedule 1 and 2 of IL-2 treatment and for patient not trated with IL-2. Conclusion: Adoptive immunotherapy with low doses of rHIL-2 is feseable and seems to be effective in inducing activated immunocompetent cells proliferation. Moreover, schedule 2 of treatment seems to be more effective in controlling minimal residual disease in HR-NB after autologous BMT.

Chimerism after paediatric stem cell transplantation H. Bilgen, F. Aydin, K. Ozdilli, F. Savran, S. Anak, S. Ozgenc, M. Carin, G. Gedikoglu, Istanbul University, Golden Horn University, Our Children Leukemia Foundation, Ist. Med.Faculty Medical Biology (Istanbul, TR) Peripheral blood samples of 78(49 boys/ 29 girls) children with a mean age of 9.5 +4.02 were studied for chimerism status using fluorescence in situ hybridization(FISH) with an X and Y chromosome probe in children who recieved sex-mismatched allogeneic BMT and using PCR techniquewith VNTR gene polymorphisms in sex-matched allogeneic BMT.There were 49 children in FISH groupand 29 children in PCR group.There were no major differences in sex,age,underlying diseases,conditioning regimens in both groups.The mean duration of follow up was 76.71 + 6.2 months.Complete chimerism was seen in 47(60.3%) of the patients and 31 patients(39.7%) showed mixed chimerism.GvHD was found in 19 (24.4%) of the patients.The overall survival was 67.7% (5 years) in the study group.There was no significant difference in overall survival of the complete chimerism and the mixed chimerism groups(72% v68%).The relapse were seen in 14.9% of the patients of the complete chimerism group and 45.2% of the patients in the mixed chimerism status relapsed. The difference is significant (p=0.003).GvHD has an impact on relapse. Only 2 ( 10.5%) patients who had GvHD had relapsed and 90.5% of the patients who had relapsed had no GvHD.There was a ternd to meaningful difference (p= 0.06).The TBI was superior to other chemotherapy containing conditioning regimens in terms of obtaining complete chimerism( p=0.056) As a result, analysis of chimerism using FISH and PCR are important methods which give information about prognosis and allows for modification to prevent relapse.

High-dose busulfan and thiotepa followed by autologous stem cell transplantation in previously irradiated medulloblastoma patients: high toxicity and lack of efficacy D. Valteau-Couanet, B. Filippini, E. Benhamou, C. Kalifa, J. Grill, O. Hartmann, Institut Gustave Roussy (Villejuif, F) We have previously demonstrated that BU-THIO and ASCT was an effective treatment for patients with a medulloblastoma relapsing locally after surgery and conventional chemotherapy. This strategy allowed to cure 80% patients with only a posterior fossa irradiation. We thus evaluated the administration of BU-THIO in patients relapsing after a conventional CNS irradiation. Patients were planned to receive BU (600 mg/m 2 ) and THIO (900 mg/m 2 ) and ASCT in treatment of a medulloblastoma relapse. Surgery of a residue and additional irradiation was performed if necessary and feasible after BU-THIO. Toxicity was compared to that of 35 patients treated during the same period without previous CNS irradiation (55 Grays on posterior fossa, 35 Grays on CNS). From 05/88 to 03/02, 15 patients were treated in the pediatric department of IGR according to this strategy. Toxicity was significantly higher than that of non irradiated patients, for thrombocytopenia < 50000/mm3 duration 56 (13 -732) and 30 days (4 -124) respectively (p=0.02), HVOD (10/15 pts and 12/35 pts respectively) (p=0.06), neurological toxicity (8/15 pts versus 3/35 pts) (p=0.001) and toxic deaths (4/15 pts versus 3/35 pts)(p=0.01). Tumor response was evaluable in 7 patients (10 with evaluable tumor, 3 toxic deaths) and consisted in 2 CR, 3 PR and 2 NR. Presently 2 of 15 patients are alive with no evidence of disease.

In conclusion, toxicity of BU-THIO was significantly more severe in the previously irradiated patients. In spite of high response rate, this strategy failed to improve prognosis patients with a metastatic relapse of a previously irradiated medulloblastoma.

Combined chemo-immunotherapy in children with ALL who relapse after allogeneic stem cell transplantation -an option to induce long term remission P. Bader, E. Koscielniak, P.-G. Schlegel, A. Kors, A. Lankester, H. Kreyenberg, P. Lang, J. Greil, D. Niethammer, T. Klingebiel, University Hospital, Olga-Hospital (Tuebingen, Stuttgart, Wuerzburg, Frankfurt, D; Utrecht, Leiden, NL) Immunotherapy has only very limited success in the treatment of relapsed ALL patients. However, outcome could be significantly improved when hematological remission is achieved by prior chemotherapy. We developed a treatment approach combining low-dose chemotherapy elements with intermittently given lowdose DLI for remission induction and consolidation. Induction is with idarubicine (IDA), vincristine (VCR), prednisolone (PRED) and PEG asparaginase. Patients not in remission after 4 weeks receive intensified chemotherapy with a 2/3 reduced DAUNO-FLAG course. Following remission, DLI is given with 1x10 6 CD 3+ cells/kg in patients with MSD, 1x10 5 CD 3+ cells/kg in MUD and 5x10 4 CD 3+ cells/kg in patients with MMFD. Chemotherapy is continued with VP 16, VCR, DEXA, mercaptopurine (PN), and MTX over a period of 2 weeks. Patients in remission receive further DLI followed by 4 weeks reintensification with VP 16, VCR, DEXA, PN and MTX. Patients in remission receive then further DLI followed by maintenance therapy with PN and MTX for 2 weeks. This course is repeated until the patients achieve either MRD negativity or relapse again. So far, 11 children (CR1, n=3; CR2, n=7; CR3, n=1) who received their transplants either from a MSD (n=3), MUD (=5) or from a MMFD (n=3) were treated. Time from transplant to relapse ranged from 62 days to 610 days (mean 270 days). In 7/11 patients CR could be achieved after four weeks induction chemotherapy, in 2/11 after an additional Flag-course and 2/11 patients did not respond to treatment. Induction chemotherapy was well tolerated in 6/9 responding patients and treatment was solely performed in the outpatient clinic. In 2/9 patients admittance to ICU was necessary because of severe infections and 1/9 patients has to be admitted because of hypoalbuminemia. No GVHD occurred. 10/11 patients are alive with a medium follow-up of 377 days (range 1614 days to 85 days). 7 of these 10 patients are in CR and 3 alive with persistent disease. 3/7 patients in hematological CR achieved MRD negativity after 76, 418, and 955 days respectively. 4/7 patients were still on treatment with measurable MRD from 1x10 -2 to 1x1E -4 . In summary, long term remission can be achieved by combined low-dose chemotherapy given intermittently with low-dose immunotherapy with moderate toxicity. This treatment strategy warrants further evaluation and optimization.

Cyclosporine-A induced severe neurological events after allogeneic haematopoietic stem cell transplantation in children D. Wojcik, M. Slociak, J. Toporski, E. Gorczynska, D. Turkiewicz, K. Kalwak, A. Chybicka, Wroclaw Medical University (Wroclaw, PL) Cyclosporine A (CsA), calcineurin inhibitor is a potent immunosuppressive agent commonly used after bone marrow and organ transplantation. Severe neurological event (SNE) of CsA is a known but rare side effect. This life-threatening condition is often heralded by prodromal symptoms, which, when properly identified, can allow treatment to be initiated. The aim of the study was to document the incidence, presentation, severity and outcome of CsA-induced SNE in children after alloHSCT. From January 1994 to November 2003 107 children in our center underwent alloHSCT (65-from HLA identical family donor, 42 from matched unrelated donor). GvHD prophylaxis consisted of CsA±MTX in all cases. Children who underwent MUD transplantation received ATG. CsA was introduced on day -1 before grafting at the initial daily dose of 3mg/kg as a continuous iv infusion, then two hour iv infusion twice daily, then orally twice daily. The expected blood CsA level ranged from 100 to 200 ng/ml and was evaluated three times a week. Six children (5,6%) -4 girls and 2 boys, age: 2-16 y, transplanted for AML (2) , ALL (2), BDA (1), Fanconi anemia (1) developed SNE. In one patient we observed the recurrence of SNE while reinstituted CsA. Five children experienced SNE after MUD transplantation (11,9%). One child out of 65 (1,5%) after the transplantation from HLA identical family donor developed SNE. All patients were free of neurological symptoms prior to the transplant. Onset of SNE varied from day +8 to +120 after alloHSCT. Three patients after MUD transplant presented grade II GvHD and were on steroids at the onset of SNE. All children developed prodromal symptoms such as headache, nausea, visual disturbances, abdominal pain followed by cortical blindness, generalized convulsions, unconsciousness and hypertension. In 2 pts mechanical ventilation was necessary. The CsA level measured in the whole blood at the onset of SNE ranged from 115 to >800 ng/ml. Other laboratory results were unspecific. CT-scans revealed unspecific pathological changes in CNS. In all pts neurological symptoms were unpredictable but reversible up to 24 hours. CsA treatment was ceased in 1 patient. The pathogenesis of CsA neurotoxicity remains unclear. Our findings document SNE in children due to CsA induced toxicity. Transplantation from MUD and concomitant use of steroids seems to be a risk factor for SNE. Its occurrence, causes and consequences should be assessed within a large, prospective study.

Cord blood -a valid option for allogeneic transplantation in a heterogeneous risk population of 15 children lacking an adequately matched donor S. Schönberger, T. Niehues, H.J. Laws, R. Meisel, G. Kögler, P. Wernet, U. Göbel, D. Dilloo, University Hospital (Duesseldorf, D) In the absence of an adequately matched related or unrelated donor, cordblood (CB) may be employed as an alternate hematopoietic stem cell (HSC) source for allogeneic transplantation. As the risk for graft-versus-host-disease (GVHD) is considerably lower in transplantation of CB compared to HSC from other sources, requirements for HLA-matching are less stringent. Moreover in contrast to CBT in adults, the potentially small cell dose is of lesser concern in children. Here we report on our single center experience of 15 CBT in pediatric patients (pts). 13 of the 15 pts suffered from malignant hematological diseases (8 acute leukemias, 2PR+6CR;2 chronic leukemias;2 secondary myelodysplastic syndromes,1 lymphoma in PR) while 2 pts were transplanted for thalassemia and aplastic anemia. Age at transplantation was 5,3; 0,3-22 (median; range) years (yrs). 5 pts received a CB with =/> 5/6 HLA-match (HLA-A,B,DR) from a related donor. The other 10 pts were transplanted with CB from an unrelated donor with an HLA-match of =/> 4/6 in 7 pts and 3/6 in 3 pts. Number of infused nucleated cells (INC) was 3,1x10 7 ; 0,9-8,5x10 7 /kg bodyweight (bw). Follow-up post transplant was 5,6; 0,2-7,5 yrs. Neutrophils engrafted (ANC>500/ul) at day 20; 11-35 post CBT. Among the patients that survived and retained their graft, the time to platelet engraftment (> 20.000/ul) was 45; 31-81 days. In none of the 6 pts that died, platelets had recovered before the time of death in spite of neutrophil engraftment in 4 pts. Primary graft failure occurred in 3 pts. All these pts were transplanted with unrelated CB and INC was <1x 10 7 /kg bw in 2 cases. Autologous reconstitution was observed in 2 pts with 1 pt still being alive and well. The 3rd pt survived after re-transplantation with unrelated bone marrow. 8/12 pts (67%) developed acute GVHD and 3/12 pts (25%) suffered from severe GVHD III°/IV°. Among those surviving beyond day 100, only 1/11 pts was affected with chronic GVHD. 5/15 pts (27%) died of transplantant-related complications including bacterial infection in 3 pts, toxoplasma gondii infection and organ toxicity each in 1 pt. 2 died of GVHD III°/IV° associated with severe viral infections. Of note only 3/13 pts with malignant diseases suffered a relapse yet only 1 pt died. With an overall survival of 58% in our 15 pts this analysis documents that even in high risk pts, CB may be a valid choice for allogeneic HSCT in children in the absence of a suitable donor.

Rhesus mismatch in paediatric allogeneic haemopoietic progenitors transplants: a retrospective study on its influence on outcome R. Ridwan, M. Ortin, S. Patel, J. Shankari, P. Fomasagaram, S. Ball, J. Marsh, J. Treleaven, The Royal Marsden Hospital, St George's Hospital Medical School (Sutton, London, UK) Recent reports suggest Rhesus (Rh) mismatch does not have any influence on the outcome of allogeneic HPT, in terms of GvHD or GvL effect, in adults. The aim of the present study is to assess whether Rh mismatch has any influence on the long term outcome of children receiving HPT, where GvHD and GvL effect are known to be less intense than in adults. We have conducted a retrospective study where all the allogeneic paediatric HPT performed at our centre between 1996 and 2003 (n=108, median age 11 ± 4.6 years) were included. 60 patients received grafts from unrelated donors (UD) and 48 from related donors (RD). Indications for HPT included ALL (75 cases), AML (12), MDS (6), CML (10), undifferentiated (3), Lymphoblastic NHL (1), HLH (1). Data on Rh mismatch were available in 81 cases, of which 14 were mismatched. The orientation of the alloimmune reaction was in donor direction (9 cases) and recipient direction (5). Study endpoints were acute and chronic GvHD (aGVHD, cGvHD), final outcome (alive/dead) and cause of death (relapse/toxic). Data on the development of aGvHD were available in 77 cases. Patients with Rh mismatch had a significantly (p<0.02) higher incidence of aGvHD and also more severe forms of the disease (p<0.03). Data on cGvHD were available in 74 cases. There was a significantly (p<0.005) higher incidence of cGvHD in Rh mismatched pairs, and the disease was significantly more severe (p<0.007). Treatment for aGvHD and cGvHD was more often necessary in cases of mismatch (p<0.04 and p<0.005, respectively). Rh mismatch did not significantly influence survival or the cause of death. Cases where the alloreactivity was orientated in the recipient direction experienced a higher incidence of aGvHD (p<0.05) and cGvHD (p<0.004). These cases also required treatment for cGvHD more often (p<0.006). Children receiving RD HPT from Rh mismatched donors had a significantly higher incidence of cGvHD (p<0.006), but no relation with aGvHD was found. When the orientation of the mismatch was against donor, the incidence of cGvHD was also higher (p<0.02). In cases of UD HPT, a relation with both aGvHD (p<0.03) and cGvHD (p<0.03) was found only in cases with orientation against recipient. Overall, Rh mismatch seems associated with a higher incidence of both acute and, specially, chronic GvHD. The reason for the lower tendency seen UD HPT might be the overall higher incidence of GvHD existing in these. This mechanism should be studied in prospective studies. advantage in haploidentical hematopoietic stem cells transplants for hematological malignancies (Ruggeri,Science 2002). Thus we have looked for the influence of this parameter on the outcome of haploidentical transplantation in 46 children with inherited disorders.In these indications, due to the young age of the patients and their clinical status at the time of transplantation, conditionning regimen is milder than used in leukemia patients leading to a higher incidence of graft rejection. In this context, the potential benefit of KIR ligand incompatibility would be of great impact.Therefore we have chosen to analyse only the first transplantation and to not take in account overall survival for these patients. Among these patients 41 were transplanted for primary immunodeficiencies,1 for metabolic disease and 4 for non malignant hematopoietic diseases.Median age at time of transplant was 21 months .Conditionning regimen consisted of Busulfan (20 or 16 mg/kg total dose respectively for children aged of less or more than 6 years ) and Cyclophosphamide (200mg/kg total dose). Rejection prevention included anti-LFA1 or anti CD2 monoclonal antibody or rabbit anti-thymocyte globulin. GvH prophylaxis was only done by transplant T-cell depletion using two different methods: CD2 and CD7 complement dependent lysis and CD 34 selection patients were divided into those with (n=16)and without (n=28) KIR ligand incompatibility with their donors. Two out of 46 patients died early after transplant. Of the remaining 44 ,18/28 (64%) and 10/16 (62%) engrafted respectively in compatible and incompatible KIR ligand groups,without any difference on the incidence of aGvHd superior to grade II. In conclusion: in this little serie of patients with inherited diseases receiving only a chemotherapy -based conditionning regimen before T cell depleted haplo-identical transplant, we do not find any advantage of KIR ligand incompatibility on the prevention of two major transplant complications (rejection and aGvHd ). We can not exclude the hypothesis that non myeloablative conditionning regimen associated with ex-vivoT-cell depletion mask the potential positive effect of KIR ligand incompatibilty. To rule out the possibility of unexpexted KIR ligand expression related to ethnical origin in our serie of patients,we are currently performing KIR ligand genotyping.

A preparatory regimen of high-dose busulphan and melphalan for autologous haematopoietic cell transplantation in childhood high-risk acute myeloblastic leukaemia, neuroblastoma and Ewing sarcoma E. Gorczynska, D. Turkiewicz, J. Toporski, K. Kalwak, A. Dyla, A. Chybicka, Wroclaw Medical University (Wroclaw, PL) The aim of the study was to evaluate retrospectively efficacy and tolerance of Busulfan (BU) Melfalan (MEL) conditioning for autologous HCT in children. Patients: BU and MEL followed by autologous hematopoietic cell transplantation (autoHCT) was applied between May 1999 and October 2003 in 56 children, 29 girls and 27 boys aged from 2 to 18 years (median 7,8). Diagnoses included: High-risk AML (26) in 1st CR (21) or beyond 1st CR (5), and solid tumors ( NBL 24, Ewing/PNET 6) in 1st CR (14) or beyond (16). Median follow-up reached 17 months (from 2 months to 5.5 yrs). Methods: BU was given orally from day -7 to -3 at a total dose of 16 mg/kg. MEL (short iv infusion) at a dose of 140mg/m 2 on day -2. Peripheral blood hematopoietic progenitors (PBPC) were transplanted in 23 out of 27 AML patients and 17 out of 30 children with solid tumors (ST). Remaining patients were grafted with bone marrow (BM) or combination of BM and PBPC due to insufficient mobilization. Median number of transplanted CD34+ cells/kg bw reached 2,8x10 6 and ranged from 0.5x10 6 to 45x10 6 . Regular assessment of AT-3 activity and supplementation to maintain activity above 80% was used as VOD prophylaxis. Results: All children recovered in granulocytes with median time to ANC>500/mm 3 of 13 days (range 10 to 112). All children with AML engrafted with platelets (PLT > 50000/mm 3 ) from day +12 to +481 (median 48). In 6 solid tumor patients no PLT engraftment was observed until relapse or death (day from +71 to +401), in remaining 23 children median time to PLT engraftment was 39 days (from 14 to 213). All pts experienced mucositis requiring TPN. Severe complications were as follows: infections (9), VOD (5, severe -2), AIHA+TTP (1), severe coagulopathy (1), late hemorrhagic cystitis BKV+ (1). In AML group, 23 children are alive in CCR (88,5%), 1 in CR after MUD BMT due to sec. NHL, 1 in 3rd CR after 2nd autoloHCT and 1 is alive in 2nd CR and awaits MUD BMT. In ST group 12 children are alive in CR (40%), 3 alive in relapse (10%) and 15 died -13 due to progression (43.4%), one -VOD, one severe coagulopathy (TRM 6.6%). Three-year EFS for AML and ST group is 0.8 and 0.2, respectively. Conclusions: In children with AML autoHCT with BU MEL conditioning is effective with limited toxicity. Efficacy of this approach in children with NBL/Ewing sarcoma is still uncertain. In our hands BU MEL resulted in lower than expected occurrence of VOD, possibly due to strict AT3 monitoring and supplementation.

either. Maintaining higher CsA levels in the first month after transplant appears to protect against aGvHD (275.9 ± 100.5 vs 229.6 ± 72.4; p<0.05).

Overall, it appears that maintaining high CsA levels within the first month after graft protects against aGvHD and later reactivation of CMV, probably by allowing a rapid engraftment and a prompt immune recovery. From the second to the sixth months, levels should be strictly kept within therapeutic range in order to avoid other viral infections. CsA levels do not seem to condition the cause of death and it would appear that maintaining low levels does not seem to promote GvL effect.

Analysis of KIR-ligand incompatibility in children with haematological malignancies after allogeneic haematopoietic cell transplantation from unrelated donors D. Turkiewicz, E. Gorczynska, J. Toporski, K. Kalwak, A. Chybicka, Wroclaw Medical University (Wroclaw, PL) NK cell alloreactivity in allogeneic hematopoietic cell transplantation (alloHCT) from killer immunoglobulin-like receptor (KIR) ligand incompatible in graft-vs-host (GvH) direction haploidentical donors is proposed to have protective effect against disease recurrence, graft vs. host disease (GvHD) and graft rejection. However, analysis of published data on transplantation from KIR ligand incompatible unrelated donors (URD) is still inconclusive. Objectives: We present a retrospective analysis of the impact of KIR ligand incompatibility on the incidence of acute GvHD and relapse in children with hematological malignancies after alloHCT from URD. Patients: Analysis of KIR repertoire was possible in 32 children after alloHCT from URD, 16 females and 16 males aged from 3 to 18 years (median 14 yrs). Indications for alloHCT included: ALL (15), AML (6), CGL (6), MDS (4), NHL (1) . Median follow-up reached 77 days (range from 15 to 1176). METHODS: Donors and recipients class I (A, B, C) and class II (DRB1, DQB1) HLA loci were identified with high resolution typing techniques. Sixteen patients were transplanted with bone marrow, 16 received peripheral blood hematopoietic cells. All children received myeloablative conditioning based on Busulfan (26), TBI (4) or Treosulfan (2) with anti-thymocyte globulin (ATG, from day -4 to -1). All children received in conditioning (day -4 to -1). GvHD prophylaxis consisted of CsA+MTX in all patients. KIR ligand compatibility was estimated according to algorithm proposed by Velardi et al. -patient lacking KIR ligand recognized by one of donor's KIR was classified as being incompatible in GvH direction. Results: KIR ligand incompatibility in GvH direction was observed in 5 out of 32 children (15.5%). Two of them presented grade I or II acute GvHD limited to skin. One child from this group died on day +55 due to infectious complications, one patient with CGL relapsed on day +384, 3 are alive with no evidence of disease 66, 70 and 215 days after HCT. Among 27 children with no KIR incompatibility in GvH direction 1 graft rejection, 3 relapses and 13 treatment-related deaths were noted. Cumulative aGvHD incidence was 48% and GvHD was the cause of death in two cases. Conclusions: KIR ligand incompatibility between donor and recipient did not prevent relapse in a child with CML. It seems, however, to have some protective effect against severe grade III and IV aGvHD.

Outcome of haematopoietic stem cell transplantation for refractory or relapsed acute leukaemia in paediatric patients: a single-centre analysis in Japan T. Tanabe, Y. Kawano, Y. Nagatoshi, J. Nagayama, Y. Shinkoda, O. Ijichi, J. Okamura, Kagoshima University, National Kyushu Cancer Center (Kagoshima, Fukuoka, J) Background: Due to the diversity of possible stem cell sources, including unrelated bone marrow and cord blood, hematopoietic stem cell transplantation (HSCT) can now be applied to almost all pediatric patients regardless of their disease condition. Although it is well known that the survival rate in patients who do not achieve complete remission (CR) is quite low, even after HSCT, we are often obliged to perform HSCT in such patients. To evaluate the usefulness of HSCT in the treatment of intractable acute leukemia, we analyzed the outcome of HSCT in patients with refractory or relapsed acute leukemia as singlecenter study. Patients and methods: We treated 28 pediatric patients with acute leukemia between 1991 and 2001 at the Section of Pediatrics of National Kyushu Cancer Center. The patients (15 males and 13 females) ranged in age from 6mo to 15 y. None were in CR. Twenty-three of the 28 were in a refractory state and five were in partial remission that had just been induced by intensive chemotherapy. The pre-transplant regimen included TBI in 24 patients and did not include TBI in the remaining 4. The stem cell sources were bone marrow in 24 patients (15 related, 9 unrelated), G-CSF-mobilized blood stem cells in two (related), and cord blood in two (unrelated). GVHD prophylaxis consisted of a single prophylaxis with cyclosporine A (CSA) or MTX in 9 patients, CSA + sMTX in 12, and tacrolimus + sMTX in 7. Results: Transplant-related mortality was 29% (8 of 28). A total of 13 patients were alive in CR for a median 620 days (range, 252-2980) , and the estimated relapse-free survival rate (RFS) was 31.2% at 3 years after HCST. Acute GVHD (grades II-IV) and chronic GVHD developed in 11 and 12 patients, respectively. There was no relationship between the occurrence of GVHD and the outcome. Four of 5 patients who were transplanted from mismatched donors (>2-loci), died in the early stage after HSCT. Conclusion: These results suggest that HSCT may be suitable for pediatric patients, even those with refractory or relapsed acute leukemia, if they have a suitable donor.

Differential analysis of cell-line specific chimerism after blood stem cell transplantation U. Koehl, O. Beck, D. Schwabe, E. Seifried, T. Klingebiel, C. Seidl, JW Goethe University, Institute of Transfusion Medicine (Frankfurt, D) Determination of donor host chimerism in PB or BM by microsattelite (STR) marker provides relevant clinical information about engraftment of donor cells after allogeneic stem cell transplantation. Lineage-specific chimerism in highly purified leukocyte subsets seems to be even more informative for detection of graft failure or relapse of the disease. We compared a singleplex in-house STR set with two commercially available multiplex STR systems (Profiler and Cofiler, ABI, Germany) in pediatric patients, two suffered from ALL, one from thalassemia and one from aplastic anemia. Chimerism was detected routinely in PB, BM and CD3 selected T-cells. In some cases the cell populations investigated included granulocytes, monocytes, Bcells, stem cells, NK cells and T-cell subsets (CD4+ and CD8+) (all purified using microbeads, AutoMacs, Myltenyi Biotec, Germany). Based on the in-house STR system only, calculation of chimerism resulted in 3-9 informative STRs per patient. Standard deviation was < 4.2% in PB (n=297), <5.5% in BM (n=46) and 6.9% (n=39) in the purified leukocyte subsets CD3+, groups. Graft failure was seen only in two patients in ATG group (p= 0,492). Incidence of CMV, bacterial and fungal infection was not different between two groups during the first transplantation year. We were able to show that ATG leads to a reduction in severe acute and chronic GvHD in comparison to patients transplanted without ATG althought there was no satistical difference. The neutrophil engraftment time may delay but, ATG did not cause an increase in CMV, bacterial and fungal infection and graft failure incidence. Our data sugget that ATG is safe and has possible positive effects as a part of the conditioning regimen in pediatric patients who have undergone allogeneic PBSCT from HLA identical donors. However, longer follow-up and larger study groups are necessary to determine maximum efficient and minimum side effect doses of ATG.

H.S. Ahn, H.J. Kang, H.S. Choi, H.Y. Shin, Seoul National University, National Cancer Center (Seoul, Goyang-si, KOR) Positive selection of CD34+ cells has been used to reduce tumor cell contamination of autograft in malignant disease. In this study, we have investigated the feasibility and efficacy of CD34+ cell transplantation in neuroblastoma. Total 19 high risk or relapsed neuroblastoma patients were enrolled. After confirming no tumor cells in bone marrow, autologous peripheral blood stem cell were mobilized with chemotherapy and G-CSF. Pheresis products underwent CD34+ selection using CliniMACS (Miltenyi Biotec) device and stored at -196'C until reinfusion. Neuroblstoma contamination of selected product was measured using real-time quantitative RT-PCR detecting tyrosinehydroxylase mRNA in some patients. CD34+ cell recovery rate and purity were median 79% (range 19-120%) and 94% (range 90.5-98%), respectively. A median of 5.7x10 6 /kg (range 1.3-36.7x10 6 /kg) CD34+ cells were infused. Median days to recover neutrophils more than 500/uL and 1,000/uL were 11 (range 8-20) and 12 (range 9-23), respectively. Median days to achieve platelet more than 50,000/uL and 100,000/uL without transfusion were 27 (range 9-180), 79 (range 10-112), respectively. The SR and EFS were 64.7% and 42.5%, respectively. Nine out of 19 patients were relapsed 8-12 months after transplantation and remained 10 patients are alive without disease. Transplantation related toxicity was tolerable and there was no TRM. The mean virtual neuroblastoma contamination value in CD34+ selected product of relapsed patients (132.7, n=5) was higher than nonrelapsed (0.1, n=6). These results show the successful engraftment without fatal transplantation-related toxicity of the CD34+ cell selective PBSCT in the neuroblastoma. Despite the CD34+ selection, high level of tumor contamination was detected in some relapsed patients, which should be proved the prognostic value in future.

Low-dose imatinib may convert the recipient with overt haematologic relapse of Ph+ CML after allogeneic MUD-BMT to complete donor chimera and induce haematologic and cytogenetic remission K. Kalwak, D. Turkiewicz, E. Gorczyñska, J. Toporski, D. Wojcik, M. Ussowicz, A. Chybicka, Wroclaw Medical University (Wroclaw, PL) Imatinib is able to induce mixed chimerism without induction of severe GvHD in patients with Ph+ CML relapsing after allogeneic hematopoietic cell transplantation. There is, however one report describing a hematologic and cytogenetic remission by imatinib in an adult patient relapsing with accelerated phase CML after second allogeneic stem cell transplant. Here we report a case of 5-year old boy with adult type CML, who relapsed after allogeneic matched unrelated donor (MUD) BMT and was given low-dose imatinib for 4 months only.

Three-year old boy with Ph+ CML in its first unstable chronic phase underwent allogeneic MUD-BMT in February 2002. The posttransplant period was uneventful without any signs of GvHD. CsA was withdrawn early on day +55 after BMT. The patient remained complete donor chimera until March 2003, when a hematologic relapse of CML was diagnosed. At that time his WBC was 19.9 x 10(9)/L and BM showed typical signs of CML chronic phase. STR-PCR analysis of hematopoietic chimerism revealed 74% and 9% autologous cells in BM and PB, respectively. BCR-ABL was positive in both first and second round PCR. Imatinib therapy (280 mg/m(2)) resulted in rapid elimination of autologous cells in PB (100% donor cells) and a significant decrease of autologous content in BM (10%) after 6 weeks of treatment. Further administration of imatinib (70 -140 mg/m(2)) induced a rapid sustained conversion to complete donor chimera both in PB and BM only after less than 4 months of therapy. Moreover, both hematologic and cytogenetic remissions were achieved and BCR-ABL became negative in first round PCR. No signs of GvHD were observed and the therapy course was uneventful. Low-dose imatinib may convert the recipient with overt hematologic posttransplant relapse of CML to complete donor chimera. To our knowledge, this is the first case of rapid hematologic and cytogenetic remission by imatinib in a very young child with CML relapse after MUD-BMT.

Dealing with the double role -experiences of parents donating haploidentical peripheral blood stem cells. A phenomenological study A. van Walraven, L. Ball, H. Koopman, R. Bredius, C. Ropes-de Jong, A. de Jong, M. Egeler, Europdonor Foundation, LUMC, Hogeschool van Utrecht (Leiden, Utrecht, NL) Hematopoietic stem cell transplantation is an effective therapy for life threatening haematological diseases. A related or unrelated volunteer usually donates stem cells. For those lacking such a donor, haplo identical PBSCT is a feasible alternative. Parents donating stem cells simultaneously and uniquely fulfil a double role. Experiences of siblings and unrelated stem-cell donors have been extensively reported but not those of parental donors. In the pediatric transplant unit, LUMC, Leiden in the study period 1997 to 2002, 23 haplo-identical PBSCT's were performed in 18 children. Fifteen eligible parents were invited to participate in the study. Thirteen parents agreed to participate. Experiences of parents were investigated using hermeneutic phenomenological principles. Data was collected through transcribed in-depth interviews conducted within the participants' home. Six mothers and 7 fathers of 11 patients (2 families where both parents had donated and one father whom had donated twice) were included. The median age of parents at donation was 38 yr (range 29-48). The median time between donation and the interview was 2.7 years (range 0.4-5.0). At the time of interview, 5 recipients were alive and well, 6 died either from infection (n=1), graft failure (n=2) or relapse (n=3). The description of the experiences and feelings were comparable to both parents of seriously ill children as well as stem cell donors, as described in current literature. There were no important differences found between maternal and paternal experiences. Central themes for all participants were 'Hope and Fear, Needs for Information', 'Do Anything for Your Child', and 'Transplant Outcome'. Hope and fear were often experienced simultaneously, leading to turmoil and confusion during all stages of the transplantation process. The need for information in relation to all aspects of transplantation and donation varied and the perception of information given was distorted because of the emotional difficulties. Parents would "do anything" for their child but often had feelings of 'powerlessness' or 'having no choice'. The phenomenon of "double role" was not strongly perceived by the parents, but their experiences were similar to both roles as described in current literature. Although haplo identical transplantation was the only chance for cure, parents considered the role of being a donor of minor importance.

B-cell recovery in a SCID patient after a second BMT following minimal myelosuppressive conditioning P. Hoogerbrugge, P. Brons, M. van der Burg, J. van Dongen, C. Weemaes, University Medical Center St. Radboud, Erasmus MC (Nijmegen, Rotterdam, NL) Patients with B negative T negative severe combined immune deficiency (B-T-SCID) generally are not conditioned prior to transplantation with marrow from an HLA-identical sibling donor. B-cell reconstitution is frequently absent following transplantation with HLA-identical marrow in B-T-SCID patients (Antoine et al. Lancet 2003; 361: 553 ) . The absence of adequate myelosuppressive conditioning prior to BMT may be a possible explanation for this lack of B-cell engraftment, therefore we performed 'minimal myelosuppressive' conditioning for a retransplantation in a child who lacked B-cell engraftment after a prior HLA-identical BMT. Casus: A female patient presented with SCID at 5 months of age. Detailed molecular analysis showed a mutation in the Artemis gene. Maternal T-cell engraftment was present. At 7 months of age, the patient received non-T-cell depleted marrow from her HLA-identical brother. After an uneventful post transplantation course, a gradual rise in T-cells of donor origin occurred, B-cell engraftment was absent and the patient still was dependent on immunoglobulin substitution. Therefore, she received a second transplant with marrow from the same donor at the age of 38 months. Conditioning consisted of busulphan 2mg/kg at days -3 and -2. This minimal conditioning did not result in neutropenia, anemia or thrombocytopenia. At 8 months after the 2nd transplant, B-lymphocytes were present in normal amounts in peripheral blood, and a bone marrow analysis revealed 32% cells of the B-lineage (CD22+) in the lymphocyte gate (pre BMT: <3%). Chimerism studies confirmed donor-origin of these cells. Mixed chimerism was present in the granulocytes, NK-cells were still of recipient origin. Immunoglobulin levels normalised, and the patient responded normal to vaccination. Conclusion: Normal B-cell function could be achieved after HLAidentical BMT following minimal myelosuppressive conditioning in a patient with B-T-SCID due to an Artemis mutation. There is no established treatment for refractory Langerhans cell histiocytosis (LCH). Allogeneic bone marrow transplantation (BMT) may be used as a salvage treatment, although transplant related mortality (TRM)is high for this patients (Akkari 2003 , Kinugava 1999 . Therefore, to reduce the TRM we decided to use treosulfan-based preparative regimen for allo-BMT in 2 children with refractory LCH, i.e. in 4 years old boy (UPN 112) and 18 months old girl (UPN 137). Both patients underwent BMT from matched sibling donors, 24 (UPN 112)and 5 months (UPN 137) after diagnosis. They were prepared for BMT with intravenous treosulfan 10 g/m 2 for 3 consecutive days in combination with etoposide 30 mg/kg and cyclophosphamide 2 x 60 mg/kg. The number of CD34 cells infused per kg of body weight was 8,8 x 10 6 for the boy, and 25 x 10 6 for the girl. GvHD prevention consisted of CsA and additionally in patient UPN 137 of "short" MTX. For monitoring of hematopoietic chimerism an automated DNA sizing technology have been applicated. There was not any regimen related toxicity. Both patients achieved engraftment (ANC>0,5 x 10 9 /L within 15 (UPN 112) and 18 (UPN 137)days, platelets>20 x 10 9 /L within 13-16 days respectively). The results of quantitative assessment of whole blood chimerism in both patients demonstrated an increasing recipient chimerism; from complete donor,(day +20) up to complete recipient genotype (day +730) in patient UPN 112; and from 20-30% (day +26) to 80-90% (day +351) in patient UPN 137 (Tab. 1). To reverse declining donor chimerism, donor lymphocyte infusion was administered twice (1,2 x 10 7 CD3 /kg) for the girl (UPN 137) (Tab. 1). Despite of mixed chimerism children remain in continuous complete clinical remission 31 (UPN 112) and 12 (UPN 137) months. Conclusion: In reported two children with refractory LCH the treosulfan-based regimen demonstrated no significant toxicity and myeloablative effect sufficient for development of immunotherapeutic effect of allo-BMT.

C. Castellini, A. Prete, F. Melchionda, M. Franzoni, R. Rondelli, A. Pession, University of Bologna (Bologna, I) Objectives: With the aim to overwork the antineoplastic effect associated with GvT, even in solid tumours, while improving safety of HSCT, reducing preparative regimens intensity, inducing a tolerance between donor and recipient immune system creating a mixed chimerism (MC) post-transplant state and optimising GvT effect by speeding up donor immune recovery, non myeloablative stem cell transplant (NST) was developed. Patients and methods: 2 patients, one affected by relapsed neuroblastoma (NB) 18 months after autologous stem cell transplantation, the other by refractory NB, underwent NST from HLA-identical sibling donor. Preparative regimen included Thiotepa (10 mg/kg; day -3) and Melphalan (120 mg/m 2 ; day -2). Source of HSC was bone marrow in both cases, as donors were <18 year old; number of reinfused CD34+ cells was 4,45 and 4,8 x 10 8 /kg of body weight, respectively. GvHD prophylaxis was actuated by Ciclosporine A , from day -1, at the dose of 2 mg/kg/day. HSC engraftment was evaluated by STR technique on peripheral blood samples. Immunological response was studied by evaluation of cytokines (IL-6, IL-8, IL-2 receptor and TNF) and lymphocitic subpopulations (LSP). Results: white blood cells (WBC) nadir was achieved on day +7 and +6, with a WBC count of 70 e 20/mm3 respectively. Preparative regimen determined a low grade acute toxicity. In both cases, reinfused HSC engrafted, on day +13 and +12. Engraftment evaluation at day +15 showed a MC in the first patient and a complete chimerism (CC) in the second; both patients showed CC on day +30 and +60. The maximum grade of acute GvHD was II, appeared 1 day before engraftment in both cases, and was treated with corticosteroid therapy (2 mg/kg/day). After a follow up of 4 and 12 months, both patients are alive and well, the first with a very good partial response (as documented by radiological and nuclear examinations) and the other in complete remission. In both patients we observed a progressive increasing of tested cytokines and expansion of LSP, in concomitance with a progressive reduction of catchment areas at MIBG scintigraphy. Conclusions: as refractory or relapsed NB is a poor prognosis pathology, often affecting patients in declined clinical conditions, NST showed to be tolerable and safe, efficacious in terms of allogeneic HSC engraftment. However, further studies and a longer follow-up are requested to evaluate NST antineoplastic, efficacy, principally mediated by GvT effect.

Sequential reduced intensity and full intensity allografting using same donor in child with chronic granulomatous disease with coexistent, significant morbidity J. A.T. Nicholson, R. Wynn, T.F. Carr, A.M Will, Royal Manchester Children's Hospital (Manchester, UK) Although reduced intensity conditioning (RIC) allogeneic BMT allows subjects with significant co-existent medical problems to undergo BMT, its utility in children with non-immunodeficiency genetic conditions is limited by a high rate of graft rejection. This rejection is often late and follows a period of successful engraftment. We report a 12 years old boy with X-linked Chronic Granulomatous Disease (CGD). His associated colitis was complicated by toxic megacolon and a high output fistula. He had required prolonged hospital admission to support his nutrition and for antibiotic therapy. He underwent a reduced intensity conditioning unrelated donor transplant using Fludarabine (150mgs/m2), Campath (1mg/kg) and Melphalan (140mgs/m 2 ) with prompt granulocyte recovery and achieved full donor chimerism. His neutrophils were >0.5 by day 10, and his platelet requirement was minimal. The transplant convalescence was complicated by CMV reactivation, managed with ganciclovir. The engrafting donor neutrophils were able to reduce nitroblue tetrazolium (NBT) (demonstrated on flow cytometry) and functionally were able to heal the fistula. At 5 months following transplant he developed (allo-) immune mediated hemolytic anaemia, thrombocytopenia and neutropenia. Chimerism fell from full donor to all recipient, despite discontinuation of the cyclosporine graft versus host disease prophylaxis (GVHD). He remained in a far better physical condition than prior to his first transplant. He therefore underwent a full intensity second BMT, after conditioning with Cyclophosphamide (120mgs/kg) and Total Body Irradiation and Campath. GVHD prophylaxis was with Cyclosporine and short course Methotrexate. Again full donor cell engraftment with functional neutrophils occurred with neutrophils >0.5 on day 14, and transfusion independence. There has been minimal GVHD and the transplant has been complicated only by CMV reactivation. This case illustrates both the utility and limitation of reduced intensity conditioning in paediatric non-immunodeficient, genetic disease transplant. Pre-transplant counseling in such circumstances should include the possibility of repeat transplant at a later date, rather than risking full ablative conditioning in a sick individual. Manipulation of chimerism with donor T cell infusion might be employed to strengthen donor engraftment after the first RIC BMT, although the graft may be very rapidly lost (and through an antibody mediated mechanism).

Autologous peripheral blood stem cell transplantation with BCVAC conditioning in childhood acute myeloid leukaemia H. J. Kang, H. Y. Shin, H. S. Choi, K. S. Han, H. S. Ahn, National Cancer Center, Seoul National University Hospital (Goyang-si, Gyeonggi-do, Seoul, KOR) Autologous peripheral blood stem cell transplantation (APBSCT) after intensifying conditioning is one of the post-remission therapeutic options in childhood AML patients without a matched family donor, but the optimal conditioning regimen has not been defined. This study was performed to evaluate the efficacy of a novel conditioning regimen without busulfan or total body irradiation. Twenty-eight children with AML underwent APBSCT with BCVAC (BCNU, etoposide, cytosine arabinoside and cyclophosphamide) conditioning regimen during first remission. The event free survival rate was 71.43% for all patients and the only cause of treatment failure was relapse. Eight male patients recurred at 1 to 11 months (median 5 months) after APBSCT. One patient remains alive with salvage therapy after relapse. With the exception of fever, mucositis and diarrhea, no serious complications occurred during APBSCT, including venoocclusive disease (VOD), and there was no transplantation related mortality. One patient developed secondary MDS after APBSCT but recovered hematologically on medication. APBSCT with BCVAC conditioning was found a safe and effective alternative option for patients with childhood AML in first remission, without a matched family donor.

Autologous and allogeneic peripheral blood stem cell transplantation as compared with bone marrow transplantation in children with leukaemia chronic GVHD and relapse rate did not differ in AML or ALL patients receiving BM or PBSC from HLA-identical siblings' donors.

Introduction: The Epstein-Barr virus (EBV) associated lymphoproliferative disease (LD) is a clinical and histological heterogeneous poly-or monoclonal B-cell proliferation. It occurs under immunosuppression, mostly after solid organ or hematopoietic stem cell transplan-tation (HSCT), and frequently results in a fatal outcome. Experience in children is limited. We present three patients with EBV-associated LD and one patient with EBV viremia after HSCT. Patients: Four children in the age from 6 to 14 years underwent matched-mismatched unrelated allogenic HSCT for leukemia (3 patients) and severe aplastic anemia. LD and/or EBV viremia were detected in the time of days 49-166. Risk factors were donor mismatch, anti-thymocyteglobuline, immunosuppression with OKT3 (anti-CD3 antibody) and graft versus host disease WHO grade III -IV. Maximal viral load (polymerase chain reaction) were measured from 1400 K/mL up to 476'684 K/mL. Two patients were treated with gancyclovir, vincristine and rituximab. The patient with EBV viremia was treated with gancyclovir and preemptive rituximab, EBV viremia subsequently decreased. One patient died of intracerebral bleeding and one patient of hemorrhagic alveolitis and graft versus host disease. The patient with EBV viremia died of relapse of their disease. One patient with LD is in complete remission 23 months after SCT. Discussion: The patients demonstrate clinical heterogeneity and a poor outcome after EBV reactivation and/or lymphoproliferative disease. Main risk factors may include the type and intensity of immunosuppression. The quantitative EBV polymerase chain reaction in plasma shows a correlation with a high viral load. Consequences may consist of cessation/reduction of immunosuppression, antiviral and/or cytostatic drugs and rituximab, a B-cell specific monoclonal antibody again CD 20. Conclusion: EBV-screening and management in children after SCT should be studied in prospective trials.

CAMPATH 1H in conditioning or reconditioning for allogeneic haematopoietic cell transplantation in childrenanalysis of outcome and early immune recovery J. Toporski, D. Turkiewicz, E. Gorczynska, K. Kalwak, R. Ryczan, B. Rybka, A. Chybicka, Wroclaw Medical University (Wroclaw, PL) CAMPATH 1H is a monoclonal antibody recognizing CD52 antigen present on almost all peripheral blood (PB) lymphocytes. It produces profound lymphopenia and therefore has been used for prevention of rejection or GvHD in high risk patients. Patients: We have included CAMPATH 1H (Alemtuzumab, Schering AG) in conditioning (7) or reconditioning (secondary graft failure -3, primary -1) for 11 children, 7 boys and 4 girls, with median age of 8.6 yrs (range 0.6 -11.8) with diagnoses of: MDS-3, AL-3, CML-2, NBL-1, Omenn S.-1, WAS-1. Methods: Ten children were grafted from an alternative donor -6 from partially matched related donor (PMRD), 4 from matched unrelated donor (MUD); 1 child was retransplanted from matched family donor (MFD). Ten children were transplanted with PB progenitors, one received bone marrow. Conditioning was myeloablative, Busulfan -based. One child with primary graft failure received CAMPATH alone. GvHD prophylaxis included immunomagnetic T-cell depletion (6 children transplanted from PMRD and 1 from MFD) and CsA+MTX (4 children transplanted from MUD). CAMPATH was given as 2 hour infusion from day -4 to -1 in a daily dose from 10 or 15mg/m2. Steroids and antihistaminic drugs were used to prevent adverse effects. Number of CD3+ T-cells, CD19+ B-cells and CD56+16+ NK cells in PB was evaluated with flow cytometer on days +30, +60 and +100. Results: Nine children developed chills, fever and rash during or after the first CAMPATH infusion which resolved after additional steroids. No life-threatening episodes were observed. No adverse reactions were seen after consecutive infusions. All children engrafted. Six children are alive with no evidence of disease from 60 to 512 days after HCT (median 209). Five patients died, from day +59 to +149 (median 133), 2 due to EBV-LPD (after T-cell depleted graft from PMRD) and 3 due to infection (2 proven, 1 probable pulmonary aspergillosis). GvHD was observed in 2 patients after T-cell add-backs due to imminent relapse in one and EBV-LPD in the other. Median number of PB T, B and NK cells on days +30, +60 and +100 is presented in a table.

Conclusion: CAMPATH-based conditioning in children requiring intensive immunoablation is feasible. No life-threatening infusionrelated episodes were noted. Neither rejection nor GvHD was observed. The use of CAMPATH results in profound immunodeficiency in terms of T and B cell reconstitution and delays NK cell recovery.

at the Short Tandem Repeats( STR) detection level . Period of observation ranged in allo sib group from 4 to 9 years and in alloMUD group 9 and 20 month. Follow up proved that in 9 cases any malignancy appeared including lymphoprolferative disease. In one case EBV and HHV8 reactivation were recorded, successfully treated with antCD20 antibody . This long lasting observation strongly suggests that ATG given with low dose of CTX and with Fluadarbin in MUD transplantation were sufficient for achieving full chimerism without a risk of cancer.

Haematopoietic stem cell transplantation is effective curative treatment in paediatric refractory/aggressive Langerhans cell histiocytosis M. Caniglia, A. Lombardi, R. Pinto, I. Rana, F. Zinno, G. Peluso, C. Rapanotti, A. Angioni, C. Roberti, B. Pinazzi, G. Isacchi, W. Arcese, G 

Although the new strategies concerning the management of LCH have been made a considerable advances on the outcome of pediatric patients, the best therapeutical approach for aggressive/refractory multi-system LCH patients remains still controversial. Antiproliferative and immunosuppressive therapy in combination with HSCT was proposed as the appropriate treatment for these poor-prognostic patients because this procedure may offer an opportunity to induce long-term remission and disease free survival. Because the appreciable morbidity and mortality of allogeneic HSCT, this strategy has therefore been reserved for the few LCH patients with a very poor prognosis. In this report we describe 4 children (14,27,96,48 months respectively old) with refractory aggressive MS-LCH, treated with allogeneic HSCT in our Institution between 2001 and 2003. Disease activity score proposed by Akkari V. et al (2003) was retrospectively calculated for all included patients at diagnosis and before HSCT as well as the Karnofsky status.All patients presented MSD progression despite chemotherapy with single or multiple agents or immunosuppressive therapy. Allogeneic HSCT was performed 10-22-72 and 47 months respectively from onset.Patient 1 and 3 received Umbelical Cord Blood Transplantation (UCBT) from HLA 4/6 and 5/6 mismatched unrelated donor. Patient 2 and 4 received Bone Marrow Transplant (BMT) from their related HLA-identical donors. Conditioning regimen including Busulfan 4 mg/kg and Fludarabin 30 mg/m2 from day -7 to day -4, Thiotepa 10 mg/kg on day -3 was used as preparative regimen. All patients received hors ATG 15 mg/Kg from day -6 to day -2 Cyclosporine A 3mg/Kg from day -1 to day + 180 and PDN 1 mg/kg until day + 30. After HSCT all patients are alive with a median follow-up of 16 months (32, 25, 8, 1) . Conditioning regimen was well tolerated without major complications. Donor engrafment was demostrated for ¾ patients (patient 4 was transplanted in Nov 03, too early for follow-up data) by PCR DNA analysis and progressive improvement of disease symptoms were observed after 18,6,and 8 months respectively. Patient 1 ad 2 are Disease free , Patient 3 presented an A-GVHD and a reduction of esophtalmous. We conclude that HSCT is a good courative treatment for these poor-prognostic patients. Selection of patients by early response to conventional chemotherapy and timing of HSCT remains controversial. D. Di Martino, M.P. Terranova, F. Scuderi, L. Scarso, S. Iacovone, C. Cermelli, A. Valetto, S. Dallorso, G. Morreale, G. Dini, G.Gaslini Institute (Genoa, Pisa, I) Immunological reconstitution post hematopoietic stem cell transplantation (HSCT) is not only a troublesome problem but also an intricate mechanism where humoral and cellular immune components are involved. This reconstitution is a slow process with a variable period of immunodeficiency that can lead to opportunistic infections. The aim of our study was to investigate if oligo-monoclonality of Immunoglobulin (Ig) heavy chain (H) repertoire after HSCT is restricted to memory B lymphocytes (CD27+/CD19+ subset) or if it is a general property of all B cells. Eight children subjected to allogeneic (n=4) and autologous (n=4) HSCT at G.Gaslini Institute were enrolled in this study. We performed an analysis of Ig H third complementarity determining region (CDR3) gene expression on CD27+B cells. CD27+ lymphocytes were selected with a high gradient magnetic separation columns (MACS) by whole lymphocyte population from peripheral blood on 180 days post HSCT. We utilized a CDR3 fingerprinting technique to analyze size distribution of polymerase chain reaction (PCR) amplified IgH CDR3 not only in memory B lymphocytes isolated from patients on 180 days after HSCT but also in whole B lymphocyte population, isolated from patients before (±12 days), on take (PMN >/= 500cells/mm 3 ) and on 180 days post HSCT. We performed two PCR utilizing: VH3 and IgM primer in first; FR3C3,4,6 and JHC primer in nested PCR. At the same time,CD27 surface antigen expression was analyzed by flow cytometry, measuring the percentage of positive cells. In healthy donors, CDR3 fingerprinting profile showed 16/20 bands, each band corresponding to a particular length of CDR3: this is a polyclonal situation. On take our patients analyzed just after transplantation show an oligoclonality and few CDR3 bands were detected. B cell repertoire reached the polyclonal situation of normal healthy donors on 180 days both in allogeneic and autologous HSCT. On 180 days after HSCT, the IgHCDR3 repertoire in both CD27+ and whole B lymphocytes are as polyclonal as in healthy controls. The median values of CD27+B cell subset analyzed by flow cytometry were 0.55% for allogeneic and 2.9% for autologous HSCT. We conclude that on 6 months post HSCT in patients subjected to allo and auto transplantation, the memory B lymphocyte compartment is recostituted as the whole population of B lymphocytes and this observation may be of importance when considering reimmunization of transplanted patients.

M. Fernández-Sanmartín, M. Izquierdo, J.M. Fernández-Navarro, A. Verdeguer, V. Castel, Hospital Infantil La Fe (Valencia, E) Introduction: Cidofovir (CDV) has proved to be effective against a wide variety of DNA viruses : HS, VZ, CMV, HHV6, adenovirus, poliomavirus. Some of them are responsible for high morbidity and mortality in the hematopoietic transplant setting. Scarce experience in children, doubts about its efficacy and potential renal and ocular toxicity limit CDV use in paediatrics. Objective: To present our experience with CDV in a paediatric group of allogeneic stem cells recipients in terms of both, efficacy and toxicity. Patients and Methods: Five patients with normal renal function and without any ocular symptomatology, received 7 courses of CDV treatment, with a total of 27 doses. Patients characteristics, CDV indication, number of courses, outcome and toxicity are shown in Table I . We administered 5mg/Kg per week for three weeks and then, every other week. In order to reduce the risk of nephrotoxicity, we provided vigorous hydration with normal saline two hours before and three hours after CDV infusion, and oral probenecid. Results: We did not observe renal toxicity defined as an impairment on plasmatic creatinine clearance and/or decrement in tubular phosphate reabsorption in any of the 27 doses administered. No patient developed ocular toxicity. Only one patient required CDV discontinuation due to gastric intolerance to probenecid. (Table I, FigI) Conclusion: Although the number of patients is not sufficient to establish significant conclusions, we find CDV to be effective and well tolerated. The possibility of treatment in an outpatient basis is an extra reason to consider CDV among the antiviral armamentarium in paediatric SCT Objectives: 1.To describe the characteristics of Acute Leukaemia patients treated with haematopoietic stem cell transplantation (SCT) 2.To analyse survival outcomes and mortality predictive factors. Method: Data source: Catalan Registry of SCT. Inclusion criteria: Patients suffering from acute lymphoblastic leukaemia (ALL) (n=637) or by acute myelogenous leukaemia (AML) (n=647) that underwent SCT in Catalonia. For survival analysis, only patients undergoing first SCT were included. Statistical analysis: actuarial method and the Wilcoxon test for univariate survival analysis and Cox regression for multivariate analysis. Results: AML in adult SCT patients shows a 67% increase from 1988-1995 to 1996-2002 , mainly due to AML 1st CR and resistant AML, whereas ALL in paediatric patients a decreasing trend is observed. Disease status at SCT is the only identified paediatric mortality predictive factor, showing a statistically significant difference: RR (relative risk) for ALL 2nd CR is 1.69 (95%CI:1.23-2.32) vs AML 1st CR. Main results are displayed in Tables 1 and 2 . Conclusions: 1.AML is increasingly frequent in SCT adult patient population. Conversely, ALL is declining among paediatric patients group. 2.Of all variables analysed, only disease status has an impact on paediatric survival patients. 3.Disease status, type of transplant, period and age are identified as predictive mortality factors in adult patients.

Treatment of relapsed acute myeloid leukaemia after allogeneic bone marrow transplantation with combination chemotherapy of cytarabine, idarubicin plus etoposide and subsequent donor leukocyte infusion: a prospective study S.J. Choi, J.H. Lee, J.H. Lee, S. Kim, M. Seol, Y.S. Lee, J.S. Lee, W.K. Kim, K.H. Lee, Asan Medical Center (Seoul, KOR) Donor leukocyte infusion (DLI) alone for relapsed AML after allogeneic BMT has been unsuccessful, with CR rate of only around 20%. Low efficacy of DLI alone in AML would be, at least in part, associated with large leukemic cell burden and rapid proliferation rate of AML that may overwhelm graft-versusleukemia effect (GVL) of DLI. Therefore, in case of relapsed AML, it would be reasonable to attempt cytoreductive chemotherapy before DLI in order to allow time for the development of GVL. However, the role of chemotherapy and subsequent DLI (Chemo-DLI) for the treatment of relapsed AML is largely unknown because of the lack of prospective studies. We prospectively evaluated the efficacy and toxicities of Chemo-DLI in treating relapsed AML after allo-BMT. The chemotherapy was administered from day -7 to -2 of DLI and consisted of cytarabine 1 g/m 2 /day continuous i.v. for 5 days, idarubicin 12 mg/m 2 /day i.v. for 3 days, and etoposide 150 mg/m 2 /day i.v. for 3 days. The donors received G-CSF 10 mg/kg/day s.c. for 4 days starting from day -3 of DLI. Peripheral blood mononuclear cells of the donors were collected and infused into the patients on day 0 and 1. No post-DLI GVHD prophylaxis was administered. Sixteen patients were enrolled. Ten patients achieved CR at a median of 30 days after DLI. Of 10 patients who achieved CR, 4 patients remain alive in CR with a median follow-up of 1,488 days (range, 771 -2,034 days). Twelve of 13 assessable patients developed acute GVHD (grade I-II = 4; grade III-IV = 8). Eight of 9 assessable patients developed chronic GVHD (limited = 2; extensive = 6). The 2 year overall survival (OS) was 31%, with a median survival duration of 215 days. Cox regression analysis showed higher leukemic blast percentage (50% or more) in the marrow before Chemo-DLI to be the only independent prognostic factor for lower OS (hazard ratio, 8.5; P = 0.009). The 2 year leukemia-free survival (LFS) for patients who achieved CR was 44%. The only clinical factor analyzed to be predictive of lower LFS was higher leukemic blasts percentage in the marrow before Chemo-DLI (P = 0.002). Our data showed that cytoreductive chemotherapy immediately followed by DLI produced a high CR rate and induced a prolonged remission in a substantial portion of the patients with relapsed AML after allogeneic BMT. High leukemic cell percentage in the marrow before Chemo-DLI was analyzed to be an adverse prognostic factor for lower OS and LFS.

A. Reichle, M. Zaiss, B. Panzer, P.J. Wild, G. Rothe, R. Andreesen, F. Hofstaedter, W. Dietmaier, University of Regensburg (Regensburg, D) Autologous transplantation in de novo AML may give insight into the nature of leukemogenesis. In the present study AML blasts, and positively selected CD34+ cells from leukapheresis products collected during first complete remission (CR) of de novo AML (n=43) following treatment according AMLCG protocol, were used for the first time to study genetic instability (GIN) in the remission hematopoiesis and in the corresponding CD34fractions. GIN was defined as microsatellite instability (MSI), >2 allelic imbalances (AIs), or MSI or AI at APC locus. Altogether 38 events at 8 of 10 tested loci were identified in CD34+ cells of 22 patients (51%), MSI (n=10), AI (n=28), no events in CD34+ cells of healthy donors. AI or MSI were observed on the following chromosome arms: 2p (n=2, hMSH2), 5q (n=9, APC), 7q (n=5, D5486, c-met), 8q (n=6, Eto), 11q (n=7, MLL), 17q (n=6, BRCA1), 17p (n=3, TP53). None of the clinical parameters at diagnosis (LDH, cytogenetics, blast counts) were correlated with GIN. In multivariate analysis GIN was an independent prognostic factor for relapse-free survival (p=0.0008). Only in 7% of the patients identical patterns of genetic instability were found in the corresponding AML blasts at diagnosis. In conclusion, hematopoiesis in patients with AML in first CR suffers frequently from genetic changes below the chromosomal level, and these changes are frequently different from those found in the corresponding AML blasts.

High-dose idarubicin and busulphan as conditioning regimen to autologous stem cell transplantation in acute myeloid leukaemia F. Ferrara, S. Palmieri, M. De Simone, M.R. D'Amico, A. Fasanaro, A. Viola, M. Annunziata, G. Mele, Cardarelli Hospital (Naples, I) In order to reduce the relapse rate after autologous stem cell transplantation (ASCT) in acute myeloid leukemia (AML) we developed an original conditioning program, named IBu, consisting of the combination of high dose idarubicin (IDA), given at 20mg/sqm as 3 days continuous infusion from day -13 to -11 and busulphan (Bu) at 4mg/kg from day -5 to -2. We report results from a series of 52 AML patients autografted in first or subsequent complete remission (CR) conditioned with IBu regimen. There were 31 males and 21 females with a median age of 50 years (16-74). Fourty-nine patients had non M3-AML in first (n=45) or second (n=3) CR (karyotype: normal in 34 patients, unfavourable in 10 patients, no mitoses in 4); four had M3-AML with t(15;17) in second (n=3) and fourth (n=1) molecular remission. All patients received peripheral blood stem cells (PBSC) collected after consolidation plus G-CSF. The median interval between CR achievement and ASCT was 3 months (2) (3) (4) (5) (6) (7) (8) . The median number of CD34+ cells infused was 6,3x10 6 /kg (2, (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) 4) . Finally, in patients aged more than 60 years (n=12), IDA and Bu were reduced to two and three days, respectively. The median number of days with granulocytes <500/cmm and of platelets <20000/cmm was 10 (7-21) and 12 (6-95), respectively. The median number of platelet and blood units transfused was 3 (1-7) and 3 (0-14), respectively. Extra-hematological toxicity mainly consisted of grade WHO III-IV stomatitis (44/52 or 85%), while 2 patients had grade III hepatic toxicity and one experienced transient hallucinations. Furthermore, most patients had FUO, while two experienced documented fungal infection. No transplant related death occurred. LVEF examination post-ASCT did not reveal any cardiac toxicity. Finally, median time of hospitalization was 29 days (22-67). After a median follow up of 12 months , 34 patients (65%) are in continuous CR, while 18 have relapsed at a median time from ASCT of 4 months (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) . Two patients died in CR for late reactivation of pulmonary aspergillosis and gastric cancer, respectively. Median overall and disease free survival have not yet been reached, as shown in the figure. Of note, in this series no patient autografted in CR1 had favourable cytogenetics and no case of transplant related mortality occurred, including 12 patients aged over 60 years. In conclusion, our data demonstrate the efficacy of the IBu regimen in patients with AML, due to a substantial reduction of relapse rate.

Allogeneic bone marrow transplantation compared with autologous peripheral blood stem cell transplantation in patients with acute myeloid leukaemia in first complete remission M. Jimenez, A. Espigares, J. Santiago, C. Fernandez, M. Capote, C. Martin, C. Herrera, J. Casaño, A. Torres, Hosp. Reina Sofia (Cordoba, E) Background: To compare the role of allogeneic bone marrow transplantation with autologous peripheral blood stem cell transplantation in patients with acute myeloid leukaemia (AML) in first complete remission(CR1)and to define the pronostic factors contributing to the patients evolution. Methods: We studied 88 patients diagnosed with AML in CR1 who underwent allogeneic bone marrow (ALO-BMT) or autologous peripheral blood stem cell transplantation (AUTO-PBSCT) between January 1990 and December 2002. Patients younger than 55 years with an HLA-identical donor received ALO-BMT (n=48) and those without donor or older than 55 years were planned for AUTO-PBSCT (n=40). The median follow up was 4,6 years. The median age was 28 years (range 4-55) in ALO-BMT and 40,5 years (range 1-64) in AUTO-PBSCT. The median time from diagnostic to transplantation was 7 months in both groups. The 2 groups were comparable with respect to sex, initial WBC count, FAB clasification, cytogenetic risk groups and number of induction courses. However the number of consolidation courses with high-dose ARA-C was different: 9 (22,5%) patients with ALO-BMT and 31 (77,5%) with AUTO-PBSCT received 2 or 3 courses (p<0.001). Patients were conditioned with BUCY 28 (31,8%) or TBI + Cyclophosphamide 60 (68,2%). Results: The 5-year probabilities of disease free survival (DFS), relapse (RP), and overall survival (OS) for ALO-BMT and AUTO-PBSCT were 50,7+7% vs 43,3+9% (p=NS), 43,7+8% vs 52,0+9% (p=NS); 48,5+8% vs 52,1+8% (p=NS) respectively. Transplant related mortality at day 100 was 10,4+8% for ALO-BMT vs 0% for AUTO-PBSCT (p=0.035). For patients undergoing AUTO-PBSCT the administration of 2 or more consolidation courses before transplant results in a significant improvement in DFS (p=0.014), RP (p=0.010) and OS (p=0.005). The multivariate analysis for the entire group reveals the following factors as favorable for DFS: age (p=0.008) and 2 or more consolidation courses (p=0.035) ; for RP: age (p=0.014) and a single induction course (p=0.023); and for OS: age (p=0.004) and 2 or more consolidation courses (p=0.01). Conclusion: 1. During first complete remission in acute myeloid leukemia, patients undergoing autologous peripheral blood stem cell and allogeneic bone marrow transplantation do not show statistically significant differences in DFS, RP and OS. 2. For patients undergoing AUTO-PBSCT the number of pretransplant consolidation courses (>2) results in better DFS, OS and RP.

Allogeneic haematopoietic stem cell transplantation during aplasia induced by salvage chemotherapy for otherwise refractory haematological malignancies M. Ditschkowski, R. Trenschel, M. Hlinka, N.K. Steckel, M. Koldehoff, A.H. Elmaagacli, D.W. Beelen, University Hospital of Essen (Essen, D) Relapse of advanced, refractory haematological malignancies immediately before intended allogeneic stem cell transplantation increases transplant related mortality and adversely influences curative options. Between 8/00 and 10/03 we treated 27 patients (pts) diagnosed with refractory relapse or progressive disease (AML n=20, CML n=4, NHL n=1, MDS n=1, ALL n=1) with salvage chemotherapy and subsequent myeloablative conditioning at the time of documented disease-free bone marrow aplasia followed by allogeneic PBSCT (n=26) or BMT (n=1). All patients (median age 39 years) had failed to achieve sustained remissions by conventional chemotherapy. Preparative salvage therapy based on cytarabine alone (n=6) or in combination with one (n=12) or two (n=7) alkylating agents. Six of these pts additionally received gemtuzumab, and two pts were treated with fludarabine or VP16 alone. Conditioning regimen consisted of TBI and cyclophosphamide (n=13) plus thiothepa (n=2), TBI and fludarabine (n=5), treosulfan and fludarabine (n=5) or treosulfan and cyclophosphamide (n=2). For GVHD prophylaxis CSA combined with alemtuzumab (n=7) or a short course MTX (n=19) or mycofenolate was applied. Donors were identical siblings (n=8), mismatched related (n=1), matched unrelated (n=17), or non-identical unrelated (n=1). Primary graft failure was observed in one case, 2 pts showed secondary graft failure. WBC counts > 1000/µl and platelets > 50 000/µl were reached at a median 15 and 24 days, respectively. Fourteen pts (52%) died from transplant-related complications which included MOF (n=11), fungal encephalitis (n=2), and suicide (n=1). In only 4 pts (15%) disease recurrence was the leading cause of death. Nine pts (33%) survive in sustained remission (median follow up 6 months) with an estimated overall and event-free survival of 19% and 16% respectively at 1 year after HSCT. In conclusion, salvage chemotherapy immediately followed by allogeneic HSCT has a high potential to induce complete and sustained remissions in refractory hematological malignancies and may thus be potentially curative even in pts with a very dismal prognosis. Advances in reducing transplant-related complications are required to further improve the outcome in this very unfavorable patient subset.

Improved overall survival in poor risk AML/MDS patients using PBSC compared to BM after allogeneic unrelated donor transplantation P861 Autologous bone marrow transplantation in first complete remission AML patients: the influence of infused TNC for engraftment and LFS differs after purged and unpurged marrow G. Milone, A. Tornello, S. Leotta, U. Consoli, P. Murgano, M. Poidomani, S. Mercurio, F. Indelicato, V. Pinto, R. Giustolisi, Ospedale Ferrarotto (Catania, I) In a single institution, 31 patients affected with AML in 1st CR received autologous bone marrow transplantation (ABMT) after BU-CY eradication. In 15 cases bone marrow cells were purged in vitro using ASTA-Z, while in 16 they were left unpurged. After transplantation a Neutrophil count > 0.5 x 10e9/l was reached in 25 days (median 8-49) and there was no difference in time to N. engraftment between the purged and unpurged groups (logrank: P=0.39). Median time for PLT engraftment was 60 days (median 17-270) and significantly longer for purged than unpurged transplants: 85 days vs. 59 days (logrank: P=0.04). When all cases were considered using the Cox regression analysis, the dose of infused Total Nucleated Cells (TNC) was an important factor for myeloid engraftment (P=0.02). A similar result was also obtained in purged patients (P=0.005) but not in the unpurged group (P=0.2). No transplant related mortality was observed while 16/31 patients relapsed between 4 and 14 months after ABMT. The Kaplan-Meier estimate of LFS for the entire group of patients is 50.5% when the median follow-up for patients still in response is 40 months. LFS is 58% in purged and 40% in unpurged groups (logrank: P=0.26). However patients receiving a TNC > median had a LFS of 28% and patients receiving TNC < median had a LFS of 65% (logrank: P=0.017). The number of infused TNC was an important factor for LFS in the stratum of unpurged marrow (P=0.05) but not in that of purged marrow (P=0.2). In multivariate Cox analysis, the number of TNC infused was a significant factor for LFS (P=0.03), independently of others factors such as FAB types that we found important in univariate analysis.

In conclusion, in our study, myeloid engraftment times of purged and unpurged transplants did not differ. After purged transplant, there is, however, a delay in platelet engraftment time. The number of infused TNC is an important factor for myeloid engraftment both when all patients are considered as a whole and in purged patients, but not in unpurged patients. The number of infused TNC is linked to a higher risk of relapse in the group of unpurged patient but not in purged patients.

GvHD and intensive chemotherapy prior to transplantation allow better leukaemic control in high risk CR1 AML after reduced-intensity conditioning HLA-identical allogeneic transplantation C. Faucher, J.M. Boiron, M. Mohty, J. Bay, V. Perreau, K. Bilger, N. Vey, A.M. Stoppa, D. Coso, P. Ladaique, D. Maraninchi, D. Blaise, Institut Paoli Calmettes, CHU Bordeaux, CRLCC Jean Perrin (Marseille, Bordeaux, Clermont-Ferrand, F) We report here a prospective investigation of HLA identical ASCT for CR1 AML patients aged 50 and/or with any comorbidity precluding standard ASCT. All patients received RIC including fludarabine (180 mg/m 2 ), busulfan (8 mg/kg) and Thymoglobuline* (10 mg/kg). The study was designed to offer allogeneic immunotherapy after achievement and maintenance of 1st CR with minimal procedure-related toxicity. For this purpose, the intensity of chemotherapy prior to ASCT was progressively increased, concomitantly to decreasing the immunosuppressive intensity of RIC. 26 pts were included [age:52 (26-60); M/F:11/15; age 50:20; previous aspergillosis:6; previous VOD:1; secondary leukemia:6; CR after 2 inductions:4; poor cytogenetics:9]. GVHD prophylaxis included [CSA alone:15; CSA+MTX:6; CSA+MMF:5) with either a BM graft (10) or PBSC (16). ASCT was performed without any previous intensive chemotherapy (Group 1, N=5), after 1 cycle of high dose cytarabine (6g/m 2 /d x 4) + idarubicine (12mg/m 2 /dx2) (HIDAC) (Group 2; N=15) or after HIDAC and an autologous PBSCT prepared with melphalan (140mg/m 2 ) (Group 3: N=6). Fludarabine was decreased from 180 to 120 mg/m 2 and Thymoglobuline* from 10 to 2.5 mg/kg. As of October 2003, (follow-up:19 m (2-59) , all patients engrafted, achieving full donor chimerism on day 60 (30-90). 6 presented grade 2 acute GVHD (Cumulative incidence (CI) = 23%) and 10 chronic GVHD (CI=42%). 2 pts died from transplant toxicity (TRM, CI=8%) and 10 relapsed (Relapse CI=38%) for an overall 2 year survival (OS) probability of 57%. All patients in group 1 relapsed and 1 is long term survivor after a second ASCT. Of the 15 pts with HIDAC alone, 5 relapsed (relapse CI: 33%), 1 died from GVHD (TRM CI: 7%) for a 2 year OS of 68%. Of the 6 patients with HIDAC and Auto PBSCT, none relapsed and 1 died from GVHD. Relapse was statistically associated with the absence of pre-graft intensive chemotherapy (p=0.02), the use of higher doses (5-7.5 mg/kg) of ATG (p=0.003) and the absence of acute and chronic GVHD (p=0.001). Longer survival was associated with the use of pre-graft intensive chemotherapy (p=0.04). We conclude that in a population of high risk CR1 AML patients, RIC ASCT is associated with a low TRM and a potent GVL effect if adequate prior chemotherapy is delivered. Prior auto PBSCT that is currently under evaluation, appears beneficial in the context of RIC ASCT, though a longer follow-up and a higher number of pts is still needed.

Autologous stem cell support of induction therapy prior to allogeneic transplantation to improve treatment delivery in patients with relapsing or secondary acute leukemia and CML blast crisis I. Hardan, A. Shimoni, A. Avigdor, A. Kneller, M. Berkowicz, P. Raanani, S.M. Stemmer, N. Shemtov, M. Yeshurun, I. Ben-Bassat, A. Nagler, Tel-Aviv University (Tel-Hashomer, IL) Patients (pt's) with relapse of acute leukemia (AL) or blast crisis of CML (BC CML) can be salvaged with an allogeneic stem cell transplantation (alloSCT). Prior to transplant a meaningful response should be achieved by salvage chemotherapy. This stage is usually associated with considerable toxicity and a prolonged pancytopenia. Administration of autologos stem cell backup (autoSC) may minimize this toxicity and enable a window for opportunity which allows performing curative allogeneic time of harvest and transplant necessitate the use of alternative aggressive approaches. Aim of our study was to confirm these preliminary data, updating the results on the 16 patients transplanted in II nd CR with PCR negative stem cells. Patients' median age was 35 years (range 9-61) and 5 were males. The median duration of first CR was 14 months (range 8-22), and induction therapy of relapse included an ATRA containing regimen in all patients but one. Eleven out of 16 patients had been treated in hematological relapse while 5 in molecular relapse. BAVC schedule was employed as conditioning regimen in all patients, followed by unpurged marrow stem cells. The median interval between II nd CR and reinfusion was 3 months (range 1-6). Seven out of 16 patients relapsed after a median of 6 months from ABMT (4-12);five of them died from disease progession, while 2 patients are currently alive in their IIIrd CR after AlloBMT. One patient presented a myelodysplasia testing PCR negative for PML-RAR alpha transcript 26 months post-ABMT, and the follow up was censored at the time of MDS diagnosis. Eight patients are in hematological and molecular CCR with a median follow up of 102 months (26-129) from transplant. Overall 10/16 patients are alive with a median follow up of 92 months from transplant (13-129). Ten-year projected probability of survival is 63% and the median has not been reached after ten years. No statistically significant difference in disease-free survival was observed neither evaluating the role of Ist CR duration (<=12 months vs >12 months), nor comparing the two different kinds of relapse (hematological vs molecular) (63% vs 40% p=n.s.). In conclusion, ABMT still seems to be an interesting approach for patients with acute promyelocytic leukemia in second molecular CR, giving a chance of long-term DFS. In our series there not seems to be an advantage in treating patients at the time of molecular relapse, but these data need further evaluation on a larger number of patients.

Haematopoietic chimerism status may predict relapse after in vitro T-cell depleted allogeneic stem cell transplantation for acute leukaemia I. Baas, W. Marijt, R. Barge, I. Starrenburg, R. Willemze, J. Falkenburg, Leiden University Medical Center (Leiden, NL) Objectives: Allogeneic stem cell transplantation (alloSCT) is an effective therapy for acute leukemia and MDS. Relapses can be treated with DLI but the success rate is low and treatment is associated with GVHD. Pre-emptive DLI may increase the likelihood of effective anti-leukemic treatment. If the risk of relapse could be estimated after alloSCT only patients at high risk for relapse could be treated with pre-emptive DLI lowering the chance of unnecessary induction of GVHD in the low risk patients. In this study we evaluated whether chimerism status after alloSCT may predict relapse. Methods: We evaluated 42 patients (27 AML, 13 ALL, 2 MDS) receiving an HLA-identical alloSCT between 1998-2001. The patients were categorized into good -or intermediate/bad risk leukemia according to age and cytogenetic abnormalities. Morphological examination and chimerism analysis of bone marrow samples were performed every 3 months for at least 2 years. Chimerism analysis was performed using X-and Y chromosome specific FISH probes (donor-recipient pair with sex difference) or by PCR analysis using primers specific for selected polymorphic short tandem repeats (donor-recipient pair of same sex). Patients were classified as complete chimeric (CC) when chimerism status was 100% donor at both 3 and 6 months post transplantation. Clinical follow up was obtained by reviewing the medical charts and ranged from 30-66 months post transplantation.

Results: At 6 months post transplantation 29 patients could be evaluated for chimerism status. 13 were not evaluable (7 due to early relapse, 5 to transplantation related mortality, 1 because of insufficient data). Of the 29 evaluable patients 9 were CC (31%) and 20 were mixed chimeric (MC) (69%). A clear difference in relapse rate was observed: in the CC-group only 1 patient relapsed (11%) while in the MC-group 7 patients relapsed (35%). 4 of the relapsed patients were characterized as having good risk leukemia, the other 4 as having intermediate/bad risk leukemia. 7 of the 9 patients in the CC-group were alive and well >30 months after transplantation. In the MC-group 10 patients died (50%) during follow up. Conclusion: For patients surviving the first 6 months after transplantation chimerism status at 6 months appeared to predict the risk for relapse which was independent from risk assessment by age and cytogenetic abnormalities. Pre-emptive DLI in these mixed chimeric patients may rescue them from subsequent relapse.

Comparison of long-term outcome after autologous transplantation of non-cryopreserved bone marrow and allogeneic haematopoietic stem cell transplantation for the treatment of high-risk adult acute lymphoblastic leukaemia J. Holowiecki, S. Giebel, J. Wojnar, M. Krawczyk-Kulis, M. Wojciechowska, L. Kachel, T. Czerw, Silesian Medical Academy (Katowice, PL) We analyzed outcome of 177 ALL patients aged 23 (16-57) years, given HSCT in 1st or subsequent complete remission (CR) at the Dept. of Haematology and BMT in Katowice, Poland, between 1991-2003 . Patients with high-risk of relapse were treated with alloHSCT (those having a suitable donor) (n=27; sibling 22, matched unrelated-5) or autologous HSCT (those without a donor) (n=101) in 1st CR. Standard-risk patients were given alloHSCT (n=26, sibling n=16,MUD n=10) or autoHSCT (n=23) in case of relapse after achieving 2nd CR. Median duration of preceding CR as well as recipient's age were comparable for both study groups. 39% of patients given alloHSCT and 15% of pts in autoHSCT group was bcr/ablpositive. For all autoHSCT and 93% of alloHSCT, bone marrow was used as a source of stem cells. In case of autoHSCT, CAV (cyclophosphamid 60mg/kg on d. -3, -2, cytarabine 2 g/m 2 d. -3, -2, -1, etoposide 800 mg/m 2 d. -3, -2) was used as preparative regimen and bone marrow was not cryopreserved but stored for 72h in 4degC. For alloHSCT, TBI/Cy (46%) or BuCy (53%) was used. The OS and LFS rates at 8 years were higher for allo-compared to autoHSCT, however the differences did not reach statistical significance (p>0.1 for all parameters) (see Table) . The outcome was comparable for alloHSCT recipients given transplant from unrelated vs. sibling donors. We conclude that although alloHSCT is a preferable option for high-risk adult ALL patients, a significant proportion of those not having a suitable donor may be successfully treated with CAV regimen followed by autologous transplant of non-cryopreserved bone marrow. diagnosed as follicular lymphoma (FCL), 59 as aggressive B-NHL, 6 as aggressive T-NHL and 8 as mantle cell lymphoma (MCL). Status at transplantation for FCL was CR 1 (stage III/IV): 8pts., CR 2: 8 pts. or sensitive disease (PR, CR>2 or responding relapse): 13 patients and for aggressive lymphoma including MCL CR 1: 25 pts., CR 2 : 16 pts., sensitive disease : 29 pts. or refractory disease: 3 patients. TRM for all patients was 3,9 % i.e. in 3/42 sensitive disease due to bacterial and/or fungal infections and in 1/33 CR 1 due to viral hepatits. So far, late complications were rare except one patient with cancer of the stomach (d+782), but no secondary hematologic disorder was seen. Relapse rate for FCL was 38 % within the median observation period of 40 months (1-113 mo) , occuring in patients transplanted in CR 1: 12,5 %, in CR 2: 37,5 % and in sensitive disease: 53 %. Overall disease free survival was 46 % without reaching a plateau curve yet (CR 1: 75 %, CR 2: 36 %, sensitive disease: 31 %). Overall survival was 52 % ( CR 1: 100 %, CR 2: 57 %, sensitive disease: 26 %). Patients with aggressive lymphoma relapsed in 37 % within a median observation time of 31 months (1-229 mo) occuring in patients transplanted in CR 1: 24 %, in CR 2: 37,5 % and in sensitive disease: 41 %. All 3 patients with chemotherapy refractory disease showed progression shortly after ASCT and died within 7 months. Overall disease free survival post transplant was 56 % with a plateau after the last relapse at 56 months (CR 1: 72 %, CR 2: 60 %, sensitive disease: 48 %). Overall survival was 66 % (CR 1: 77 %, CR 2: 92 %, sensitive disease: 49 %). Excluding patients with refractory disease, ASCT for non-Hodgkin´s lymphoma was a manageable and a beneficial treatment that led in more than half of the patients to a long lasting disease free survival.

Intensified full-dose induction therapy (HD-machop radiotherapy) followed by consolidation with BAVC regimen and autologous stem cell transplantation as front-line therapeutic approach for high-risk aggressive non-Hodgkin lymphoma F. Zaja, A. Sperotto, V. Tomadini, F. Patriarca, C. Filì, F. Kikic, D. Damiani, R. Mestroni, M. Tiribelli, R. Fanin, Udine University Hospital (Udine, I) Background and objectives. Patients with high-intermediate risk and high risk non-Hodgkin's lymphoma , according to the International Prognostic Index, have a poor outcome, with a 5year projected survival of 40% and 30%, respectively. The aim of the present study was to investigate the role of intensified third generation regimen followed by autologous stem cell transplantation in these patients. Design and methods. Twenty-five consecutive patients with high-intermediate risk (15 patients) and high-risk (10 patients) non-Hodgkin's lymphomas were selected for this prospective study. They were treated with an intensified third-generation regimen consisting of HD-MACHOP, radiotherapy to bulky or localised residual disease, and autologous stem cell transplantation. Results. After induction therapy (HD-MACHOP radiotherapy), 10 (40%) patients were in complete remission, 8 (32%) in partial remission, and 7 (28%) did not respond. Fifteen patients, 7 (46.5%) in complete remission and 8 (43.5%) in partial remission, underwent autologous stem cell transplantation. After transplant, 13 (87.0%) patients were in complete remission, 1 (6.5%) remained in partial remission, and 1 (6.5%) had disease progression. No transplant-related death was recorded. Disease-free survival and event-free survival for the entire population were 86.0% and 61.5%, respectively. Interpretation and Conclusion. This preliminary analysis indicates that intensive induction treatment is feasible and is a good mobilizer since an adequate peripheral blood stem cell harvest was reached in 17 (85.0%) patients. Moreover, 15 patients (60%) were able to complete the therapeutic program with transplant, with a relapse rate of 12.0%.

Allogeneic stem cell transplantation for patients with refractory anaemia with matched related and unrelated donors: T-cell depletion and reduced-intensity regimens are associated with an increased relapse risk T. de Witte, R. Brand, A. van Biezen, P. Guardiola, J. Cornelissen, A. Gratwohl, M. Boogaerts, V. Runde, R. Arnold, J.-L. Harrousseau, A. Fauser, G. Mufti, D. Niederwieser on behalf for the EBMT, CLWP-MDS subcommittee Summary: Transplantation with matched allogeneic donors is the curative treatment of choice for patients with MDS. Early transplantation before transformation to advanced stages of MDS or AML results usually in a 50% EFS, but the high transplant-related mortality has precludes a general application of alloSCT for patients with RA. This analysis evaluated the impact of recipient age, transplant year, early transplantation, cytogenetic characteristics, T-cell depletion, type of donor, and the intensity of the conditioning regimen on the outcome of transplantation with matched allogeneic donors. The study population consisted of 184 patients, 131 of whom have been transplanted with an HLA-identical sibling. None of the patients had progressed to more advanced stages of MDS. None of the patients had received intensive chemotherapy prior to the transplant conditioning. 53 Patients received T cell depleted grafts and 24 patients received a transplantation after a reduced intensity conditioning. 33 Patients were <20 years, 77 patients between 20 and 40 years, and 74 patients >40 years. Transplant periods: 37 patients before 1993, 45 patients in period 1993 -1996, 47 patients in 1997-1999 , and 55 patients more recently. 40 Patients have been transplanted within 6 months after diagnosis, 67 between 6-12 months, and 75 after 12 months. Cytogenetic data were available for 87 patients, 54 of whom had cytogenetic abnormalities. The overall 5-year survival was 53%. Patients transplanted with matched related and unrelated donors have a survival of 54% and 48% resp. Younger age was associated with better survival of 78% if <20 years, but no significant differences were observed in patients older than 20 years. T-cell depletion was associated with inferior outcome: 43% with and 57% without T-cell depletion resp. The transplantation outcome has improved in time. The survival was 35% when transplanted before 1993, but 57%, 54% and 78% when transplanted in more recent periods. The presence of chromosomal aberrations did not influence the outcome. Reduced intensity conditioning resulted in a low TRM of 16%, but the relapse risk increased to 54% (p=0.0001) and the EFS was lower 36% vs 51% after standard conditioning (p=0.09). Multivariate analysis using hazard Cox model with age, transplant year, type of donor, disease duration prior to transplant, and type of conditioning as variables showed an improved survival in recent years (p=0.01). T-cell depletion and the use of alternative donors resulted in inferior outcome. Less intensive conditioning resulted in an increased relapse risk (p=0.01). More recent transplantation, the use of sibling donors and reduced intensity regimens resulted in a lower TRM. This data show that alloSCT results in excellent outcome nowadays, even at older age and including alternative matched donors. The high relapse rate observed after reduced intensity conditioning warrants prospective studies.

Shariati Hospital (Teheran, IR) Objective: As there is inconsistency with regard to the out come using allogeneic and autologous peripheral blood stem cells (PBSCT) compared to bone marrow (BMT) in children with leukemia, we did a retrospective analysis in children patients with AML, ALL, CML transplanted from 1993 to

/L was 11 and 14 days V.s 13 and 17 days in BMT respectively

At present 50 out of 75 are alive (66%) and 25 pts died due to Hemorrhagic stroke, ARDS, VOD, severe GVHD, HUS and relapse.21/75 pts relapsed. Relapse rate was 30

ALL pts appeared 60% in BMT group vs.77.8% in PBSC group, cGVHD 20% in BMT and no cGVHD in PBSC. aGVHD in AML pts appeared 77.8% in BMT group vs.71.4% in PBSC group and cGVHD 22.2% in BMT vs.14.3% in PBSC and aGVHD in CML pts developed 66% in BMT group vs. 100% in PBSC and no cGVHD. Conclusion: PBSCT in childhood AML and ALL was consistent with significant faster ANC and platelet recovery in allogeneic PBSCT and risk of aGVHD was more than BMT. However

For GvHD prophylaxis all pts. received CSA based regimens and furthermore ATG-S ™ 40 -90mg/kg. For analysis patients were divided according to their remission status at HSCT in good risk (CR1;RA)(n=25) and poor risk (>CR1/RA)(n=91) and the last group according to their graft source Diagnosis are: AML/ sAML (99) and MDS (17)

/25 pts. (100%) in the good risk group and 80/91 pts. (85%) in the poor risk group achieved CR. In the good riskgroup 9/25 pts. (38%) and in the poor risk-group 46/91 (50%) died of infection

In the poor risk group aGvHD II°-IV° occurred in 9/35 pts. after BMT (25%) and in 21/55 pts. after PBSCT (37%)

At a median follow up of 1070 days (101-2899) 64/116 pts. with myeloid malignancies are alive resulting in an OS of 55%. No influence of matching was obtained. Conclusion: PBSC as the graft source in matched and as well in mismatched URD transplantation compares favourable to BM in OS and RFS in poor risk patients with myeloid malignancies. The reduced relapse rate seem to be related to an

University Hospital Reina Sofia (Cordoba, E) Introduction: Allogeneic stem cell transplantation (SCT) is a potentially curative option for high risk B-lineage acute leukemia (B-ALL) but resurgence of leukemia B cells is still the main cause of treatment failure. In this study, we set out to determine the impact clinical factors on normal B-cell lymphopoiesis restoration and leukemia B cells detection after SCT in 30 consecutive patients for B-ALL

All patients received myeloablative conditioning, based on FTBI (n=15 in ALL, n=2 in AML), busulfan (n=21, n=23) or treosulfan (n=5, n=5). CTX was administered to 44 pts, VP16 to 36 pts, melphalan to 17 pts and fludarabine to 5 pts. ATG was also given before alternative HSCT. 28/71 children relapsed after HSCT. Children who relapsed after HSCT showed higher in vitro resistance of leukemic blasts to dexamethasone (1.4-fold, p=0.016), mercaptopurine (1.4-fold, p=0.03) and thioguanine (3.5-fold, p=0.002). No significant differences were found for other drugs, however a trend towards higher resistance of blasts of patients who relapsed after HSCT to all tested drugs with the possible exception of thiotepa. 29 pts died: 22 at relapse (including 1 with secondary malignancy), 6 from TRM (2 GvHD, 2 MOF, 1 VOD, 1 HUS/TTP) and 1 from viral infection

Independent prognostic factors by Cox multivariate analysis were: TBIconditioning

Hospital Clinic (Barcelona, E) Background: Stem-cell transplantation (SCT) is an extended practice in the management of acute myeloid leukemia (AML)

52% males; median age: 35, range: 8-64) underwent SCT (autologous, 30 cases; allogeneic, 62) in an advanced phase of the disease (> 1st CR) in a single institution. Patients were stratified according to tumoral burden at the time of SCT (CR2, n=50; low-tumoral burden, <20% BM blasts, n=22; hightumoral burden, > 20% BM blasts, n=20)

In 35% of equivocal cases tricolor flow cytometry was used to establish diagnosis and rule out follicular, marginal zone or small lymphocytic lymphoma. Majority of pts (62%) received CHOP, COP or Chlorambucil (33%) or regimens containing high-dose methotrexate or cytosine arabinoside (5%) as initial chemotherapy. Patients with localised disease usually involving the Waldeyer's ring received involved field irradiation (IF-RT) as a consolidation. 17% of pts were given rituximab in addition to chemotherapy. 14% of all pts (n=16) proceeded to high-dose chemotherapy (BEAM) and autologous hematopoietic cell transplantation (autoHCT)

There was a trend for better OS of pts who were given rituximab. Conclusion: Our data on

The number of DexaBEAMs given prior to autologous stem cell transplantation seems to have no effect on outcome in relapsed and refractory non-Hodgkin lymphoma

D) Introduction: Autologous stem cell transplantation (ASCT) achieves long-term survival in patients (pts) with NHL. We studied the outcome of ASCT after DexaBEAM for relapsed and refractory NHL treated at the University of Leipzig between 1994 and 2003. The response to DexaBEAM and the no. of cycles given prior to ASCT were analysed. Patients and Methods: 167 pts were treated on an intention-totreat-basis with DexaBEAM (dexamethasone, BCNU, ectoposide, Ara-C, melphalan) to be followed by ASCT. 97/167 pts (58%) (66m, 31f) received ASCT. Median age was 47 y (range 19-63). 42 pts (43%) had indolent (ind) and 55 (57%) aggressive (agg) lymphomas. Of these, 13 pts (13%) had primary refractory disease, 54 (56%) PR and 30 (31%) first relapse. After salvage DexaBEAM, CR, PR, and progressive disease were present in 42 pts (43%), 47 (49%), and 8 (8%) respectively. Pts in CR and PR were considered chemosensitive, pts with refractory disease chemo-resistant. 28 pts (29%) (ind, n= 13, agg, n= 15) received one DexaBEAM, 69 pts (71%) two cycles prior to ASCT. Results: Median time interval between chemotherapy and ASCT was 56 days (range 10-153)

Median interval between ASCT and relapse was 75 d. RI at 3 y for pts in PR, CR was 49%, 44% respectively (P= 0.9479). OS, DSF tended to be better and RI lower for pts with ind lymphomas

The no. of DexaBEAMs given prior to ASCT had no effect on OS, DSF, and RI (P= 0.464, P= 0.312, P= 0.109 respectively). Less than 60 d interval between chemotherapy and ASCT was associated with improved DSF (P=0.058) and less RI

Chemo-sensitive lymphomas are a prerequisite for long-term survival and DSF after ASCT

Outcome is not significantly different for PR or CR prior to ASCT

DexaBEAMs given seems to have no effect on outcome

Results: Primary hypothyroidism was detected in 52% of patients; it was transient in 7/26 and more frequent in patients who received Busulfan (p=0.1). Hypergonadotropic hypogonadism was seen in 78% of patients who reached puberty (14/19 girls, 15/18 boys), indepently of whether they were prepubertal or not at HCT. While LH and FSH were high in 100% of affected girls, only germinal line was affected in boys; the 3 boys with high LH levels had normal serum testosterone levels and spontaneous virilization. Excluding the 5 patients with final height at onset, a mean height decrease of 0.65 standard deviation score (sds) was observed at the end of the follow-up. These height decrease was higher in patients who received TBI (0.92+/-0.88 vs 0.26+/-0.93 sds; p<0.01) or Cy (0.82+/-0.87 vs 0.35+/-1.06 sds; p=0.07)as conditioning treatment. Height decrease was greater in patients with hypothyroidism, but was onmy statistically significant in the first year after HCT, probably as substitutive therapy was stablished thereafter. Patients with high or normal LH levels had a growth decrease of 0.92+/-0.63 and 0.30+/-0.78, respectively (p<0.05). No differences were found between sexes or type of HCT. Conclusion: Prevalence of hypothiroidism was high. Although often subclinical, it affects growth pattern if not treated. Hypergonadotropic hypogonadism also had a high incidence and was not prevented by prepubertal stage at HCT.

Late toxicity after autologous stem cell transplantation for non-Hodgkin lymphoma patients: a competing risks analysis N. Mounier, G. Sergent, R. Ruiz-Soto, P. Brice, C. Hennequin, J. Marolleau, E. De Kerviller, J. Briere, C. Gisselbrecht, Hôpital St. Louis (Paris, F) ASCT has evolved as fundamental in the treatment of high risk or relapsing aggressive NHL, none the less the late toxicity (3 months after ASCT) associated to this therapy has to be assessed as well as the risk of secondary malignancies. Among the NHL patients transplanted at our institution between [1993] [1994] [1995] [1996] [1997] [1998] [1999] [2000] [2001] 158 corresponded to aggressive NHL. At diagnosis median age was 46 years (18-69), male/female 98/60, IPI 2-3 in 39 (25%) and 31 (20%) had marrow infiltration. 64 pts. (40%) were transplanted as consolidation of first line therapy and the median number of progressions before ASCT were 1 (1 -3) . CR or CRu was achieved in 115 (73%), PR in 35 (22%) and 8 (5%) were in SD. 38 (24%) received TBI as part of the conditioning. In 148 cases (94%) PBSC were used as source of stem cells. With a mean follow up of 3 years, the OS rate was 61 ± 9 % and DFS 55 ± 8 %. The IPI had no predictive value either in front-line patients nor in relapse patients treated with ASCT. 8 malignancies were diagnosed: 6 solid tumors, 1 myelodysplastic syndrome and 1 AML (after 30 and 3 months respectively) with a cumulative incidence of secondary cancer at 3 years of 37%. Late toxicity affected 43 pts. (27%), that associated to infections was the most frequent (1 0 ). We also observed neurological (7), pulmonary (4), cardiovascular (4), digestive (5), endocrine (3), urogenital (2) , hepatic (1) , musculoskeletal (2) , ophthalmic (2) , ORL (1) and systemic symptoms (3) with a cumulative incidence of late toxicity at 3 years of 2%. As grade 3-4 toxicities were considered 33 pts (20%) but none of them died as consequence of it. Nine pts (6%) presented cardiac toxicity as left ventricular dysfunction but only 3 (2%) referred associated symptoms and required treatment. When taking into account the competing risks, Multivariate analysis revealed age to associated to relapse (RR= 1.02), the number of progressions to late toxicity (RR= 2.68) and secondary cancer (RR= 5.68) , and the use of mitoxantrone in the conditioning regimen to late toxicity (RR= 2.98) .In conclusion, the results of this cohort study confirmed the great efficacy of ASCT in NHL patients. ASCT. In addition, ASCT carries little risk of late toxicity and when presented is not life threatening.

Central nervous system involvement with seizures in patients undergoing haematopoietic stem cell transplantation. Single-centre experience in more than 500 patients S. Guidi, A. Gaudenzi, C. Nozzoli, G. Muscas, A. Vannucchi, M. Paganini, L. Lombardini, R. Saccardi, G. Pellicanò, M. Bonolis, A. Bosi, Ospedale di Careggi (Florence, I) Neurotoxicity in hematopoietic stem cells transplantation (HSCT) recipients may be due to infections, disease relapses, pharmacological toxicity, vascular accidents, metabolic encefalopathies. CNS symptoms incidence is reported from 11% to 70% of HSCT patients. Ciclosporin-A (CyA) has been advocated as the cause of a wide array of neurological symptoms, ranging from the headache, restlessness and tremor, vision change up to seizures and coma. MRI studies usually demonstrate reversible brain lesions mainly confined to the white matter. Aim of the study: We have retrospectively reviewed for neurological complications with seizures 284 autologous and 223 allogeneic HSCT performed between 1996 and September 2003,at the Florence BMT Unit. We evaluated the role of laboratory analysis, electroencephalogram (EEG) examination and neuroimaging techniques MRI in clarifying the pathogenesis of seizures. Patients: No autologous HSCT patients showed seizures instead of 15 /223 (6,7%) of allogeneic HSCT recipients, age 14-45, 5 male and 10 female, CML 6, AML 4, NHL LB 2, ALL 2, Idiopathic myelofibrosis 1, 6 early and 9 advanced, MUD 12 and familiar 3, PBSC 4 and BM 11, 14/15 matched , ablative conditioning regimen 14 and RIC 1. All 15 patients who developed neurological sympthoms were receiving Cya treatment as GvHD prophilaxis and ten methylprednisolon more than 1 mg/kg as therapy of GvHD also. Methods: All 15 patients were evaluated by a neurologist for clinical and liquor examination, blood chemistry and Cya blood levels were screened, EEG, TC and/or MRI were done whenever it was possible. Results: CyA neurotoxicity was diagnosed in 10/15 patients all with normal blood levels, one patient was diagnosed affected by HHV6 encephalitis, another one by HSV encephalitis, three were suspected, but not proven to have had seizures CyA related one with status epilecticus. 3 patients are alive in cGvHD treatment, 12 died. 9 deaths were non relapse mortality and three because disease progression. CyA CNS toxicity is reversible just stopping treatment and MRI is the most helpful technique to confirm the diagnostic suspicion especially by FLAIR. Conclusion: Despite the literature in our experience seizures are indicative of poor prognosis mainly because our cases are mostly on GVHD treatment or GvHD related and 10/15 pts were receiving high dose steroids.

Low toxicity with a fludarabine-based conditioning regimen in haematopoetic stem cell transplantation for children with mucopolysaccharidosis (M. Hurler) L. Grigull, A. Beilken, A. Sander, T. Luecke, M. Schrappe, K. Welte, A. Das, H. Schmid, K. Sykora, Medical School Hannover (Hannover, D) Hurler syndrome (MPS1H) is a progressive inborn error of mucopolysaccharide metabolism that leads to premature death by the age of 10 years. Allogenic stem cell transplantation (SCT) can provide a life-long correction of the enzymatic deficiency. However, more than 70% of the patients do not have a matched sibling donor. Here, we report on five children with MPS1H transplanted with a fludarabine based conditioning regimen. Patients: By the time of SCT the five children were 10 months to 3.75 years old. MPS1H was diagnosed clinically and by leukocyte alpha-iduronidase activity. In patient 1, a matched family donor was identified, in patients 2 and 3 the mothers were stem cell donors. In patients 4 and 5, an unrelated SCT was performed. The preparative regimen consisted of bulsulfan, fludarabine and anti-thymocyte globuline. For HLA-mismatch transplantations, melphalan was added. Results: Four patients received a CD-34+ selected graft (mean CD34+ cell dose 32.6x10 6 /kg, range 19.5-54) without immunosuppression after SCT. The mean CD3+ cell dose was 3.4x10 4 /kg (range 1. 3-5.4 ). PMN > 0.5/nl was seen after 15. 3 days (range 10-21) , platelets > 50/nl were observed after 16.5 days (range 9-25). One child received a full marrow and cyclosporine until day +120. She engrafted with PMN > 0.5nl after 16 days and platelet > 50/nl after 26 days. All five children are alive 7 to 35 months after SCT and in ambulatory care. No toxicity > grade II and no GvH > grade I occurred. In patient 2, a subdural hematoma on day 6 after SCT necessitated surgical removal, but the further post-transplant course remained uneventful. In patients 4 and 5, antiviral therapy was initiated for CMV reactivation. All patients show sustained engraftment. In three children, complete donor chimerism was achieved; patients 4 and 5 have mixed chimerism. In three patients, donorlymphocyte infusions (DLI) were given due to decreasing donor chimerism. Conclusion: SCT is the therapy of choice in children with MPS1H. Our preliminary results show that a fludarabine based conditionig regimen in combination with a CD34-selected graft is safe, non-toxic and effective in achieving stable engraftment.

Allogeneic haematopoietic stem cell transplantation in children with myelodysplastic syndrome in Poland D. Wojcik, J. Toporski, E. Gorczynska, D. Turkiewicz, K. Kalwak, M. Slociak, W. Pietras, A. Chybicka, K. Drabko, J. Kowalczyk, M. Leda, J. Wachowiak, K. Pajdosz, Wroclaw Medical University, Lublin Medical University, Poznan Medical University (Wroclaw, Lublin, Poznan, PL) We present the results of 40 HSCT in 34 children with MDS between 1995 and November 2003. There were 15 girls and 19 boys, aged from 0,2 to 17,5 y (median 7,1) with RA -5, RARS -2, RAEB -6, RAEB-t -8, JMML -13. Sixteen transplant procedures were performed in 13 patients from matched family donor (MFD). An alternative donor was used in 24 transplant procedures in 21 children (partially matched related donor PMRD -14/11, matched unrelated donor MUD -10/10). Median time from the diagnosis to allogeneic hematopoietic stem cell transplantation (alloHSCT) was 7 months (from 1 month to 4,5 years). Bone marrow was used as a source of cell in 22 transplants, and peripheral blood in 17 procedures. One child was grafted with marrow and peripheral blood cells. Conditioning regimen in the vast majority of patients consisted of Busulfan + Cyclophosphamide with Melphalan or Etoposide. Children transplanted with graft from an alternative donor received in additional ATG (MUD) or ATG+OKT3 or Campath-1H (PMRD). Oral Busulfan was substituted with i.v. Treosulfan in four children. One patient was conditioned with fTBI+Etoposide. Children transplanted from PMRD received positively selected CD34+ (CliniMACS immunomagnetic selection). GvHD prophylaxis consisted of CsA and Methothrexate. PMRD transplanted children received T-cell depleted graft as the only GvHD prophylaxis. Primary engraftment achieved 32 (94%) of 34 children. One child grafted from MFD failed to engraft and one engrafted after the second transplant. Median time to ANC >500/mm 3 was 15 days (range 0.5 to 57), and platelets count >50000/mm 3 was 27 days (range 11 to 78). Seventeen children (50%) are alive with median observation time of 35 months (range 2 to 85 months). Survival with regard to the type of the donor: 6/13 with MFD, 7/11 with PMRD and 4/10 with MUD grafted children. Seventeen patients died -3 due to relapse (9%) and 14 (41%) of procedure related complications (aGvHD -6, cGvHD -1, infection -3, CNS bleeding -2, VOD -1 and graft failure -1). GvHD still remains the single most reliable reason for procedure related mortality. Genomic HLA Class-I and II matching and more intense immunosuppression might result in lower GvHD incidence, eventually improving the event free survival. However, wider use of alternative donors (in our group 24 children out of 34, 70%) and the need for more aggressive immunosuppression (e.g. T-cell depletion) may increase the number of infectious complications.A. Rodriguez-Villa, J.J. Ortega, M.A. Diaz, I. Badell, A. Martinez-Rubio, M. Maldonado, A. Verdeguer, E. Bureo, P. Gomez-Garcia, J.M. Perez-Hurtado, J. Estella, E. Gonzalez-Valentin, Hospital Reina Sofia, Hospital Vall d'Hebron, Hospital Niño Jesus, Hospital Sant Pau, Hospital La Paz, Hospital Ramon y Cajal, Hospital La Fe, Hospital Marques de Valdecilla, Hospital Virgen del Rocio, Hospital San Juan de Dios, Hospital Carlos Haya (Cordoba, Barcelona, Madrid, Valencia, Santander, Sevilla, Malaga, E) The Spanish Pediatric Hematopoietic Stem Cell Transplantation Registry (RETMON: Registro Español de Trasplante de Médula Osea en Niños) arose in February 1994, associated to the Spanish Pediatric Bone Marrow Transplantation Group (GETMON: Grupo Español de Trasplante de Médula Osea en Niños). Centers belonging to GETMON annually report data on their transplant activity to RETMON employing our specially designed forms. Since May 1976 to March 2003, 18 spanish center reported  2287 hematopoietic stem cell transplantations carried out to  2085 patients aged less than 19 years. Of them, 1324 (57,9%) were autologous transplantations and 961(42,1%) were allogeneic . In the auto-transplants group, bone marrow progenitors source (BM) was employed in 508 cases (38,36%) and peripheral stem cell source (PB) , alone or combined with BM, was used in 813 cases(59,63%). In the last 5 years, the use of PB for autografts has increased from 33,1% to 86,9%. Indications for the auto-transplants were hematologic malignancies in 758 cases ( 57,2%) and solid tumors in the remaining 552 cases (42,8%). Out of the 961 allo-transplants, 648(67,4%) were performed employing identical related donors and alternative donors were used in the remaining 313 cases (32,6%). Over the last decade, the percentage of alternative donors has increased from 10,3% to more than 35%. Allo-transplants were performed using BM in 716 cases (74,5%), PB cells in 148 cases (15,4%), umbilical cord blood in 83 cases (8,63%) and BM plus PB in 14 cases (1,45%) . Indications for the allo-transplants were malignant disorders in 735 cases (76,4%) and non malignant disorders in 227 cases (23,6%). Actuarial 100-day transplant related mortality (TRM) was 12,06% for the whole group; 6,3% for the autografts, 12,2% for the allorelated group and 35,8% for the allografts from alternative donors. Conclusions: This report shows the current trends in pediatric hematopoietic stem cell transplantation in Spain and provide rational basis for patient counselling and health care planning to improve the quality of medical research.

A retrospective study on the influence of cyclosporin levels on GvL, viral infections and overall outcome in paediatric allogeneic HPT R. Ridwan, M. Ortin, S. Patel, J. Shankari, P. Fomasagaram, R. Sinha, S. Ball, J. Marsh, J. Treleaven, The Royal Marsden Hospital, St. George´s Hospital Medical School (Sutton, London, UK) Maintaining cyclosporin-A (CsA) levels in the lower limits of the therapeutic range is a common policy in transplant practice. The aim of this approach is to promote GvL effect. With the aim of assessing whether post-HPT CsA levels have an influence on the promotion of GvL effect or whether it might allow a higher rate of viral infections, we have conducted a retrospective study where all the allogeneic paediatric HPT performed at our centre between 1996 and 2003 (n=108, median age 11 ± 4.6 years) were included. 60 patients received grafts from unrelated donors (UD) and 48 from related donors (RD). Indications for HPT included ALL (75 cases), AML (12), MDS (6), CML (10), undifferentiated (3), Lymphoblastic NHL (1), HLH (1) . Overall, 29 cases of active viral infections (including 12 episodes of CMV reactivation) were seen. CsA levels during the first six months were not significantly different between patients who had or did not have active viral infections (232.79 ± 55.6 vs. 244.28 ± 60.2). However, patients receiving RD HPT had a tendency to suffer viral infections when the CsA levels had been higher (p<0.06) during the first 6 months. Interestingly, children with lower CsA levels during the first month experienced a higher incidence of CMV reactivation (188 ± 47.9 vs 258 ± 66.3; p<0.05). These tendencies were not present in UD HPT. Overall, 42 patients have died, 25 of toxic death and 15 of relapse. No significant differences were seen in the overall outcome or in the cause of death between patients with higher or lower CsA levels. When we examined our series according to the donor origin or the type of disease, no differences were seen CD4+CD3+, CD8+CD3+, CD56+CD3-, CD14+, CD34+ and CD19+ cells. Using both, the in-house and the multiplex STR system gave comparable quantitative results. Our results indicate that molecular assessment of chimerism can be performed with a wide variety of STR loci either by single or multiplex PCR. Quantitative determination of cell-line specific chimerism should facilitate therapeutic interventions. Supported by "Hilfe für Krebskranke Kinder Frankfurt e.V."

12 Gy FTBI versus 13, 2 Gy FTBI in preparative regimen for allogeneic BMT from matched sibling donors in children with ALL in II CR J. Wachowiak, D. Boruczkowski, J. Malicki, G. Kosicka, M. Leda, A. Pieczonka, G. Stryczynska, K. Marcinkowski University of Med. Sciences, Poznan Cancer Center (Poznan, PL) In children with ALL in II CR the antileukemic effect of allogeneic BMT (allo-BMT) needs further improvement. Therefore, it was investigated, whether in those children the total dose of fractionated total body irradiation (FTBI) (12Gy versus 13,2Gy) and its source (Cobalt-60 unit versus linear accelerator) demonstrate an impact on allo-BMT results in terms of regimen related mortality (RRM) and probability of leukemia free survival (pLFS). From 1993 to 2003 the BMT was performed in 26 children with ALL in II CR. Median age was 10 years (yrs). In all patients (pts) FTBI was given in 8 fractions during 4 days (2 fractions/day) to a total dose of 12 Gy in 14 children, and 13,2 Gy in 12 children. Dose to lungs was reduced to 9,4 Gy. Nine pts transplanted between 1993-1997 have been irradiated with Co-60 unit, while 17 pts transplanted after 1997 with linear accelerator (15 MeV photons). In half of pts FTBI was followed by VP 60 mg/kg i.v. on day 3, and in second half of them by CY 60 mg/kg/day i.v. on days -3 and -2. Twenty four pts have been transplanted from HLA-identical sibling, and 2 from syngeneic twin. For GvHD prevention cyclosporin alone was used in majority of pts. After syngeneic BMT no GvHD prophylaxis was given. Engraftment was achieved in all pts. No one child died because of transplant related complication, and the leukemia relapse was the exclusive cause of treatment failure. After 6 yrs pLFS for 14 children given 12 Gy FTBI was 0,58, while 0,72 in the group of 12 pts prepared for BMT with 13,2 Gy. For 9 pts irradiated with Co-60 the 5 yrs pLFS was 0,56, and 0,66 for 17 pts irradiated with linear accelerator. In relation to both dose and source of radiation, among 17 children conditioned with linear accelerator the 4 yrs pLFS was 0,73 in 9 pts given 13,2 FTBI, and 0,63 in 8 pts obtained 12 Gy FTBI. Three out of 9 children irradiated with Co-60 were given 13,2 Gy FTBI and 2 of them are alive in CCR (70 and 72 months), while in 6 pts receiving 12 Gy FTBI the 4 yrs pLFS was only 0,5. In children undergoing allo-BMT for ALL in II CR the 13,2 Gy in compare with the 12 Gy FTBI doesn't increase the risk of RRM and improve the pLFS, especially when performed with linear accelerator. Supported by grant KBN 4 PO5E 108 18.

Survival of children with high-risk neuroblastoma after conventional treatment followed by high-dose chemotherapy and autologous stem cell transplantation G. Caldas, A.F. Lacerda, A. Neto, E. Vieira, F. Pereira, M.J. Ribeiro, M. Chagas, A. Ambrosio, N. Miranda, F. Leal-da-Costa, J.L. Passos-Coelho, M. Abecasis, Instituto Português de Oncologia Lisboa (Lisbon, P) Introduction: Neuroblastoma (NBL) is the most common extracranial solid tumor in children. About 65% are metastatic at diagnosis, with an expected survival below 15% at 5 years when only standard dose chemotherapy is used. High dose chemotherapy (HDCT) followed by autologous stem cell transplantation (ASCT) for high risk NBL (HR-NBL) improves the outcome of these patients (pts). Methods: We performed a retrospective analysis of 16 pediatric pts (14 males, 2 females) with HR-NBL, submitted to HDCT-ASCT between June 96 and July 02, in our Institute. Results: All pts were stage 4; mean age at diagnosis was 2.5 y (0. 3 -4.8) . Primary location was adrenal in 13 (81%), thoracic in 3 (29%). Six out of 13 (46%) were MYCN amplified; 7 out of 12 (58%) showed 1p deletion. All pts but one were treated before HDCT-ASCT according to the SFOP protocol for HR-NBL; one infant was treated following the guidelines of INES 99. Fourteen pts were submitted to surgery. Pre transplant disease status was CR1-9 pts, CR2-1 pt, CR3-1 pt, VGPR-2 pts and PR-3 pts. The HDCT regimen used was Busulfan (16 mg/kg > 5 y or 600 mg/m 2 < 6 y) + Melphalan (160 mg/m 2 ) in 15 pts and CEM (carboplatin, etoposide and melphalan) in one (due to congenital liver disease). There were no major toxic complications of ASCT. Post HDCT-ASCT treatment was completed with 13-cis retinoic acid in 6 pts and local radiotherapy in 3 others. Post HDCT-ASCT status was CR in 12 pts and progressive disease (PD) in 4 pts. Relapse occurred in 6 pts. Median time to relapse/progression after ASCT was 7 m (9±7m); we found a significant difference between pts with MYCN amplification and/or del 1p (4/7 pts; median 3 m, 4±4 m) and those without (4/6pts; median 18 m, 18±8 m). At present 9/16 pts are alive, 6 in CR and 3 in PD. The median follow-up is 32 m (11 -100 m), with an event free survival of 36% and an overall survival of 56% at 3 years Conclusions: High dose chemotherapy is feasible with low toxicity. A significant increase in overall survival and event free survival was observed, when matched with published historical controls.

ATG as part of the preparative regimen for paediatric allogeneic peripheral blood stem cell transplantation A. Kupesiz, G. Tezcan, V. Hazar, M.A Yesilipek, Akdeniz University Medical School (Antalya, TR) Severe acute graft-versus host disease (GvHD) is one of the major complications after stem cell transplantation. Treatment of severe GvHD is difficult and the condition is often fatal. Recently, antithymocyte globulin (ATG) has been added as a part of preparative regimen (in vivo T cell depletion) to prevent graft rejection and GvHD after stem cell transplantation . The aim of the study is to determine the effect of ATG on engrafment, infection and GvHD in children who underwent peripheral blood stem cell transplantation (PBSCT). Of the 26 children in the study, 25 had a non malignant disease and one had malignant disease. ATG(Fresenius) administered three different doses as a part of the conditioning regimen to 15 children with hemoglobinopathy (10 children), Fanconi aplastic anemia (FAA, one child), aplastic anemia (3 children) and amegakaryocytic thrombocytopenia (one child). In the other 11 patients ATG was not used (10 hemoglobinopathy, one ALL). Male/Female ratio was 5/15 and 8/3 in ATG and non-ATG groups, respectively. All of the donors were matched siblings or parents.GvHD prophylaxis consisted of CSA/MTX (only CSA in FAA patients).In ATG group, adverse events were noticed in 73% of the patients and included fever, headache, dispnea, chills, vomiting, hypotension. Engraftment occured in all of the patients in both groups. The median time to neutrophil engraftment (>0,5 x10 9 /L) was 14 days (range, 10-20) in the ATG group and 12 days (range, 9-14) in the non-ATG group (p= 0,035). Platelet engraftment (>20 x 10 9 /L) was reached for the ATG group after a median of 22 days (range, 8-48) and for the non-ATG group after 17 days (range, 10-31) (p= 0,28). Acute GvHD grade I-IV was observed in 13% of the ATG group and 27% non-ATG group (p= 0,62). Severe grade III/IV GvHD occurred at none of the patients in ATG group but 18% of the non-ATG group (p= 0,169). Chronic GvHD was seen in 6% of the ATG and 27% of the non-ATG group (p= 0,279). Extensive cGvHD was not observed in both P847 CMV reactivation in paediatric HPT: A retrospective study on risk factors and efficacy of secondary prophylaxis S. Patel, M. Ortin, P. Fomasagaram, J. Shankari, R. Ridwan, D. Lancaster, J. Treleaven, S. Ball, The Royal Marsden Hospital, St. George´s Hospital Medical School (Sutton, London, UK) CMV causes significant morbidity and mortality after allogeneic haemopoietic progenitors transplant (HPT). CMV reactivation is known to be less frequent in children receiving HPT than in adults. The aim of this study is to determine the factors influencing CMV reactivation, subsequent relapses, and organ disease in children receiving HPT. From 1996, 108 children (median age 11 ± 4.6years) received HPT [60 from unrelated (UD), 48 from related donors (RD)] at our centre. Indications included ALL (75), AML (12), MDS (6), CML (10), undifferentiated (3), NHL (1), HLH (1). Pre-HPT serology for CMV was performed by ELISA. Post-HPT CMV monitoring was performed by pp-65 antigen test (DEAFF). Study endpoints were incidence of reactivation, influence of donor and recipient status, timing post transplant, incidence of organ disease and influence of HPT-related complications. Of 41 CMV positive pairs (11 double positive, 10 single recipient positive, 20 single donor positive), 17 patients had CMV reactivation (positive antigenemia). Recipient status was significantly (p<0.0001) more relevant for reactivation than donor status. Reactivations were significantly (p<0.05) more frequent in single positive recipient than in double positive pairs. Data on evolution were available on 14/17 cases. These episodes appeared at a median of 44 ± 31.6 days after transplant. Only one of these cases developed organ disease. All episodes were treated for an average of 17.7 ± 17;9 days. There were 5 relapses that occurred at a mean of 20 ± 61 days after the first episode. 4 had received UD HPT and 1 RD HPT. 2 cases were double positives and 3 single recipient positives. No relation between incidence of relapse and timing of the first episode or duration of its treatment were found. However, all cases were treated for GvHD. 2/5 cases suffered organ disease. Both cases had received UD HPT and were single recipient S230 positives. One died of CMV disease. Secondary prophylaxis had been followed by 4 cases. The development of organ disease was less frequent in this group (1/4, p<0 .05) although it did not significantly prevent relapse of antigenemia. Conclusion: The group at highest risk of CMV reactivation in paediatric HPT is positive recipient with negative donor. Disease is more common at relapse in patients receiving UD HPT with negative donor. Secondary prophylaxis and monitoring with molecular techniques might be appropriate for this group to prevent/anticipate disease.

Correlation of immune reconstitution and survival in children undergoing allogeneic haematopoietic stem cell transplantation U. Koehl, K. Bochennek, S.Y. Zimmermann, R. Esser, C. Andreas, J. Soerensen, H. Gruettner, A. Ackermann, D. Schwabe, T. Klingebiel, T. Lehrnbecher, University of Frankfurt (Frankfurt, D) Introduction: The speed of the immune reconstitution seems to play an important role in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). A delayed reconstitution is related to a high risk of infection and relapse. In this study, we evaluated the reconstitution of different lymphocyte subsets in children undergoing HSCT in very short intervals and correlated the data with one-year survival. Patients and Methods: Immune reconstitution was analyzed in 24 patients undergoing allogeneic HSCT for ALL (n=13), AML (n=3), CLL (n=1), MDS (n=3), aplastic anemia (n=2) and thalassemia (n=2). Stem cell source included unmanipulated bone marrow (BM) and peripheral blood stem cells (PBSC) (n=5 and n=7, respectively) and CD34 selected BM and PBSC (n=2 and n=10, respectively). Lymphocyte subtypes in peripheral blood were assessed by four-coloured-flow cytometric analysis (Coulter Epics XL, Coulter, Germany). CD19+ B-cells, CD4+CD3+ Thelper cells, CD8+CD3+ cytotoxic cells and CD56+CD3-NKcells were determined weekly during the first three months after HSCT, then every other week for three months, followed by a 4week-interval for at least 2 years. Data were compared to agematched normal values (Comans, J Pediatr 1997; 130:388) . Results: Fifty percent of the patients reached the age-matched 5th percentile of NK cells after 4 weeks following HSCT, whereas the age-matched 5th percentile of cytotoxic T-cells, of B-cells and of helper T-cells was reached by half of the patients after 7, 14, and 27 weeks, respectively. The reconstitution of helper and cytotoxic T-cells was significantly faster in patients receiving PBSC compared with patients with BM or CD34 selected transplants. In contrast, B-cell reconstitution occurred earliest in patients receiving BM or CD34 selected PBSC. When correlating one-year survival and the maximum cell count of lymphocyte subsets measured within the first 6 months after HSCT, the only significance was found for CD8+CD3+ (12 survivors out of 13 patients with CD8+CD3+ counts >500/µl vs 5 survivors out of 11 patients with CD8+CD3+ counts <500/µl; P=.01). Conclusion: The preliminary results of our pilot study underline the importance of immune reconstitution on the outcome of children undergoing HSCT. Further data are needed to clarify the impact of the speed of recovery of the different lymphocyte subsets on treatment-related morbidity and mortality.

Non-radiotherapy-based conditioning with stem cell transplantation from alternative donors for children with refractory severe aplastic anaemia C. Urban, M. Benesch, K.W. Sykora, W. Schwinger, H. Lackner, E. Kloibhofer, University Children's Hospital, University of Hannover (Graz, A; Hannover, D) Intensive conditioning with the inclusion of total body/lymphoid irradiation (TBI/TLI) is widely used to prevent graft rejection in patients with refractory severe aplastic anemia (SAA) receiving allografts, particularly from alternative donors and/or after CD34+-selection. However, with this approach transplant related complications are also increased with raised concern of longterm irradiation side effects, especially in children. We here present three children with transfusion-dependent SAA not responding to multiple immunosuppressive courses, who were allografted after conditioning with fludarabine-based regimens. One of these patients previously received two ''megadose'' CD34+-purified peripheral blood stem cell transplantations (PBSCT) from a matched unrelated donor (MUD) with TLI 5 Gy for the first transplant and had late graft failure on day +585 and day +850, respectively. Age at stem cell transplantation (SCT) was 11, 16 and 11 years. The interval between diagnosis and SCT was 39, 156 and 4 months, all patients were heavily pretransfused. Conditioning included fludarabine 30 mg/m2 x6 days. In addition patient 1 (failing 2 previous grafts) received thiotepa (TT) 10 mg/kg and Campath 1H 10 mg/m 2 x6 days, patient 2 cyclophosphamide (CY) 60 mg/kg x2 days, TT 7.5 mg/kg x2 days and OKT-3 0.1 mg/kg for 23 days and patient 3 CY 60 mg/kg x2 days and ATG Fresenius 30 mg/kg x3 days. Stem cell graft was unmanipulated marrow (3.18 x 10 6 CD34+/kg) from the same unrelated donor for the patient who failed the two previous CD34+-purified PBSCT and CD34+-purified PBSC from an unrelated donor (9.62 x 10 6 CD34+/kg) and from a haploidentical related donor (8.59 x 10 6 CD34+/kg), respectively in the two other patients. Graft-versushost disease (GVHD) prophylaxis with cyclosporin A and mycophenolate mofetil was only given to the patient with the unmanipulated marrow graft. None of the patients developed GVHD. Engraftment was prompt with neutrophils >0.5 x 10 9 /l on day 11, 9 and 10 and platelets >30 x 10 9 /l on day 18, 19 and 22. Follow-up is 22, 34, and 6 months, respectively. All patients have normal counts with complete donor chimerism. We conclude that fludarabine-based conditioning is powerfully immunosuppressive and may be used for children with refractory SAA transplanted from alternative donors and/or with CD34+purified grafts even after rejection from previous SCT with TLI. Increased sensitive for cross-linking agents characterises Fanconi Anaemia patients and BMT is curative approach constitutes a high risk of transplant related mortality during HSCt. In 1994 we started a program of FA children transplantation with reduced intensity conditioning (RIC) . -7 patient received HSCt from sibling donors (allosib) ) (5 boys and 2 girls, age from 5 to 14 years), for conditioning Cyclophosphamide (CTX) 4 x 20 mg , and ATG were used. -3 patient received HSCt from matched unrelated donor (alloMUD) (2 boys and 1 girls, age from 0.5 to 3.5 years) for conditioning Fludarabine 5 x 30 mg/m 2 ., CTX 4 x 10 mg /kg .b.w. and ATG were used. The aim of the use of ATG was to decrease the number of lymphocytes to the extent securing immunosuppression. Usually, a cumulative dose of 25 mg/kg b.w. was given in five consecutive days in allosib and 10-20 mg/kg b.w. in four consecutive days in alloMUD transplantation . In allo sib group toxicity >= 2 grade (WHO) in 5 cases and GvHD>= 2 grade in 1 case were seen. In alloMUD group toxicity >=2 grade ( WHO) in 2 cases and aGvHD >= 2 grade in 2 cases were seen. In alloMUD group one child died due to infection in +28 day. All patients reconstituted haematologicaly and were fully chimeric transplantation at the optimal timing. We studied the possible benefit of supporting salvage therapy with autoSC. Seventeen pt's with relapsing acute leukemia (n=10 AML-8, ALL-2), secondary leukemia (n-3) and myeloid blast crisis of CML (n-4) were enrolled (study group). The induction therapy included antracyclin and ARA-C in dosages adjusted for age and disease stage. Forty-eight hours after the completion of the ARA-C therapy autoSC graft that was cryopreserved in remission (acute leukemia) or in chronic phase (CML) was infused (1.3 -2.5 x 10 6 CD34+ cells/kg). G-CSF was added from day +4 post autoSC infusions. An historical group of 66 pt's with AL or BC CML who had undergone slavage therapy without autoSC support serve as the control group. Results: Sixteen of the 17 patients of the study group achieved a PMN count of > 0.5x10 9 /L on day 7 to 13 (median =10 days) from the autoSC infusion, (12 days from completion of the salvage therapy), in contrast to day 19 to 27 (median=22 days) in the control group (p<0.005). platelet recovery (>20x10 9 /L) was observed on the fifteen day in the study group compared to day 25 in the control group (medians, p<0.01). Sixteen out of the 17 pt's of the study group were able to proceed to alloSCT from matched related ( 8) or unrelated (8) donors. All pt's achieved a complete or very good partial response to the salvage therapy. With a median follow up of 13 months, eleven of the 17 pt's (65%) with AL (8) and CML (3) are alive in complete remission. Conclusions: AutoSC support for induction therapy shortens pancytopenia and thus reduces toxicity. This approach enables the administration of the entire therapeutic program for patients with relapsing/secondary leukemia and BC CML, and thus may improve long term results of therapy in this very high risk group of pt's. Naturally, this approach requires forethought and the preparation and cryopreservation of an autoSC graft at first CR in AL and CP CML.

High-dose melphalan is an effective salvage therapy in AML patients relapsing after autologous PBSCT G. Bug, J. Atta, S. Klein, B. Hertenstein, L. Bergmann, S. Boehrer, D. Hoelzer, H. Martin, Klinikum der Johann Wolfgang Goethe-Universität, Medizinische Hochschule (Frankfurt, Hannover, D) Background and Aim: AML patients relapsing after autologous transplant are treated rather heterogeneously than by standardized protocols. In our center conventional salvage chemotherapy includes mitoxantrone, topotecane, cytarabine (MTC, Bergmann et al., Onkologie 2003, 26 (5) , 86). The aim is to achieve a subsequent remission and proceed to allogeneic transplant with reasonably low toxicity. However, CR rates are below 50% and overall longterm survival is poor. In an attempt to improve results, we initiated a pilot study using high dose melphalan (HD-Mel) followed by autologous PBSC support as salvage therapy to induce complete remission. Patients and Methods: In a pilot study 8 AML patients (median age 57, range 46-60 years) relapsing after a previous autologous transplant (n=6) or chemotherapy (n=2) received HD-Mel (140-200 mg/m²) with auto-PBSC as salvage therapy. Six of 8 patients had received MTC chemotherapy 27-41 days prior to HD-Mel with 5/6 refractory to MTC, while 2/8 reveived only salvage HD-Mel without preceeding other chemotherapy. Results: All 8/8 patients including those 5 refractory to MTC achieved CR after salvage with HD-Mel and auto-PBSC support. Median cumulative duration of neutropenia was >6 weeks in the 5 pts. refractory to MTC but only 2 weeks in the other pts. Subsequent CR lasted 2 -7 months, thereby facilitating further allogeneic Tx. Five patients proceeded to MUD allo-Tx in CR after 1,7 -3,1 months with 2 remaining in CCR after 6 and 28 months, while 3 died after MUD-Tx in CR due to infection and/or GvHD. Three pts. did not reiceive a consolidating allo-Tx, relapsed again after 2, 5 and 7 months and eventually died. Conclusions: HD-Mel is highly effective in inducing complete remission in relapsed AML patients even if refractory to MCT chemotherapy, a finding not previously described. Cumulative toxicity after subsequent MUD-transplant was low after immediate HD-Mel but high due to prolonged neutropenia after failing preceeding attempts with MTC chemotherapy. Thus we propose to reinduce remission using immediate HD-melphalan in relapsing AML patients with stored autologous PBSC.

Allogeneic transplantation of Bcr-Abl positive acute lymphoblastic leukaemia is associated with a low incidence of relapse

Objective: The outcome of BCR-ABL positive acute lymhoblastic leukemia (ALL) treated with conventional chemotherapy is poor. Despite the advent of imatinib the overall survival has not changed due to the early development of resistance. Patients: From 1992 to 2002 29 BCR-ABL positive ALL patients received allogeneic bone marrow or stem cell transplantation at our center. The median age was 37,7 years. Twelve patients had an identical sibling donor, twelve had an unrelated donor and five patients were transplanted haploidentical from a related donor. Twelve patients were transplanted in first remission, 17 in second or higher remission or in refractory disease. Six patients were transplanted with reduced intensity conditioning because of advanced age and poor medical status. Twenty-three patients received a standard intensity conditioning based on 12 Gy of total body irradiation plus cyclophosphamide (60 mg/kg) or etoposide (60 mg/kg). Pretransplant therapy included imatinib in 8 patients. This resulted in a complete molecular remission in 2 patients and a partial molecular remission in 4 patients. Two patients were refractory to imatinib. Results: Concerning engraftment no adverse effect of imatinib therapy were observed. The median time to recovery of neutrophiles was 21 days. Transplant related mortality (TRM) at 1 year was 27%, overall mortality 34%. The relapse rate was 7% (2/29). These two patients were transplanted with refractory leukemia and died shortly afterwards. None of the patients transplanted in 1st remission has suffered a relapse. In addition the overall survival at 1 year was significantly higher in those patients transplanted in 1st remission (p=0,0184). The probability of overall survival was 70% for patients transplanted in 1st CR vs. 35% (p=0.05). A matched pair analysis of 29 BCR-ABL negative ALL patients transplanted at our center revealed no difference in long term survival -51% vs. 58% (p=0.761). Conclusion: As non relapse mortality was the major cause of death in BCR-ABL positive ALL after allogeneic transplantation the focus should lie on reducing short and long term toxicity. Therefore pretransplant-therapy with imatinib to induce a state of minimal residual disease followed by reduced intensity conditioning may be an option to improve TRM without compromising cure rates.

Bone marrow angiogenesis reportedly is increased in various hematologic disorders including acute lymphatic and myeloid leukemias. In these patients the bone marrow microvessel density (BM-MVD) appears to be associated with an unfavorable prognosis. In the present study, we have retrospectively analysed the BM-MVD (at diagnosis) in 31 patients with acute myeloid leukemia (AML) (median age: 38 years; range: 21-53 years;f:m-ratio: 1:1,4) who underwent conventional chemotherapy and consecutive allogeneic bone marrow transplantation (BMT). The median BM-MVD at diagnosis was 30 mm² (range: 17-48/mm²) and thus was significantly higher compared to controls (n=9; BM-MVD: median 7/mm², range 2-11/mm²; p<0.05). In patients who failed to achieve a complete remission (CR) in response to induction chemotherapy, the BM-MVD was significantly higher (median: 41.5/mm²) at diagnosis than in patients who entered CR (median: 28.5/mm2, p<0.05). In addition, patients with high BM-MVD (>30mm 2 ) had a significantly shorter overall survival compared to patients with a lower BM-MVD (<30 mm 2 , p<0.05). Moreover, patients with a high BM-MVD (>30mm 2 ) were found to have a significantly higher risk of relapse (p<0.05). In 4 patients in whom a continuous complete remission was documented after BMT, the BM-MVD levels were analysed at diagnosis as well as between day +80 and day +100 after BMT. In all 4 patients, the BM-MVD was found to decrease in response to BMT until day 100 (p<0.05). Together, our data suggest that the BM-MVD is a prognostic parameter concerning survival in patients with AML undergoing allogeneic BMT.

S. Capria, D. Diverio, M. Ribersani, M. Breccia, E. Baldacci, F. Simone, A.P. Iori, F. Mandelli, G. Meloni, University La Sapienza (Rome, I) In 1997 our group published an experience on 15 APL patients autografted in IInd CR, suggesting that ABMT with PML-RARalfa negative stem cells is likely to result in prolonged clinical and molecular remissions, whereas patients who test PCR+ at the

A. Athanasiadou, K. Stamatopoulos, I. Sakellari, I. Zorbas, M. Gaitatzi, A. Fassas, A. Anagnostopoulos, G. Papanicolaou Hospital (Thessalonika, GR) Leukemic relapse post allo-HCT is usually of recipient cell origin; there are rare cases of leukemia developing in donor cells. Donor cell relapse is generally identified using cytogenetic and molecular markers. We report a 37-year-old female with Ph (+) ALL who underwent allo-HCT (in first complete hematological, cytogenetic and molecular remission) from her HLA-matched brother. Six months after HCT, RT-PCR analysis documented the reappearance of BCR-ABL (e1a2) chimeric transcript; four months later, a bone marrow aspirate revealed overt leukemia relapse. FISH analysis of the "relapse" sample demonstrated complete donor (male) chimerism as well as the presence of the BCR-ABL chimeric gene. VNTR analysis demonstrated allelic disparities in patient samples pre-and post-transplantation at loci 3'HPR/apoB and VWF1; in contrast, allelic identity was identified between donor DNA and patient DNA samples posttransplantation. Segregation analysis demonstrated that pretransplantation the patient was heterozygous at the HA-2 minor histocompatibility locus (HA-2M/V), whereas post-transplantation she was homozygous for the V allele and thus identical with the donor (also homozygous for the HA-2 V allele). To our knowledge, this is the first case demonstrating BCR-ABL (+) ALL developing in donor cells after allogeneic-HCT for Ph(+) ALL. From 1987 to 1996 we performed a trial of in -vivo / ex -vivo T cell depletion at Ulm University under the auspices of the Campath Users Group. The cohort of patients (pts) consisted of 27 males and 14 females, median age 38y (range 18 -51y). The diagnoses were AML CR1 42 pts and ALL CR1 1pt. The conditioning regimen consisted of TBI 12Gy and CTX 120mg / kg. Rejection prophylaxis was performed with Campath 1G 20mg / d from d -11 to d -7, the bone marrows were depleted of T cells with Campath 1M and donor serum. No further GvHDprophylaxis was given posttransplant. No graft rejections were observed, the actuarial risk of developing acute GvHD I -IIO was 23%, no patient developed grade III or IV GvHD. The probability of developing chronic GvHD was 9%. After a median follow -up of 108 mo (range 75 -192 mo) 65% of pts are alive and in remission. The actuarial risk of relapse is 16%, 19% of pts have died of transplant -related complications. Opportunistic infections were the main cause of death (CMV -IP 1pt, P.carinii -IP 1pt, VZV -IP 1pt). 3 pts developed secondary malignaNcies all of them EBV -associated. 1 pt died of EBV -associated lymphoproliferative disease and 2 pts presented with EBVassociated Hodgkin´s disease. The latter patients were successfully treated with radiotherapy and chemotherapy respectively and are in remission of both AML and Hodgkin´s disease. These data demonstrate that in -vivo / ex -vivo T cell depletion can successfully prevent both graft rejection and acute and chronic GvHD in patients with standard -risk acute leukaemias in CR1 without increasing the risks of graft rejection or relapse.. Due to the profound and persistent cellular immune defect careful long -term monitoring of these pts is mandatory.

Haemopoietic progenitor transplantation in the treatment of Philadelphia positive acute lymphoblastic leukaemia: a single-centre study on prognostic factors R. Sinha, M. Ortin, S. Patel, R. Ridwan, P. Somasegarem, J. Shankari, S. Mellor, J. Swansbury, J. Treleaven, Royal Marsden Hospital (Sutton, UK) Philadelphia positive ALL (Ph+ALL) is an aggressive from of leukaemia with a high incidence of relapse. Allogenic HPT is the only effective treatment known although the disease free survival is still very poor (30-40%). The aim of this study is to identify factors associated with poor outcome after transplant. Eighteen cases of Ph+ALL with a median age of 8.5 ± 5.2 years underwent HPT at The Royal Marsden Hospital. In all cases the diagnosis was confirmed by both FISH and RT-PCR. All but two cases presented as CD10+ ALL, one was a T cell ALL. The majority had a presenting white cell count (WCC) of less than 50; 6 cases presented with hyperleucocytosis. All cases commenced ALL induction with the aim of performing an allogenic HPT as soon as there was a donor available post induction. The median time from diagnosis to transplant was 6±7.9 months. Data on HPT were available in 15 of 18 cases. These 15 cases received an allogenic graft, 12 from unrelated and 3 from related donors. In all cases conditioning comprised TBI plus either cyclophosphamide (12 cases) or etoposide (3 cases). Unrelated Donor HPT recipients also received campath as part of conditioning. Data on outcome were available in 16 cases. After a median follow-up of 19 ± 14.9 months, 5 patients are alive and disease free, and 11 have died (3 toxic deaths, 8 relapses). There was a lower WCC and shorter time to transplant (average 7 months compared to 11 months) in patients who did not relapse post HPT compared with those who did, although these differences are not statistically significant. There was a tendency towards non-relapse in patients who were conditioned with etoposide and TBI compared to those that were not (p<0.06). All current survivors have a straightforward genetic translocation with no overall chromosomal gain or loss. Conclusion: Ph+ALL remains an aggressive disease despite HPT, however patients with WCC<50 have a better outcome, as well as those with a typical 9:22 translocation without overall chromosomal gain or loss. No other prognostic factors were identified. Etoposide plus TBI seems to be a better overall form of conditioning in terms of outcome.

Results: After a median follow-up of 106 months (range 3.5-172), outcome of patients is summarized in the following table. ALLO-SCT OS (3-yr) : 40%(SE:10), 21%(SE:10) and 12%(SE:9) for 2nd CR(n=25), <20 %blasts (n=19), and >20% blasts (n=18), respectively. Relapse risk: 31%(SE:13)*, 52%(SE:14), and 65%(SE:18) for 2nd CR, <20% blasts, and >20% blasts, respectively AUTO-SCT OS (3-yr) : 28%(SE:9) for 2nd CR (n=25) Relapse risk: 59%(SE:12)* for 2nd CR *p=0.04 Following allo-SCT, prognostic factors for a lower risk of relapse were the status of disease at SCT (second CR vs active disease, p=0.02), a longer duration of first CR (>12 months, p=0.02), and development of acute GVHD (p=0.05). With regard to autologous SCT, acute promyelocytic leukemia (APL) was the only variable that showed a trend (p=0.1) for a lower risk of relapse. Conclusions: As compared to auto-SCT, allo-SCT is associated with a lower risk of relapse in patients with AML in second CR, although the high toxicity of the procedure may overcome its benefit. Therefore, the treatment of choice for patients with advanced AML should be individualized according to specific risk of the disease and patient's characteristics. It is supposed, that high percentage (%) of CD34+ cell in autologous peripheral blood stem cell (PBSC) products correlate with relapse rate in AML patients (pts.) (N Feller at al, Leukemia 2003) . We analyzed correlation between the % of CD34+ cells in peripheral blood and relapse incidence in AML pts. Patients and methods: We assessed 25 pts. with de novo AML (13 F, 12M), age 18-58 (med.43) from the period 1995-2003 irrespective whether pts. were transplanted or not (21 transplant and 4 non-transplant pts). All pts. were treated with consistent protocol: the first cycle of chemotherapy consisted of cytarabine (100 mg/m 2 /day, 1.-7.) and idarubicin (12mg/m 2 /day, 1.-3), the second and third cycles included cytarabine (2x3000 mg/m 2 /day, 1.-4.) and mitoxantrone (12 mg/m 2 /day, 5.-7.). After reaching complete remission and when the granulocyte count had risen to 0.5 x10 9 /l, filgrastim (10ug/m 2 /day) was started in all patients. The % of CD34+ cells in blood was measured after the granulocyte count reached 1.0 x 10 9 /l. Results: The highest measured % of CD34+ cells in blood was correlated with EFS and OS. A high % of CD34+ cells in blood significantly correlated with shorter EFS. At a cut-off level of 0,72% CD34+ cells, which is the median percentage of all measurement (range 0,18-6,6%), the EFS was significantly lower (p=0,05) in the group of pts. with more than 0.72% CD34+ cells compared with less than 0.72% CD34+cells at a median FU of 35 months . The difference in OS was not significant (P = 0.14). The relapse rate at a median EFS of 39 months (15-100) was significantly (P = 0.04) higher in the group of pts. with more than 0.72% of CD34+ cells compared with the group with less than 0.72% CD34+ cells (84% vs 34%). The total number of CD34+ cells/kg in PBSC products correlated with EFS: differences were significant (p=0.0336) at a cut-off level of 3 x 106 CD34+ cells/kg (88% vs 59%). Conclusion: Although no MRD data are available in our study, we assume that massive mobilization of CD34+ cells and high frequency of relapses in the subgroup of pts. with CD34 more than 0.72% indicate an insufficient in vivo purging, as well as low chemotherapeutic bone marrow toxicity. It is concluded that a high percentage of CD34+ cells in blood and also a high total number of CD34+ cells/kg in PBSC products are a poor prognostic factors for pts. with AML. This subgroup of pts. might benefit from a more intensive chemoterapeutic treatment.

Granulocytic sarcoma in childhood acute myelogenous leukaemia: clinical characteristics and management H. Kook, C. Oh, S.Y. Kim, S.J. Kim, H. Noh, H. Nam, T. Hwang, Chonnam National University Hospital (Gwangju, KOR) Granulocytic sarcoma (GS), an extramedullary tumor consisting of primitive myeloid cells is rare manifestation of acute myeloblastic leukemia. However, GS can occasionally precede the development of systemic leukemia by weeks to years. The objectives of this study were to describe the frequency, clinical characteristics and survival of AML children with GS from a single Korean institute. Retrospective review of all the AML children who presented between January 1995 and June 2003 was undertaken. GS occurred in 9 children among 118 AML patients (incidence = 7.6%). the age at the diagnosis of AML were ranged from 8 months to 13 years (median age = 82 months) with equal sexual distribution. The site of GS were scalp (n=4), paranasal sinuses (n=1), skull (n=3), external auditory canal (n=1), spinal epidura (n=1), and spinal intramedulla (n=1). The symptoms related with GS were scalp mass(n=4), paraparesis(n=3), facial nerve palsy(n=3), hearing impairment(n=2), exophthalmos(n=1). In the case with spinal epidural mass, GS preceded the diagnosis of AML by 15 months. Cytogenetics were available in 8 cases, and t(8;21) was found in 5. Surgical decompression and radiotherapy along with chemotherapy were given to 2 patients with spinal involvement. However, both patients succumbed to treatment-related toxicities. Seven cases received stem cell transplantations (3, allogeneic bone marrow; 4, autologous peripheral blood). 5-yr EFS was 35% by Kaplan-Meier survival curve of the 9 patients. Among 7 transplanted patients 3 allografted patients are alive (86 mo, 5 mo, 1 mo), while 3 of 4 autografted patients were either died or relapsed. In conclusion, GS should be considered in cases with or even without AML. Early diagnosis and multimodality management, preferentially allogeneic stem cell transplant, should be attempted to achieve a long-lasting cure of this rare presentation.

Haematopoietic stem cell transplantation in treatment of adult patients with acute myeloid leukaemia in single-centre experience: results and risk factor analysis L. Gil, A. Czyz, A. Lojko, K. Sawinski, M. Kozlowska-Skrzypczak, M. Komarnicki, K. Marcinkowski University (Poznan, PL) Hematopoietic stem cell transplantation (HSCT) is accepted form of post-remission treatment in adults with acute leukemias. We present results of allogeneic and autologous HSCT in 89 pts with AML, followed-up for 1-152 months. According to transplantation method all pts were divided into 3 groups. In allogeneic group 32 pts (median age 30, range 15-47 yrs) were conditioned with high dose chemotherapy (HD-allo): BuCy2 (25) or BuCy2+VP16 (7). Twenty five pts were in CR1, 2 in CR2 and 5 in relapsed/refractory disease. PBSC received 5 pts and 27 were grafted with bone marrow HSC. Transplanted material contained 3.2(1.2-5.1)x10 6 /kg CD34+ cells. Fifteen pts (median age 51, r 30-65 yrs) were treated with reduced intensity conditioning (RICallo): fludarabine-based (11) or low-dose TBI (4). This group comprised 10 pts in CR1, 3 in CR2 and 2 with refractory disease. Patients were grafted with PBSC, containing 3.6(1-6.1)x10 6 /kg CD34+. Autologous HSCT was applied to 42 pts (median age 37, r 16-56 yrs). Thirty five pts were in CR1, 5 in CR2 and 2 in more advanced stage. For conditioning BuCy2 (36) or BuCy2+VP16 (6) were given. Eleven pts were transplanted with PBSC and 31 with bone marrow stem cells; graft contained median 2,7(2-3,9)x10 6 /kg CD34+ cells.

Results: hematopoietic recovery occurred in 94% in HD-allo and 93% in auto-HSCT groups. All pts in RIC-allo group achieved mixed or complete donor chimerism by day +90 after transplant. Mean survival and probabilities of 5 yrs-overall survival (pOS), relapse and non-relapse mortality (Kaplan-Meier method) with respect to transplantation method, are shown in Table. In Cox model, for whole group (n=89), the stage of the disease >=CR2 was the only independent prognostic factor for OS by multivariate analysis (p=0.032; hazard ratio (HR)=1.5). Factors predicting OS by multivariate analysis in respective subgroups were: in HD-allo group, aGVHD>=2 (p=0.047, HR=0.54), cGVHD (p=0.0007, HR=3.62), disease stage >=CR2 (p=0.012, HR=1.97); and in auto-HSCT group, relapse after HSCT (p=0.013, HR=1.72). In RIC-allo group, although being nondiscriminative, cGHVD was the strongest independent factor (p=0.09, HR=1.92).In conclusion, results of HSCT in adult AML are similar, regardless of the method of transplantation The relapsed ALL have a poor prognosis and only 10% of these pts are alive at five years from relapse. So would be very important to explore new protocols of reinduction chemotherapy in order to perform a subsequent allogenic transplant in a condition of remission. Here we resume our experience in this setting using Liposomal Daunorubicin (Daunoxome-DNX) and Aracytin (Ara-C) as reinduction chemotherapy before Matched Unrelated Bone Marrow Transplantation (MUD-BMT). Patients: 10 relapsed ALL pts (8 B phenotype, 2 T phenotype), 5 males and 5 females, median age 28 yrs (range 17-48); 7/10 pts were at first relapse and 3/10 at second or subsequent relapse. All pts (10/10) had one or more adverse prognostic factors at diagnosis and all of them had been treated with antracyclines with a median cumulative dose of 233 mg/m 2 (range 55-336). All pts had received a reinduction chemotherapy before transplant with DNX 80 (6/10) or 100 (4/10) mg/m 2 , days 1-3 plus Ara-C 2 g/m 2 , days 1-5. Eight pts (80%) achieved a complete remission (CR) and 1/10 (10%) a partial remission (PR) for an overall response rate of 90%; one pt was resistant to reinduction therapy. DNX plus Ara-C has been well tolerate without cardiotoxicity. All pts in CR or PR obtained a fast hematologic recovery. MUD transplant was performed after a median of 3 mths (range 1-5) from DNX based reinduction chemotherapy. At the time of transplant 7/10 (70%) pts were still in complete remission, 2 were relapsed, 1 was resistant. All pts received Total Body Irradiation plus cyclophosphamide as conditioning regimen and immunosuppression with cyclosporin and metotrexate. 10/10 (100%) pts achieved a CR after transplant. The median follow-up from transplant was 10 mths (range 3-41); 4/10 pts (40%) are alive and 6/10 (60%) are death: 3/6 for TRM in a condition of CR (acute GVHD in 2 pts and infection in 1 pt) and 3/6 for leukemia relapse. The OS from diagnosis was 50% at 38 mths. The OS from MUD-BMT was 50% at 9 mths. The RFS after MUD-BMT was 65% at 6 mths. These data, taking into account the small number of cases, would confirme that: a) despite the adverse prognosis of relapsed ALL pts, MUD-BMT can rescue a part of them; b) TRM is still the most important problem and needs to be reduced; c) DNX and Ara-C might be a good and well tolerated reinduction chemotherapy for poor-risk ALL that allows to reach the transplantation procedure in a condition of remission. In years 2000-2003, 16 leukaemic patients were transplanted from MUDonors -All patients received conditioning regimen based on Busulphan+Cyclophosphamide (total dose of Bu 16mg/kg; Cy 120 -200 mg/kg) with addition of ATG (12 cases ) or Campath (4 cases ). * ATG cases ( 3 females, 9 males, median age 23 y, 10 ALL, 1 AML, 1 CML, 9 in CR1, 2 in CR2, 1 in chronic phase) received total dose 10mg/kg of ATG. Regimen related toxicity (RRT) >2 was seen in 6 (50%) patients;

number of CD34+ cells transplanted ranged from 1,2 to 10,6 x 10 6 /kg ( median 6,4); hematological recovery was recorded: ANC: 12 patients (100%; mean day +11); PLT: 10 patients (83%; day+20); acute GvH >II in 3 patients (25%); reactivation of viral infections was diagnosed in 3 patients (25%); 5 patients (42%) are alive from 455 to 1315 (median 766 ) days post transplant, four in complete remission (CR) and complete chimerism. Death cause : 4 infections, 1 relapse, 1 aGvHD. * Campath cases ( all male, median age 23y, 3 ALL, 1 AML, 3 in CR1, 1 in CR2 ) received total absolute dose of 20 mg Campath. RRT>2 was seen in 1 case (25%); number of CD34+ cells transplanted ranged from 3,2 to 11,4 x 10 6 /kg ( median 10,0 ); hematological recovery was seen in all patients: ANC: mean day +20, PLT: +18; there was no acute GvH >II case ; reactivation of viral infection was seen in 1 patient ( EBV reactivation with lymphoproliferative syndrome ); all patients were alive from 63 to 238 days (median 158) post transplant with CR and complete chimerism. Summary: in both groups pace of hematological recovery was comparable and all patients achieved full chimerism. The differences in toxicity, aGvHD and survival suggest further study on the effectiveness of Campath and ATG in conditioning of MUD transplants. Since 1999, 39 acute myeloid leukemia (AML) patients in I CR received PBSCT after BU-CY conditioning regimen. All patients received high-dose hydroxyurea plus daunorubicin, etoposide and cytarabine (DCE schedule) as induction treatment, and daunorubicin and cytarabine (DIA schedule) as consolidation, according to AML12 EORTC-GIMEMA Protocol. Twenty-three patients were males and 16 females, median age was 41 years (range 17-61). The median number of CD34+ cells reinfused was 2.7x10 6 /Kg (range 2-54x10 6 /Kg). Recovery of PMN (>0.5X109/L) was observed after a median of 14 days (range 11-17) from transplant and it was stable during the time for all patients. Seven/39 patients are not evaluable for PLTS reconstitution (>50x10 9 /L) because of one died (on d+52) for toxicity, two relapsed 4 months after transplant without PLTS recovery and four are still thrombocitopenic in complete remission 1, 2, 2 and 4 months after autograft. Among the 32 evaluable patients, the PLTS recovery was observed after a median of 20 days (range 11-209). Six/32 patients showed a late PLTS recovery at +90, +115, +120, +140, +153 and +209 days from transplant, while 26 patients showed the PLTS reconstitution after a median of 18 days (range S242 11-70) after autograft. In 15/32 (47%) patients a marked decrease of platelets count, (below 50x10 9 /L) was observed after a median of 54 days post transplant (range 26-77). The median minimum value achieved was 30x10 9 /L (range 10-39x10 9 /L) and the thrombocytopenia lasted for a median of 31 days (range 10-174). Among the 32 patients a stable PLTS reconstitution (>50x10 9 /L) was achieved after a median of 80 days (range 11-209) post autograft. Has been widely reported that PBSCT is characterized by a fast recovery of PMN and PLTS compared to autologous bone marrow; however, our experience shows that the PLTS recovery is unsustained, because can give a rapid rise followed by a secondary fall in the most of patients. This has to be taken in the account when a program with IL2 is planned after an autograft.

The impact of the source of stem cells on the outcome of autologous haematopoietic progenitor transplantation (HPT) in patients with AML J. Berlanga, D. Gallardo, D. Benéitez, M. Hermosilla, M. Encuentra, Institut Català d'Oncologia (Barcelona, E) Introduction: There has been concern about mobilisation of myeloid leukemia cells with G-CSF in AML, autologous HPT could be associated with a higher risk of relapse if peripheral blood instead of bone marrow, is used as a progenitor source. Methods: In a single institution 25 patients diagnosed with AML and undergoing an autologous HPT were studied. From June-95 to February-98 eleven HPT were performed with BM cells, and from March-98 to December-01 fourteen HTP were performed with PB cells. The median age were 42 (17-62) and 44 (17-62), the time from diagnosis to transplant were 5 months in both groups, the patients in 1st/2nd CR were 11/0 and 12/2 respectively. There were two patients with favourable cytogenetics in the BM group and none in the PB group. Results: The median time to achieve a neutrophil count > 1x10 9 /L were 41(23-193) and 13 (11-384) (p<0.001) for BM and PB groups, and platelet count >20x10 9 /L were 59 (37-1266) and 22 (11-384) (p=0.002). The incidence of documented infections were 6/11 (55%) for BM group and 2/12 (14%) for PB group (p=0.081). Amphotericin B administration was required in 55% (6/11) and 7% (1/14) patients from BM and PB groups respectively (p=0.007). Graft-failure was diagnosed in none patient with BM and in 1 patient with PB transplant. The transplant related mortality were 0% for both groups. At a median follow-up of 76 (7-102) months for BM and 15 (2-46) months for PB patients (p=0.009), the EFS were 64% and 16% respectively (p=0.019). The relapse rate were 36% (4/11) and 71% (10/14) (p=0.116), and the OS were 73% and 42% for HPT with BM and PB stem cells (p=NS). Conclusion: The use of BM as a source of hematopoietic progenitor cells resulted in higher toxicity than PB, however it seemed to be associated with a lower relapse rate. Randomised studies should be performed in order to identify the best source of progenitor cells for autologous transplant in AML. Conditioning regimens were Bu+Cy, Bu+Cy+VP16, Bu+Cy+HD Ara-C, Ida+Bu+Cy, TACC, TAACC, BEAM. After different programs CH from 50% to 92% (an average 74%) AML pts and from 55% to 91% (an average 75%) ALL pts have achieved CR. 10-years relapse free survival (RFS) standard risk (SR) and high risk (HR)AML pts with high dose CH is 35% and 18% accordingly. 10-years RFS SR and HR ALL pts with high dose CH is 30% and 15% accordingly. 10-years RFS AL CR1 pts after allogeneic SCT is 60%. 10-years RFS SR and HR AML CR1 pts after autologous SCT is 71% and 40% accordingly. 10years RFS SR and HR ALL CR1 pts after autologous SCT is 46% and 17% accordingly. SCT in group AL pts with early relapse, PR, CR2 was not effective. Overall 5-years survival AL pts without CR1 after SCT is 7%. Intensity of chemotherapy during induction CR, number of consolidation courses, intensity of chemotherapy and especially SCT were more significant than such standard prognostic factors as age, subtypes of AL, high level of leukocytes, CH courses and time to CR in relation achievement of CR, duration of CR, relapse and OS. We conclude that SCT improve result CH in AL pts. The best time for SCT is CR1. Autologous SCT pts HR ALL have not advantage over the high dose CH. Pts with a poor response to conventional chemotherapy having bad prognosis after SCT. Patients with relapsed and refractory acute lymphoblastic leukaemia (ALL) have a poor prognosis and a little chance to obtain complete remission and receive bone marrow transplantation. We describe 13 patients (8 female and 5 male; median age 29.6) with relapsed or refractory ALL, who received fludarabine-based regimens (2 -FLAG: fludarabine, cytarabine, G-CSF; 3 -FLAG-Mit: fludarabine, cytarabine, G-CSF, mitoxantrone; 8 -FLAM: fludarabine, cytarabine, mitoxantrone). The immunological types of leukaemia were following: null -1, pro-T -1, pre-T -1, common -3, pro-B -1, pre-B -6. Three patients belonged to very high risk group with Philadelphia chromosome. Complete remission (CR) was achieved by 6 patients (41.6%). The median duration of ANC < 0.5 G/l was 23. 2 days (14-31) , of PLT< 20.0 G/l was 26.6 days (19-45). One patient died early because of severe pneumonia. Toxicity III/IV included infection (69%) and mucositis (15%). Three patients who obtained CR underwent allogeneic bone marrow transplantation (AlloBMT) and one autologous peripheral blood progenitor cells transplantation (AutoPBPCT). Four patients relapsed (1 after AlloBMT, 1 after AutoPBPCT and two waiting on transplantation); two patients (after alloBMT) are still in CR (37 and 8 months). We conclude that fludarabine-based regimens are very valuable therapeutic option for refractory and relapsed ALL.

H. Horst, A. Humpe, R. Schoch, R. Siebert, S. Gesk, M. Kneba, D. Hoelzer, N. Goekbuget, University Schleswig-Holstein, University Frankfurt (Kiel, Frankfurt, D) In adult T acute lymphoblastic leukemia (T-ALL) the results of first line therapy could signifi-cantly be improved. In the GMALLstudy 5/93 the relapse-free survival rate at five years was 51 % (Goekbuget et al, Blood 2001, 98:802a) . After relapse, however, treatment options are very limited. Experimental data showed promising results for arabinosylguanine (araG, GW506U78), a new nucleoside analogue that is particularly active in Tlymphocytic cells.An observational study showed a complete remission (CR) rate after one or two courses of GW506U78 monotherapy in 9 of 16 patients with T-ALL. Treatment was well tolerated with slight bone marrow (BM) suppression and increase of liver parameters being the most frequent side effects (Goekbuget et al, Hematol J 2003, 4:S266) . We report the selective cytotoxic effect of GW506U78 in two heavily pretreated patients who relapsed after allo SCT. Main characteristics of the patients are shown in the table. Patient #1 was transplanted in CR after the 2nd relapse. Two months after a matched unrelated donor (MUD) SCT he showed complete donor chimerism. Four months after SCT a relapse of the T-ALL was diagnosed in the BM. At this time a mixed chimerism with only 37.7 % donor cells was diagnosed. After salvage therapy with one cycle of GW506U78 the patient reached a CR. The BM fully recovered and showed complete donor chimerism (>95 %, without recipient signal). Two months later the patient relapsed again and died of the ALL. Patient #2 was transplanted in CR after the 1st relapse of a T-ALL. Three months after MUD SCT a relapse of the T-ALL was diagnosed. Surprisingly besides the T lymphoblasts an additional malignant population comprising of myeloid blasts (POX + EST positive) was detected in the BM. After salvage therapy with one cycle of GW506U78 the BM was almost completely depleted of lymphoblasts, the amount of myeloid blasts, however, was not affected by GW506U78. Cytogenetically the per-sisting leukemia showed the same chromosomal alteration (del 17p) as the T-ALL at the time of first relapse. The patient died one month later from the finally uncontrolled leukemia. Our findings indicate that GW506U78 is highly effective even in heavily pretreated patients with a relapse of T-ALL after MUD SCT. In our patients the cytotoxic effect, however, was restricted to the T-ALL population while myeloid blasts with the same cytogenetic aberration as the T-ALL were not affected by treatment with GW506U78. The aim of this study was to evaluate Ga-67 scintigraphy, performed before an at day +100 after stem cell transplant (SCT) as means to predict ocutcome in patients with high grade Non Hodgkin´s lymphoma (HGNHL). Sixty-four patients with HG NHL were retrospectively examined. Fifty five (85,9%) had Diffuse B Large Cell (DBLCNHL) and 9(14,1%) had Burkitt lymphoma. Sixty two patients recieved autologous (ASCT) and 2 allogeneic (AlloSCT) from macthed related donor. Fifty three patients underwent 67Ga scintigraphy in the evaluation prior to SCT and 59 on day +100 after infusion. Whole body planar imaging and single photon emission computed tomography (SPECT) of regions of interes were performed at 72 hours after injection of 259 MBq (7 mCi) of 67Ga citrate. Regarding pretrasplant evaluation, 33(86,8%) patients with negative and 5(13,2%) patients with positive 67Ga scans were in CR at last follow up (p<0.01). In addition, 5(13,2%) patients with negative and 10 (66,7%) patients with positive 67Ga prior to SCT relapsed after trasplantation (p<0,01). Statistically significant differences in terms of OS(91,58% vs 10,11% at 77 months respectively; p<0.01) and DFS(86% vs 28% at 77 months, respectivily; p<0,01) were observed between patients with positive and negative 67Ga scans. Concerning posttransplant Ga-67, 38(82,6%) of patients with negative and 8(61,5%) patients with positive 67Ga scans remained in CR at last follow up (p=0.03). In addiction, 8(17,4%) patients with negative and 5(38,5%) patients with a positive result relapsed (p=0,03). Again, statistically significant differences were observed in terms of OS(75,56% vs 26,67% at 77 months respectivily; p<0.01) and DFS(75,23% vs 53,71% at 77 months respectivily; p<0.03) between patients with positive and negative 67Ga scans. Analizing the 8 patients with a positive result posttransplant who remained in CR, 3 of them had Ga-67 positive at bone, 2 recived radioterapy and remained on CR with negative gallium and 3 had been considered as a false positive result. In conclusion, 67Ga scintigraphy performed either before or at day +100 after hematopoietic stem cell transplantation is a significant prognostic factor among patients with high grade Non Hodgkin Lymphoma. Special attention should be paid to Ga-67 positive in some areas as bone or hiliar captation because they could be false positive results. The prognosis of non-Hodgkin's lymphoma (NHL) patients who relapse after autologous stem cell transplantation (ASCT) is considered poor. There are no standard treatment modalities for these patients. We retrospectively analysed treatment, prognostic factors and outcome in 115 consecutive NHL patients transplanted 1991-2000 in Finnish centres and who had relapsed by April 2001. There were 39 females and 76 males with a median age of 49 years at ASCT. Histology included large cell B 51 (44%), follicular 25 (22%), mantle cell 15 (13%), T cell 15 (13%) or other 9 (8%) subtype. The median time from ASCT to relapse was 7 months. At relapse LD was elevated in 68 patients (59 %) and 74 of the patients (64 %) had stage III-IV disease. Altogether 95 (83%) patients received salvage treatment. Of these patients 24 (25%) achieved CR and 29 (31%) PR. Rituximab was given to 11 patients. Four patients received an allogeneic transplant and three 2nd ASCT. Overall survival was 8 months (range 0-84) and the estimated 4-year survival was 22%. In univariate analysis factors associated with survival were histological subtype, Ann Arbor stage at relapse, IPI and LD at relapse, Rituximab treatment at relapse, time from ASCT to relapse and response to salvage treatment. In multivariate analysis factors predicting survival were normal LD at relapse (p=0.002), response to salvage treatment (p<0.001) and time from ASCT to relapse >7months (p=0.017). In general, the prognosis of patients who relapse after ASCT is poor. However, many patients will response to current therapies, and some may enjoy prolonged survival. Rituximab seems to be a useful adjunct to patients who relapse after ASCT. On the other hand, for patients who relapse early or have high-risk features at relapse, novel treatment regimens are needed.

Introduction: Combination chemotherapy can cure patients with Non-Hodgkin's lymphoma (NHL), but those with treatment failure or relapse still have a poor prognosis. High-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) can improve the outcome of these patients. Chemosensitivity and achievement of minimal disease status prior o HDCT are important prognostic factors in NHL patients treated with these regimens. We therefore evaluated an intensified salvage program with a final myeloablative course. Patients and Methods: Inclusion criteria were age 18-65 years, histologically proven primary progressive or relapsed aggressive NHL and eligibility for HDCT. Treatment program consisted of two cycles DHAP (dexamethasone, cytarabine, cisplatin); patients with CR or PR received cyclophosphamide (4g/m 2 ) followed PBSC harvest; methotrexate (8g/m 2 ) plus vincristine (1,4mg/m 2 ) and etoposide (2g/m 2 ). The final myeloablative course was BEAM followed by ASCT. Results: 57 patients (median aged 43 years, range 24-65) were enrolled: 23 (40%) patients were refractory to pimary therapy and 34 (60%) patients had relapse of NHL. At 22 months of median follow-up (range 1-71 months) results are as follows: the response rate (RR) at the final evaluation (100 days after transplantation) was 45,6% (31,6% CR, 14% PR). Toxicity was tolerable. The freedom from treatment failure (FFTF) and overall survival (OS) were 20% and 40% for all patients, respectively. FFTF and OS for patients with relapse were: 38% and 58%; for progressive disease: 8% and 20%, respectively. Conclusion: We conclude that this regimen is feasible, tolerable and effective in patients with relapsed NHL. In contrast the results in patients with progressive disease are unsatisfactory. This program is currently being modified by addition of rituximab for patients with relapsed aggressive NHL. For patients with progress after primary polychemotherapy other treatment regimens have to be investigated.

In order to improve the outcome of patients with follicular lymphoma (FL), we designed a double intensive regimen supported by autologous stem cells transplantation. Study design was: i) 2 monthly cycles of C2H2OP (Cyclophosphamide: 3g/m 2 D1; Adriamycin: 75 mg/m 2 D1; Vincristin: 2 mg D1 and Prednisone 100 mg/d D1-D5) and GM-CSF each followed by peripheral blood stem cells collection (PBSC1 and 2); ii) first intensification with melphalan 140 mg/m 2 (Mel 140) supported by PBSC1 and GM-CSF; iii) bone marrow harvest; iv) second intensification with Cyclophosphamide (120 mg/kg) and TBI (12 Gy, 3 fractions) (Cy-TBI) supported by the bone marrow harvest, PBSC2 and GM-CSF, v) interferon-a (3.106 UI, 3d/weeks). From 02/1994 to 10/1995, 36 patients (pts) have been enrolled in this study. Main pts characteristics were: median age = 47y (range 25-59); gender = 24 males and 12 females, previously untreated = 26; first relapse = 10; stage III = 7; stage IV = 29; bulky disease = 10; high LDH = 9. Mel 140 was performed in 35/36 pts. Median duration of neutropenia (< 0.5. 10 9 /L) and thrombopenia (< 20. 10 9 /L) were 7 days (4-17) and 1 day (0-15), respectively. 50 % of pts experienced a mean time of 1 day with fever (0-6). Cy-TBI was performed in 35 pts. Median duration of neutropenia and thrombopenia were 12 days (9-32) and 9 days (0-60), respectively. 90 % of pts experienced a mean time of 4 days with fever (0-15). There was no treatment related mortality. IFN-a was started in 19 pts, and mean time of treatment was 12 months (1.5-25) in 12 pts. Complete response after each phase of treatment, was obtained in 11/36; 22/35 and 33/35 after C2H2OP, Mel 140 and CY-TBI, respectively. Overall complete response rate was 94 % (34/36) at the end of the procedure. Second tumours were observed in 3 pts (1 MDS, 1 in situ melanoma, 1 breast cancer). With a median follow-up of 80 months, disease free survival and overall survival are 60% and 83%, respectively. Thus, with a long median follow up, this study shows that tandem transplant for FL is safe and could be curative. Objectives: Several studies have shown that dose intensification of etoposide from 800 to 1600 mg/m 2 in BEAM regimen is not associated with greater early toxicity, but information regarding the impact of the escalated etoposide dose on late toxicity or survival is lacking, and it constitutes the main objective of the present single-centre sequential-cohort analysis. Methods: 131 consecutive lymphoma patients (pt) treated with the BEAM regimen and autologous stem-cell transplantation (ASCT) were included in the study. Patients were subdivided into indolent lymphomas (IL) (28 pt), aggressive lymphomas (AL) (50 pt) and Hodgkin s disease (HD) (53 pt). Mantle-cell, lymphoblastic and Burkitt s lymphoma cases were excluded. Two sequential cohorts were identified for comparison: from May/90 to April/95, 67 patients received BEAM at standard doses (sBEAM), while from May/95 to June/99, 64 subsequent patients received escalated doses of etoposide (from 800 to 1600 mg/m2) within the BEAM protocol (eBEAM). Results: Concerning neutrophil and platelet recovery, comparison stratified for stem cell source (stratified Kaplan-Meier) showed no significant difference between sBEAM and eBEAM groups. Early (within 100 days) and late transplantrelated mortality were similar in both groups (3% and 6% vs 7.8% and 6.2% in sBEAM and eBEAM groups, respectively), as were the incidence of secondary malignancies (3.9% vs 4% at five years). Considering IL patients separately, progression of disease after transplant was significantly lower in eBEAM group as compared to sBEAM (7% vs 43% of patients, respectively), although this was not associated with an improvement in survival, due to a higher toxic mortality in the eBEAM group. In HD cases, the 5-year event-free survival (EFS) and overall survival (OS) was better in the eBEAM group (70% and 77%, respectively) as compared to sBEAM (58% and 69%, respectively), but the difference did not reach statistical significance. In AL patients, we did not find any significant difference in response rates or survival between either group. Conclusions: The data derived from this study does not indicate that the escalated etoposide doses in the BEAM conditioning regimen improves the results of conventional BEAM for ASCT in lymphoma patients, although results in IL and HD are encouraging and may warrant further studies.

B-DLCL patients after ASCT failure have a very poor prognosis, salvage chemotherapy or a second transplantation cannot substantially modify the very poor outcome which is characterized by a median overall survival (OS) of 3 months. From 1999 to 2003 we observed 23 B-DLCL patients after ASCT failure. Fourteen patients (60.8%) were eligible for our salvage protocol in which inclusion criteria were: B-DLCL CD20+, P.S (WHO)= 0-2, age < 70 years, relapse or progressive disease (PD) after ASCT, measurable disease, absence of severe organ dysfunction, previous doxorubicin cumulative dose > 300 mg/sm, no previous Rituximab (R) therapy or CNS involvement. The median age was 47 (28-66) years; the P.S. (WHO) was 0-1 in 9 patients and 2 in five; the disease status was 12 relapses and 2 PD after ASCT. The DC-R + GM-CSF schema (every three weeks) consisting in: R 375 mg/sm day 1 and 15, Caelyx 30 mg/sm and cyclophosphamide 750 mg/sm day 1, GM-CSF 150 mg/day from day 5 until neutrophils recovery. Patients showing disease progression after two courses were excluded while the responders received two more courses; patients achieving complete remission (CR) after 4 courses did not receive any further treatment. All 14 patients received the planned treatment and were evaluable for response: the overall response rate (ORR) was 64% with 8 CR (57%) , 5 patients showed a PD after 1-2 courses. The toxicity (WHO) consisted in: grade III-IV neutropenia in 8 patients (57%) and thrombocytopenia in 2 patients (14%), grade I-II infectious in 2 patients and grade IV (pneumonia) in one patient. With a median follow up of 17.7 months the 2-years projected event-free survival and OS were 36% and 34%, 7 out of 8 patients in CR are alive and disease-free at +3, +4, + 9, +14, +21, +32, +50 months from the end of salvage therapy and 1 patients died at + 20 months of meningococcal meningitis. Our experience shows that this is an effective salvage treatment for B-DLCL after ASCT failure; indeed the follow up > 12 months in 4/14 (28%) patients in CR suggests a chance of cure.

F. Gaudio, V. Pavone, A. Guarini, T. Perrone, P. Curci, A. Giordano, R. De Francesco, V. Liso, University of Bari (Bari, I) Diffuse large cells lymphomas (DLCL) constitute about 30% of all non Hodgkin lymphoma (NHL). Initial therapy of DLCL with antracycline containing chemotherapy regimens cures approximately 40-50% of patients (pts). However about 40% of pts either will be refractory to initial therapy or will relapse from a clinical complete response. The International prognostic index (IPI) is a validated scoring system predictive of survival in de novo DLCL. Pts with aggressive NHL, mainly with poor IPI, may require intensive and extensive therapy. Objectives: The current study was conducted to analyse the outcome of pts with DLCL treated with high dose chemotherapy (HDC) and autologous stem cell transplantation (ASCT) in partial remission (PR) after ProMACE/CytaBOM first line treatment. Methods: From march 1994 thirty pts (median age 45 years) in partial remission to first line regimen consisted in ProMACE/CytaBOM protocol underwent HDC. Stage III-IV was in 25 pts (83,3%), B-symptoms in 19 (63,3%); High LDH serum level in 15 (50%); High beta2microgl. serum level in 13 (43,3%); extranodal disease in 15 (50%); bulky disease in 19 (63,3%); bone marrow involvement in 16 (53,3%). IPI score was 2 or 3 in 17 (56,6%). Conditioning regimen consisted of BEAM (carmustine, etoposide, cytarabine and melphalan) in all patients. Results: 21 pts (70%) achieved complete remission, 4 pts (13,3%) remained in PR, 5 pts (16,6%) had progressive disease. One patient died for an acute distress respiratory syndrome (TRM:3,3%) With a median follow-up of 75 months, 13 pts (43,3%) were alive without disease; 7 (23,3%) relapsed (median time 20 months), 12 (40%) died mainly due to disease progression (9 pts). With a median follow-up of 75 months, the overall survival (OS) rate was 54% and the event free survival (EFS) rate was 46% ( fig.1) . In univariate analysis, only bone marrow involvement and bulky disease at diagnosis significantly influenced OS and EFS (p<0.05). Conclusion: Pts with DLCL in partial remission after ProMACE/CytaBOM regimen may achieve prolonged OS and EFS by HDC and ASCT. Bulky disease an bone marrow involvement negatively influence the outcome of this subset of pts. More investigative programs, including prospective randomized trials, are needed to assess the efficacy of different HDC approach (double transplant or high dose sequential chemotherapy and ASCT in pts with DLCL with adverse prognostic factors mainly in early phase of treatment plane.

A. Reichle, A. Berand, E. Holler, R. Andreesen, University Regensburg (Regensburg, D) Feasibility and high efficacy of repetitive dose-intensive chemoimmuno-therapy in relapsed and refractory aggressive NHL (adjusted IPI at relapse 2 and 3) was proven by double-induction followed by tandem stem cell transplantation including a treosulfan-based conditioning regimen. For cytoreduction and stem cell mobilisation, 2 cycles VIPE were applied followed by two identical cycles of high-dose chemotherapy (HD-CT) consisting of treosulfan 14 g/m 2 iv day -4 to day -2, carboplatin 300 mg/m 2 iv day -4 to day -2 and etoposide 500 mg/m 2 iv day -2 to day -4. In B-NHLs each cycle was combined with rituximab 375 mg/m 2 . So far, 23 patients (pts) mean 49 years (range 22-65), stage III: n=3, stage IV: n=20, have been enrolled, 5 pts with early relapse (within 6 months), 10 with refractory disease and no available matched related or unrelated donor, 8 pts with late relapse (> 6 months). All patients with NHL received previously CHOP-based CT. Histology revealed diffuse-large cell lymphoma (n=17), Hodgkins' lymphoma (n=3), and pleomorphic T-cell lymphoma (n=3). Only one stem cell mobilization was necessary to collect sufficient CD34+ cells for two transplantations. Median hematologic recovery (> 1.0 leukocytes/nl and platelets >20/nl) after 1st and 2nd HD-CT was achieved by day 10 (8-11). No therapy-related death occurred. CTC °III and °IV nonhematologic toxicities were as follows: 9 of 23 pts after 1st HD-CT had °III toxicities (infection, vomiting, enteritis, stomatitis, diarrhea), after 2nd HD-CT 9 of 21 pts, respectively. Complete remission (16 of 20 pts, 80%) was achieved after doubleinduction (n=1), 1st HD-CT (n=9), and 2nd HD-CT (n=9), PR in 4 S247 pts, and no pt had progressive disease. CR after doubleinduction and 1st HD-CT was followed by continuous CR (cCR, 6 to 26 months, mean 11,5 months) in 9 of 11 cases (82%), after 2nd HD-CT in 5 of 9 cases (55%). At a median observation time of 11 months 20 pts (87%) are alive, and event-free survival at 2 years is 57%. Tandem treosulfan-based high-dose CT is feasible with manageable toxicity profile. CR and cCR rates argue in favor for a dose-response relationship even in high-risk patients with aggressive lymphoma.

Ifosfamide, carboplatin and etoposide for autologous stem cell transplantation in non-Hodgkin lymphomas L. Nassi, L. Rigacci, S. Guidi, R. Alterini, V. Carrai, C. Nozzoli, R. Saccardi, L. Lombardini, A.M. Vannucchi, A. Bosi, Ospedale di Careggi (Florence, I) Objective: Several conditioning regimens are used for autologous stem cell transplantation (ASCT) in non-Hodgkin's lymphomas (NHL). Since March 1994 we treated NHL patients (pts) undergoing ASCT with ifosfamide, carboplatin and etoposide (ICE) as conditioning regimen. Methods: We analysed 73 consecutive NHL pts who underwent ASCT using ICE as conditioning regimen. 37 pts were male, 36 female. A high-grade histology was diagnosed in 51 pts (70%), a low-grade one in 22 pts (30%). 26 pts (36%) were in Ann Arbor stage I and II at diagnosis, 47 (64%) in stage III and IV. 28 pts (38%) suffered B-symptoms, bulky disease was present in 30 pts (41%); performance status was 0 in 67%, 1 in 24%, 2 in 8%, 3 in 1%. LDH value at diagnosis was evaluable only in 51 pts; 43% of them had a pathologic LDH value. In the evaluable subset IPI at diagnosis was 0 in 11 pts; 1 in 21; 2 in 16; 3 in 3. First-line therapy was a CHOP-like regimen in 35%, a third generation regimen in 62%; radiotherapy and high-dose sequential therapy were used in one pts respectively. The median age at ASCT was 39 years (range 18-63); the median interval between diagnosis and ASCT was 13,1 months (range 4,5-133,2). Status at ASCT was first complete remission (CR) in 56%; more than first CR in 22%; partial remission in 16%; refractory disease in 6%. The source of stem cells was peripheral blood in 47 pts; bone marrow in 8; both in 18. ICE regimen included ifosfamide 3 g/m 2 on days -6 to -3, carboplatin 500 mg/m 2 and etoposide 300 mg/m 2 X 2 on days -6 to -4 . Bacterial and fungal prophylaxis was routinely performed. Results: ICE regimen was well tolerated by most pts. All of them had nausea and emesis, but only 7 needed total parenteral nutrition. The median time to engrafment was 11 days for absolute neutrophil count (>500), 12 days for platelets (20.000). The median hospitalisation was 21 days. 82% had fever during aplasia (median 1 day; range 1-7 days). The actuarial median time to treatment failure (TTF) was not reached after 5 years (54%). The actuarial overall survival after 5 years was 81%. At the univariate analysis sex, the presence of B-symptoms and the status at ASCT were statistically associated with overall survival. There was no transplantation related mortality.Conclusions: This retrospective analysis shows a low toxicity and a good efficacy in terms of TTF. We can conclude that ICE is a safe and effective conditioning regimen for ASCT in non-Hodgkin's lymphomas.

M. Magni, M. Di Nicola, C. Carlo-Stella, L. Devizzi, A. Guidetti, P. Matteucci, A. Assanelli, F. Ravagnani, M. Gianni, Istituto Nazionale Per Lo Studio E La cura dei tum (Milan, I) The role of autologous stem cell transplantation (ASCT) in indolent lymphoma is still controversial. From 1997 to 2001, we performed ASCT with in vivo purged hematopoietic progenitor cells (HPCs) in 22 patients with early relapsed or resistant follicular lymphoma (FL), using a response-adapted sequence of conventional/debulking chemotherapy, rituximab-supplemented high-dose chemotherapy and ASCT. After the initial standarddose phase, all patients received high-dose cyclophosphamide (7 g/sm) and rituximab, and underwent HPC harvest with minimal residual disease (MRD) assessment by PCR amplification of bcl-2/IgH or clonal IgH rearrangement. The 12 patients who failed to harvest MRD negative HPCs after high-dose cyclophosphamide were treated with high-dose cytarabine (1.5 to 2 g/sm every 12 hours for 6 consecutive days), underwent a second HPC harvest with PCR analysis, and received a tandem autograft (one cycle of melphalan at 180 mg/sm, and one final cycle of either highdose BEAM or mitoxantrone plus melphalan, at 60 and 180 mg/sm, respectively). The remaining 10 patients with postcyclophosphamide PCR-negative harvests, received two additional courses of standard-dose dexaBEAM before the final single myeloablative phase (either BEAM or mitoxantrone plus melphalan). After initial debulking (APO or DHAP), 21/22 patients responded but none achieved a molecular response. A molecular analysis performed after high-dose cyclophosphamide showed molecular remission in 9/22 patients. After completion of the entire chemotherapeutic program, 21/22 patients achieved a molecular complete remission and 22/22 achieved a complete clinical remission. No fatal or life-threatening toxicities were observed. With a median follow-up of 38 months, and a lead follow up of 71 months, 19/22 evaluable patients remain in molecular and complete clinical remission. The overall survival (OS) and event-free survival (EFS) rates at 71 months are 94% and 87%, respectively. These results compare favorably with the 58% OS and 56% EFS observed in 43 age-matched historical controls treated with standard-dose chemotherapy at our Institution.

R-DHAP -sequential high-dose chemotherapy with autologous stem cell support in combination with the anti-CD20 antibody Rituximab in relapsed aggressive non-Hodgkin lymphoma M. Sieniawski, J.0. Staak, H. Scheuß, J.P. Glossmann, V. Diehl, A. Engert, A. Josting, University Hospital Cologne (Cologne, D) Introduction: Combination chemotherapy can cure patients (pts) with Non-Hodgkin`s lymphoma (NHL), but those with relapse still have a poor prognosis. High-dose chemotherapy (HDCT) with autologous stem cell support (ASCT) can improve the outcome of these pts as shown in the precursor study with response rates of 46% (32% CR, 14% PR) and FFTF/OS of 20%/40% at the final evaluation (Data recently published). Chemosensitivity and achievement of minimal disease status prior to HDCT are important prognostic factors in NHL pts treated with HDCT. Rituximab demonstrated encouraging activity in aggressive NHL and showed low toxicity in the setting of combined immunochemotherapy. Patients and methods: We modified the intensified salvage program by addition of rituximab to the chemotherapy cycles followed by a final myeloablative course with stem cell reinfusion. Eligibility criteria include pts with age 18-65 years and eligible for HDCT with histologically proven CD20+ relapsed NHL. Treatment program consists of two cycles DHAP (dexamethasone, cytarabine, cisplatin) plus rituximab (375mg/m²); pts with PR or CR receive cyclophosphamide (4g/m 2 ) plus rituximab followed by PBSC harvest; methotrexate 8g/m 2 and vincristine 1,4mg/m 2 plus rituximab; and etoposide 2g/m 2 plus rituximab. The final myeloablative course is BEAM plus rituximab followed by ASCT. Results: 17 pts (median age 56 years, range 22-65) with relapsed aggressive NHL have been enrolled (stage I/II: 4, stage III/IV: 9, NA: 4). All pts had CHOP or CHOP-like regimens as first-line therapy. The median time to progression was 10,5 months. This chemoimmunotherapy combination regimen was well tolerated in all pts without side effects exceeding the toxicity expected from chemotherapy alone. 16/17 pts were available for restaging after 2 cycles DHAP with 2 CR, 10 PR, 2 SD and 2 PD. Treatment was discontinued in one pt due to development of severe heart failure. At final respose evaluation from 11 pts. 5 were in CR, 3 in PR and 3 were not avaible. Conclusion: The preliminary results suggest feasibilty and safety of this study with overall response of 67% (42% complete remission and 25% partial remission); toxicity was tolerable. The combination regimen allows effective mobilization of stem cells and the tolerability of the final myeloablative BEAM was not affected by rapid sequential administration of DHAP and high doses of cyclophosphamide, methotrexate and etoposide, each in combination with rituximab.

Allogeneic stem cell transplantation as salvage therapy for patients with lymphoma F. Zaja, C. Skert, A. Sperotto, F. Patriarca, C. Filì, M. Cerno, S. Prosdocimo, A. Geromin, D. Damiani, R. Fanin, Udine University Hospital (Udine, I) Background and objectives. Despite the therapeutic progress made in the management of lymphoma, some patients with Hodgkin's lymphoma and most patients with non-Hodgkin's lymphoma do not achieve lymphoma eradication with conventional therapies and die of progressive disease. The use of allogeneic stem cell transplantation (SCT) may represent an important salvage treatment, particularly for younger patients. Design and methods. We here report the results of a retrospective analysis performed on 28 consecutive patients with primary refractory or relapsed lymphoma who underwent allogeneic SCT. There were 6 cases of diffuse large B-cell lymphoma, 6 of peripheral T-cell lymphoma unspecified, 8 of follicular lymphoma, 3 of chronic lymphocytic leukemia and 5 of Hodgkin's lymphoma. At the time of transplant, 17 patients were chemosensitive and 11 chemorefractory. Twenty patients underwent SCT with an HLA full-matched sibling donor and 8 with a full-matched unrelated donor. Eighteen patients received a conventional conditioning regimen and 10 a reduced intensity conditioning (RIC) regimen. Results. After SCT, 16 out 21 evaluable patients (76%) achieved a complete remission, 4 had a partial remission and 1 was resistant to transplant. Seven patients could not been evaluated for response because of early treatment-related mortality (TRM). A better complete remission rate was observed in patients with chemoresponsive disease (87% vs 50%) and who underwent conventional transplant (100% vs 44%). The overall TRM rate was 32% (9 patients): 44% (8/18) in the conventional group and 10% (1/10) in the RIC group. One patient died 4 months after SCT because of lymphoma progression. The 2-year projected event-free survival and overall survival are 49.5% and 62.5%, respectively. Interpretation and Conclusion. This preliminary analysis indicates that allogeneic SCT (either with conventional conditioning regimen or RIC regimen) is a feasible and effective salvage therapy in nearly 50% of patients with lymphoma and encourages its use in cases with no other curative conventional therapeutic options.

J. Czyz, A. Hellmann, L. Kachel, J. Holowiecki, J. Gozdzik, J. Hansz, M. Osowiecki, J. Walewski, W. Jurczak, A. Skotnicki, W. Knopinska-Posluszny, Medical University of Gdansk, Silesian Medical University, University of Medical Science, Maria Sklodowska-Curie Memorial Centre of Oncology, Collegium Medicum Jagiellonian University (Gdansk, Katowice, Poznan, Warsaw, Cracow, PL) Among 341 patients treated with ASCT and reported to the data base of the Polish Lymphoma Research Group (PLRG) we identified 35 who received double transplantation (11 tandem ASCT, in 14 cases the second transplant was performed after relapse of the first) -the clinical characteristic of the patient is summarized in the Table. All but one patient engrafted after second ASCT. The median regeneration time after the first and the second transplant were: 16 (range 7 -39) and 17 (range 10 -50) days for platelets (>50x10G/l); 13 (range 7 -39) and 14 (range 9 -25) days for granulocytes (>0.5 G/l), no statistical differences were observed. Fifteen (54%) patients received G/GM-CSF after the first and 25 (71%) after the second transplant. Only one toxic death was observed, eight patients died due to progression of HD after 100 days, one case of secondary AML was reported. To asses the influence of double ASCT on overall survival, the patients were matched 1:3 with single transplants reported to the data base of PLRG. The groups were balanced according to the: age (median; <40, >40), symptoms, status at ASCT, number of chemotherapy lines (0-2, >2), histology, response to previous chemotherapy and LDH. Six-years overall survival was 72% for double transplant ASCT (95% CI; 49% -94%) and 63% (95% CI; 50% -76%) for single transplant, no statistical difference was found (p=0.39 log-rank test.

Impact of a treatment with rituximab before autologous stem cell transplantation on haematological reconstitution F. Bouchand, N. Mounier, J. Manson, D. Nabet, J. Larghero, P. Brice, I. Madelaine-Chambrin, J. Brière, J.-P. Marolleau, C. Gisselbrecht, Hôpital Saint-Louis (Paris, F) Treatment with rituximab is widely used for all types of B cell non Hodgkin's lymphomas (NHL) before autologous stem cell transplantation (ASCT) with the goal of increasing CR rate and allowing in vivo purging. We aimed to assess whether or not this procedure affects haematological reconstitution. Among the 39 CD20+ NHL patients (M/F=21/12, median age : 55) treated with chemotherapy and rituximab who underwent ASCT with peripheral blood stem cells in our department from Jul 1999 to Nov 2002, 33 were alive without relapse at one year. We report S249 the results obtained in these patients and compare them with those of a control group. 10 patients had diffuse large B cell NHL, 12 small cell follicular, 8 mantle-cell and 3 not otherwise specified. 15 were treated in first CR, 18 in relapse. Before ASCT, the IPI score was 0-1 for 50% and 2-3 for 50%. 93% patients received rituximab with a concommittant chemotherapy, 7% received sequential rituximab after chemotherapy. At time of harvest, all were in CR. TBI was part of the conditioning regimen in 42%. After graft, median day to ANC>0.5 G/L was 11 (10-20) and platelets>50 G/L was 18 (10-381). After 100 days, 79% had none or only 1 haematological lineage completely reconstituted.After one year, Hb count was normal in 33%, leukocytes count in 82%, platelets count in 61%. 27% of patients had a normal blood count, 33% had 2 lineages completely reconstituted, 27% : 1 lineage and 12% : none. This purged group was fully matched with an unpurged ASCT group with the same NHL histological type, number of CD34+ frozen cells, bone marrow involvement at diagnosis, number of relapses, IPI and conditioning regimen. The comparison of the 2 groups showed no significant difference between median days to ANC>1 G/L and platelets>20 G/L. Median day to platelets>50 G/L was significantly higher in purged group (18 vs 14, p=0.024), like median day to platelets>100 G/L (38 vs 19, p=0.004). After 100 days, median counts of leukocytes were respectively 3.6 and 4.75 G/L for purged and unpurged groups (p=0.017), ANC values were 1.71 and 3.1 G/L (p<0.002) but no febrile neutropenia was observed. Other blood counts were not different. After one year, no significant difference was observed. In conclusion, in vivo purging by anti CD20 does not lengthen duration of cytopenia following ASCT. However, after 100 days, leukocytes counts (especially ANC) are lower than in a control group. After one year, blood counts are similar to a control group. ; melphalan, 140 mg/m 2 x 1) as conditioning regimen before ASCT. Median age at transplant in patients in the BEAM group was significantly higher than in the CBV group (P = 0.001). Likewise, more patients in the BEAM group had active disease at time of ASCT than in the CBV group (P = 0.05). The remaining pretransplant characteristics were similar in both groups of patients. Results: Median time to PMN >0.5 x10 9 /L and to >20 platelets x10 9 /L was similar in both groups. In terms of RRT, stomatitis was the most frequent complication observed in both groups of patients. There was a higher incidence of hepatic venoocclusive disease (VOD) in those patients receiving CBV (11% vs. 0%, P = 0.05). There were 20 (19%) transplant-related deaths, 18 in the CBV and 2 in the BEAM group. Respiratory infections (10 patients, 6 with pneumonia) and VOD (4 patients) were the most frequent causes of death. Early transplant-related mortality (early TRM) (before day 100 after ASCT) was higher in patients receiving CBV (22% vs. 7%). Overall cumulative incidence of TRM (before and after day 100) was 25% and 7% in the CBV and in the BEAM group, respectively (P = 0.06). Conclusions: CBV is associated with a higher incidence of hepatic VOD and a higher TRM than BEAM when used as conditioning regimen for patients with lymphoma undergoing ASCT.

Autografting followed by dose-reduced conditioning and allogeneic haematopoietic stem cell transplantation in poor risk malignant lymphoma M. Haenel, F. Kroschinsky, U. Platzbecker, G. Geissler, C. Thiede, G. Ehninger, M. Bornhaeuser, Klinikum Chemnitz, University Hospital (Chemnitz, Dresden, D) We investigated the efficacy and toxicity of a tandem transplantation consisting of high-dose therapy (HDT) with autologous hematopoietic stem cell transplantation (HSCT) followed by allografting after a dose-reduced conditioning in patients (pts) with poor risk malignant lymphoma. Between 11/99 and 10/03 a total of 16 pts (11 men, 5 women, aged 25-55 years, median 46 years) with relapsed (n= 8) or refractory (n=8) diseases (2 Hodgkin's lymphoma, 3 indolent NHL, 11 aggressive NHL) were treated. Eleven pts (69%) had relapsed after a prior autologous HSCT. A median of 102 days (59-163) after HDT with BU/CY/VP16 (n=7), BEAM (n=7) or carboplatin-based regimens (n=2) and subsequent autografting 9 pts in complete remission (CR) received a dose-reduced conditioning consisting of fludarabine combined with 2 Gy TBI (n=7) or cyclophosphamide (n=2). 7 pts with a partial remission (PR) were treated with fludarabine and melphalan. This was followed by allografts from related (n=7) or matched (n=4) and mismatched unrelated donors (n=5). Immunosuppression consisted of cyclosporin A (CsA) and mycophenolate mofetil (MMF) with (n=2) or without methotrexate (MTX)(n=6) or in combination with Campath (total dose 50 mg, days -5 to -1)(n=7). One pt received CsA/MTX with prednisolone. After allografting all pts engrafted, complete donor chimerism (defined as > 95% donor cell engraftment) was observed in 15 pts after a median time of 19 days (9-84). Acute GvHD grade III-IV occurred in 6 pts (38%), and 6 pts (38%) developed chronic GvHD (all extensive disease). With a median follow-up of 25 months (1-45) 5 pts are alive in CR. Two pts relapsed (n=2) and were successfully treated by donor lymphocyte infusion (n=1) or withdrawal of immunosuppression (n=1). Two pts died of progressive lymphoma (after PR). Seven pts died from refractory GvHD (n=2) or opportunistic infections (n = 5). We observed a day 100 mortality rate of 14%. However, the probability for transplant related mortality (TRM) within 1 year after allografting was 60%, corresponding to probabilities of event-free survival (EFS) and overall survival (OS) after 2 years of 27% and 37%, respectively. In conclusion, the sequential auto-allotransplant seems to be an interesting option for pts with poor risk malignant lymphoma compared to conventional allogeneic transplantation. A reduction of the high TRM rate appears achievable by transplantation of less intensive pretreated pts.

High-dose thiotepa, melphalan and carboplatin followed by autologous peripheral blood stem cell transplantation in patients with lymphoma -a retrospective evaluation T. Demirer, E. Soydan, T. Fen, O. Ilhan, M. Ayli, M. Arat, M. Ozcan, N. Gunel, O. Arslan, Y. Genc, A. Uysal, R. Haznedar, S. Buyukberber, G. Gurman, N. Ustaer, B. Hazar, G. Ozet, H. Akan, Ankara University Faculty of Medicine, Ankara Oncology Hospital, Ankara Numune Education and Research Hospital, Gazi University Faculty of Medicine, Gaziantep University Faculty of Medicine, Mersin University Faculty of Medicine (Ankara, Gaziantep, Mersin, TR) The purpose of this evaluation was to investigate the efficacy of high-dose chemotherapy (HDC) with thiotepa, melphalan and carboplatin (TMCb), and of autologous peripheral blood stem cell (PBSC) infusion in patients with aggressive non-Hodgkin's lymphoma (NHL) or Hodgkin's disease (HD). Forty-two patients, 23 with intermediate-grade NHL and 19 with HD received thiotepa (500 mg/m 2 ), melphalan (100 mg/m 2 ) and carboplatin (1050-1350 mg/m 2 ) followed by autologous PBSC infusion. Of 21 patients with more advanced disease, 4 had primary refractory disease, 1 was in complete remission (CR)-2, 11 were in first refractory relapse, and 5 were in beyond first relapse. Of 21 patients with less advanced disease, 2 were in CR-1, 4 were in CR-2, and 15 were in first responding relapse. Fourteen patients (33%) had received prior radiotherapy (RT) prohibiting a total-body irradiation (TBI)-based conditioning regimen. The projected 2-year probabilities of survival, event-free survival and relapse for all patients were 0.65, 0.60, and 0.21 (0.85, 0.80, and 0.10 for patients with less advanced disease and 0.47, 0.42, and 0.33 for patients with more advanced disease). Grade 3-4 regimen-related toxicities (RRT) occured in 5 of 42 (12%) patients and death due to grade-4 RRT occured in only 1 (2.5%) patient. These preliminary data suggest that high dose TMCb followed by autologous PBSC transplantation is well tolerated and an effective regimen in patients with intermediate-grade NHL or HD and may be comparable to some previously used regimens including TBI-based regimens.

The impact of rituximab in stem cell mobilisation and engraftment in high-grade non-Hodgkin lymphoma P. Kaloyannidis, C. Kartsios, I. Sakellari, C. Vadikolia, M. Marantidou, A. Anagnostopoulos, A. Fassas, The George Papanicolaou General Hospital (Thessalonica, GR) Rituximab, a safe and efficient anti-CD20 monoclonal antibody, is increasingly used in B-high grade NHL (HG-NHL) treatment. Although, stem cell yield and function is not adversely affected by Rituximab treatment in indolent and mantle cell lymphomas, its role remains unclear for pts with HG-NHL. We retrospectively studied 28 HG-NHL pts who underwent stem cell mobilization (SCM), after chemotherapy (ChT) plus GCSF (27/28) or GCSF alone. Thirteen pts aged 48 (15-57) years were treated with 2(1-6) doses of Rituximab at 375mg/m 2 (R group), 50 (7-180) days prior to SCM. They had also received 2 (1-4) lines of ChT; 4 had been also irradiated. At SCM, 11 had refractory disease and 2 were in CR2. Fifteen pts (Non-R group) aged 35 (13-57) years received 2 (1-3) ChT lines (5 pts plus irradiation), 75 (21-365) days prior to SCM. Disease status was refractory: 13, CR1: 1, CR2: 1. In the R-group, SCM was successful for all patients. In the non-R group, a sufficient stem cell number was collected in 12/15 pts after the 1st attempt and in 3 pts after a 2nd SCM (2 pts) or additional bone marrow harvest (1pt). Total dose of CD34+ cells and per day-collected yield was higher for the non-R group (4.5 x10 6 /Kg vs 3.1 x10 6 /Kg, p: 0.02, 1.5 x10 6 /Kg/day vs 0.76 x10 6 /Kg/day, p: 0.03). In multivariate analysis none of the analyzed variables (age, disease status, lines of previous ChT, and Rituximab administration) was associated with CD34+ total dose or CD34+/day collected yield. Eventually, 8/13 pts from the R group and 13/15 pts from the Non-R group were transplanted. Neutrophils and platelet recovery was similar for the 2 groups (R group: day NEU>1,000/mm 3 : +11, day PLT>25,000/mm 3 : +13, Non-R group: day NEU>1,000/mm 3 : +12, day PLT>25,000/mm 3 :

Our study suggests that prior treatment with Rituximab might adversely affect stem cell collection in this cohort of pts. Neutropenia is a recently known phenomenon post Rituximab administration.In our series Rituximab did not influence neutrophil engraftment. As most centers include Rituximab in HG-NHL treatment, more studies are required to assess its impact on SCM and engraftment.

Tandem autologous stem cell transplantation or nonmyeloablative allogeneic stem cell transplantation following autologous stem cell transplantation for transformed lowgrade lymphoma V. Dubruille, N. Morineau, P. Moreau, T. Guillaume, B. Mahe, P. Chevalier, J. Harousseau, N. Milpied, Hotel Dieu (Nantes, F) Indolent lymphoma may develop disease transformation to an aggressive lymphoma. The response to chemotherapy and the outcome of these patients is poor. From January 1997 to august 2003, 21 patients, age 37-60 yo (median 50yo), were included in a program consisting of induction chemotherapy with "CHOP like" regimen followed in responding patients (partial or complete response) by either a tandem autologous transplant or more recently by an autologous transplant followed by nonmyeloanlative allogeneic stem-cell transplantation (NMA SCT) according to whether or not the patient had an HLA identical sibling. The tandem autologous transplant program has already been described (Haematologica 2002 Mar; 87:333) . For patients with a donor, the conditioning regimen for autograft was "BEAM" and the conditioning regimen for NMA SCT consisted of an association of fludarabine, busulfan and antithymocyte globulin with cyclosporine as graft versus host disease (GVHD) prophylaxis and granulocyte colony-stimulating factor (G-CSF) mobilised peripheral blood stem-cell as graft. Of 16 patients without a donor, 1 relapsed after the first autograft and 15 received the planned tandem autologous transplant. 11 patients are alive in complete remission with a median follow-up of 42 months (6-87 months); 4 relapsed after the end of procedure and died in median of 13 months (9-23 months); no toxic death. Of 5 patients with a donor, 5 underwent the NMA SCT, all engrafted. 1 relapsed at 2 months and died of acute GVHD after donor lymphocyte infusion. 4 are alive in response with a median follow-up of 14 months (8-32 months). Overall, both tandem autologous and autologous followed by NMA SCT are feasible, well tolerated and effective.A longer follow-up and a larger number of patients with a donor are needed to fully evaluate risks and benefits of NMA SCT in this setting as compared to tandem autologous transplantation. N.B. Mikhaylova, L.S. Zoubarovskaya, A.Y. Zaritsky, B.V. Afanasyev, SPb State I. Pavlov Medical University (St. Petersburg, RUS) High-dose chemotherapy with autologous hematopoietic stem cell transplantation (AHSCT) could improve survival of high-risk lymphoma patients. Nevertheless, the indications for this therapeutic approach and the optimal time are not clear yet. The goal: to reveal the impact of some prognostic factors on the outcome and long-term results of AHSCT in patient with primarily resistant and relapsed lymphoma and to evaluate the role of AHSCT as early consolidation treatment. Methods: 54 patients (34-Hodgkin's disease (HD), stage IIIB and IV, and 21-highgrade non Hodgkin's lymphoma (NHL)) received dexaBEAM/DHAP as debulking therapy for primarily resistance or relapses. Additional 11 NHL patients received dexaBEAM followed by high-dose therapy with AHSCT as a part of first-line treatment. Conditioning regimens were BEAM or CBV. The disease status at the moment of AHSCT, response to debulking therapy, IPSS, bulky disease, pretreatment status, the time of transplantation were evaluate as prognostic factors influenced on 10-year survival. The control group consists of 40 NHL and 120 HD patients received conventional treatment. Results: The 10year survival of chemosensitive patients, responding to debulking therapy was 60% in HD to compare with 8 % in resistant group (p=0,047) and 64% and 25 % in NHD (p=0, 02) 

Choice of adequate risk adapted strategy in Hodgkin disease W.J. Jurczak, A. Zaluska, D. Krochmalczyk, B. Piatkowska -Jakubas, A.B. Skotnicki, Collegium Medicum UJ (Cracow, PL) We would like to summarize our experience in treating Hodgkin Disese (HD) in nearly 100 patients at our Department during the last 5 years. There is little dispute abut the efficiency of chemotherapy combined with IFRT in a low and intermediate risk groups (I /II clinical stage with or without risk factors). Following EORTC experience we gave those patients EBVP and ABV chemotherapy with subsequent radiotherapy achieving at 4 years the EFS of 100 and 80% respectively (OS 100% and 96 %). Primary resistance or early relapses although rare, also occurred in this group: they were best predicted by inadequate response to the first 3 cycles of chemotherapy; all patients were successfully salvaged by II line regimens followed by high dose chemotherapy (BEAM) with auto-SCT. So far none of the 39 treated patients died in disease progression; however one of them was lost due to cardiac complications secondary to anthracyclin toxicity. In advanced disease (II BX -IV clinical stage), the choice of adequate therapy remains challenging. Although the efficiency of escalated BEACOPP regimen is out of question, and -as it was proven by GHSG -it prolongs OS by decreasing the number of primary resistances, we found it difficult to persuade all patients to upfront intensive chemotherapy. We offered it to anyone with 3 or more risk factors according to GHSG. 7 out of 22 patients preferred ABV: it was particularly common among young women concerned about their fertility and active professionals who chose to work during therapy. 3 of them later agreed to consolidate the first CR with BEAM conditioned auto-SCT. The 4 year EFS and OS is in the whole group 65 and 95% respectively. Auto-SCT is a reasonably safe procedure in HD (no transplant related mortality in 30 patients). The EFS and OS were 80% and 90% respectively: five out of six relapses were clustered in 9 patients transplanted with primary resistance.

M. Sieniawski, J.P. Glossmann, J.O. Staak, L. Nogova, J. Kisro, C. Rudolph, M. Reiser, C. Scheid, A. Engert, V. Diehl, A. Josting, University Hospital Cologne, University Hospital Luebeck, Carl-Thiem-Hospital (Cologne, Luebeck, Cottbus, D) Background: Patients with primary progressive or refractory Hodgkin`s (HD) or aggressive Non-Hodgkin`s lymphoma (NHL) have a particularly poor prognosis. Here we report the results of autologous tandem transplantation in these patients. Patients and Methods: Patients aged 18-55 years with primary progressive or refractory relapsed HD and aggressive NHL were included. Progressive disease was defined as progression during induction treatment or within 90 days after the end of treatment. . Patients with bulky disease (> 5cm) or residual lymphoma 30 days after the second HDCT therapy additionally S252 received involved field radiotherapy. Results: Twenty-five patients were included (HD=10, NHL=15). PBSC harvest with more than 4 x 10 6 CD34+cells/kg was successful in all patients. The median age was 34 years (range 22-51 years). Six patients (24%) had stage II disease, 3 patients (12%) stage III and 16 patients (64%) stage IV disease. Two patients with HD achieved a CR and 5 patients a PR, resulting in an overall response rate (RR) of 70% for HD patients. Three patients (30%) had a treatment failure including two deaths due to peri-transplant complications. Five patients with aggressive NHL were in CR, two patients in PR (RR 46%). From the 8 patients (56%) with treatment failure, three had progressive disease, five died from peri-transplant complications. Freedom from treatment failure (FFTF) and overall survival (OS) for all patients after 12 months was 28% and 40%, respectively. There was no outcome difference patients with HD and patients with NHL. Conclusion: Tandem HDCT followed by ASCT is feasible in this poor prognostic patients.

H. Minigo, R. Vrhovac, A. Planinc-Peraica, S. Ostojic Kolonic, R. Kusec, D. Radic-Kristo, I. Kardum-Skelin, D. Sustercic, B. Jaksic, Clinical Hospital Merkur (Zagreb, HR) Introduction: Although chemotherapy and/or irradiation provide cure for many patients with Hodgkin's Disease (HD) some do not acheive remission after initial treatment or relapse later during the course of the disease. We present our 8-years' experience in the treatment of these patients with intensive chemotherapy followed by autologous stem cell transplantation (ABMT). Objectives: To evaluate ABMT in this subset of patients by assessing its: 1) toxicity, in terms of transplant related mortality (TRM), hematopoietic recovery and need for transfusion support. 2) efficacy, in terms of CR, PR and NR achieved, and long term efficacy expressed as disease free (DFS) and overall survival (OS). Patients and methods: From February 1995 until November 2003 a total of 45 patients with HD (24 male, 21 female; age 18-60, median 28), received BEAM myeloablative treatment followed by ABMT. Two patients were transplanted in CR, all others had active disease at time of transplantation. All patients received heavy prior treatment with a median of 2 different lines of chemotherapy (range 1-6) and a median of 8 chemotherapeutic cycles (range 2-15). An average of 8.02 (range 1.03-32.6, SD 9.8) x 10 6 /kg CD34+ cells was reinfused and G-CSF (5µg/kg) was administered during the leukopenic period to all patients (median 9 days). Results: Median time to WBC recovery (>1x10 9 /L) was 10 days (range 6-26), while platelets recovered (>20x10 9 /L) in a median of 11 days (range 5-30). An average of 16.5 platelet doses (range 0-77, SD 16.3) and 366.1 mL of RBC concentrate (range 0-1990, SD 486.9) was administered. A median of 2 febrile days (range 0-20) was observed. Of all patients, 38 achieved CR (84.4%) and 6 achieved PR (13.3%). One patient died during the pancytopenic period (TRM 2.2%). At 8 years from transplantation, projected OS is 76.0% and DFS is 62.5%. Conclusions: Given that all patients received heavy prior treatment and had active disease at time of transplantation, ASCT toxicity can be considered as acceptable. A very high remission rate was achieved (CR+PR 97.7%). We conclude that BEAM with ASCT is a very effective treatment for patients with relapsed or refractory HD.

Allogeneic haematopoietic stem cell transplantation in patients with non-aggressive lymphoproliferative malignancies -single-centre experiences I.W. Blau, W.U. Knauf, O. Marinets, K. Rieger, T. Fietz, E. Thiel, Charite', Campus Benjamin Franklin (Berlin, D) Allogeneic hematopoietic stem cell transplantation (HSCT) appears to be an important approach also in the treatment of patients with nonaggressive lymphoproliferative diseases due to its curative potential. We studied 18 patients (4 female, 14 male) which underwent related (MRD)(12) and unrelated (MUD)(6) HSCT after reduced toxicity (14) and standard myeloablative conditioning (4). The median age was 48 (18-62) years. Five patients were treated for chronic lymphocytic leukemia (CLL, Binet C), 7 patients for multiple myeloma (MM) and six for indolent lymphoma (IL). All of them received intensive courses of chemotherapy before HSCT. We used a reduced toxicity conditioning regimen with fludarabin/treosulfan (14 patients)group A, and a standard TBI/cyclophosphamid conditioning regimen (4 patients) -group B. GVHD prophylaxis consisted of cyclosporin A, methotrexate (and Thymoglobuline® in MUD HSCT). Hematopoietic engraftment was estimated by chimerism analysis based of 8 different STR loci (Promega). At day 28 post HSCT 12/18 patients had mixed chimerism, median percent of donor cells was 92 (50-95) in peripheral blood. 60% of patients achieved full chimerism up to day 100 in bone marrow and peripheral blood. Four CLL patients with a matched unrelated donor and two MM patients kept mixed chimerism with high level of donor cells (90-95%) without clinical signs of relapse at the time of observation. Thus, stable long term mixed chimerism is a frequent phenomenon in patients after reduced toxicity conditioning regimen with indolent lymphoproliferative disorders and might be of relevance for graft versus leukemia effect in such kind of patients. Fourteen patients are alive with a median follow up of 15,5 (3-48) month. Seven patients (35%) are in excellent conditions and returned to their previously jobs without any immunosuppressive drugs. Results of our study suggest that allogeneic HSCT is a safety treatment option to cure patients with lymphoprolifarative disorders.

In vivo purging with hyper-CVAD plus rituximab in newly diagnosed mantle cell lymphoma E. Benedetti, F. Papineschi, F. Caracciolo, S. Galimberti, G. Cervetti, G. Castiglioni, S. Mechelli, P. Scatena, M. Pelosini, M. Petrini, University of Pisa (Pisa, I) Previously untreated mantle cell lymphoma (MCL) are consistently associated with poor prognosis when treated with CHOP-like regimens. Typically the CR rate is 20-30%, median FFS = 10-16 months and median OS = 3 years. In the attempt to improve outcome we used a high dose intensity regimen such as

Hyper-CVAD (HCVAD) with autologous stem cell transplant. Our goal was to collect disease-free stem cells using in vivo purging with Rituximab. Nine patients entered the study. Patients were apheresed after 2nd course of HCVAD (1st LPH set) and if apheresis (LPH) were PCR positive (Bcl1+/JH+) a second set of LPH were performed after completion of 4th cycle. To perform an "in vivo" purging Rituximab 375 mg/m 2 was added at day +1 and +9 after last dose of ARA-C; GCSF 10µg/kg was commenced on day +5 until LPH was ultimated. A median 5.0 (range 3.2-7.3)x10 6 CD34/kg were collected during the 1st set of LPH and a median of 4.5 (2.9-8.4)x10 6 CD34/kg during the 2nd set. Three/9 patients (6, 7, 8) were to be considered nonmobilizers. Four patients (1,2,3 and 4) needed a 2nd set of LPH because of 1st LPH set contamination. Two patients (3, 4) with 2 more cycles (4 complete HCVAD) reached PCR negativity in BM and one collected PCR negative SC. Only one patient (9) collected tumour free stem cell having BM PCR+ pre-in vivo purging. Bone marrow (BM) PCR negative patients collected tumour free SC. All BM-PCR positive patients collected contaminated SC even though exposed to in vivo purging, all except one who collected negative SC. Three patients have been thus far transplanted and longer follow up and a bigger recruitment of patients is needed. So far only in one patient purging in vivo with Rituximab 375mg/m2 added to high doses ARA-C was successful and allowed tumour free graft collection. In order to increase in vivo purging Rituximab is now added to every single cycle to try to further reduce bone marrow contamination. Furthermore if patients are BM positive right before 2nd cycle it is not worth to LPH patients because time and money consuming in respect to the poor results obtained, and LPH is directly postponed at the end of 4th cycle.

High-dose sequential followed by autologous bone marrow transplantation as salvage treatment in advanced Hodgkin's disease M.T. Delamain, R.C. Baldissera, K.B.B. Pagnano, G.B. Oliveira, I. Lorand-Metze, A.C. Vigorito, F.J.P. Aranha, K.A.B. Eid, E.C.M. Miranda, C.A. De Souza, Hematology and Blood Transfusion Center / UNICAMP (Campinas, BR) ABMT has been proposed as a salvage treatment of resistant / refractory HD. HDS using Cy 7g/m 2 for debulking and PBPC mobilization followed by MTX 8g/m 2 and then VP-16 2g/m 2 before ABMT, described by Milan group, seems to be effective in these patients. We report the results obtained using this procedure in 31 patients with HD who failed conventional therapy or relapsed within 24 months of conventional chemo +/-radiotherapy. Patients were treated with HDS followed by ABMT receiving BEAM (BCNU, Etoposide, Ara-C and Melphalan) as conditioning regimen. The median age was 25 years (12-61), 20 male (64,5%) and 11 female (35,5%). The histology at diagnosis showed: 19 (61,3%) nodular sclerosis; 9 (29%) mixed cellularity; 2 (6,5%) lymphocyte-depleted and 1 (3,2%) lymphocyte-rich HD. Bulky disease was presented in 15/31 (49%) patients and 6/31 (20%) had bone marrow infiltration. The Overall survival (OS) and disease free survival (DFS) were 49% (n=31) and 60% (n=15), respectively, in 1825 days. The OS was 64% for patients with 0-1 prognostic factors at diagnosis and 30% beyond two (P=0.07). Bulky disease, histology type and bone marrow involvement did not correlate with poor outcome. Fifteen patients died, 8/15 due to progressive disease (53%), 5/15 due to toxicity after the HDS (33%) and 2/15 due to toxicity after ABMT (13%).Status presens for alive patients after a median time of 783 days (range 50-1929) from transplant is: CR 11 ( 35,5%), partial response 2 (6,5%) and 4 in progressive disease (12,9%). We conclude that HDS followed by ABMT is an effective salvage regimen for patients with resistant/refractory Hodgkin's disease and probably induces a long and stable CR mainly in chemosensitive patients. Patients presenting >2 prognostic factors at diagnosis presented worst outcome.

High-and low-risk high-grade NHL patients display no significant survival difference in terms of long-term followup after up-front high-dose chemotherapy with autologous stem cell support H. Bertz, R. Zeiser, W. Lange, D. Frauenschuh, S. Fetscher, C.F. Waller, J. Finke, Freiburg University Medical Center (Freiburg, D) Although preliminary data from randomised studies suggest an increase in disease-free survival (DFS) and overall survival (OS), the long-term benefit from high dose chemotherapy (HDCT) with autologous stem cell transplantation (aSCT) as part of the initial treatment for patients with high grade Non-Hodgkins-Lymphoma (hgNHL) is still not clear. The purpose of this study was to evaluate the long-term benefit from high dose chemotherapy (HDCT) with autologous stem cell transplantation (aSCT), as part of the initial treatment for patients with chemosensitive hgNHL stratified according to the ageadjusted International Prognostic Index (aaIPI). Eligible were 34 consecutive patients with hgNHL, risk factors according to aaIPI and bulky disease (10 pts), who responded to first line therapy. 23/34 pts (67.6%) had two or three risk factors with respect to the aaIPI. All pts received HDCT with aSCT after a minimum of six weeks of VACOP-B standard therapy (alternating VP-16, doxorubicin, cyclophosphamide, vincristin, prednisolone and bleomycin) and VIP-E (VP-16, ifosphamide, cisplatin, epirubicin) for mobilisation in first remission. After aSCT 32 pts (94%) achieved complete remission. Two pts achieving only partial remission (PR) died due to progressive disease as well as 2 pts relapsing 1 and 6 months after aSCT. Two additional pts relapsed after 31 and 55 months at the former side of bulky disease and received a second aSCT or an allogeneic matched unrelated donor SCT in PR. The cumulative incidence of relapse at a medium follow-up of 9.5 years is 12.5% for 32/34 pts achieving CR. It is of note, that no treatment related mortality occurred. 27/34 pts are in sustained CR with a DFS of 79% at a median follow up of 114 months (range 98-130) and an OS of 79%. The 23 pts with more than one RF according to aaIPI showed identical results as the 11 pts with 0/1 RF. The results suggest that HDCT with aSCT improves long-term outcome in pts with hg NHL in first CR after standard chemotherapy as compared to historic populations. Furthermore, this study demonstrates that long-term PFS and OS after HDCT and aSCT were comparable in patients with 0/1 or 2/3 risk factors according to the aaIPI.

Non-myeloablative stem cell transplantation for mantle cell lymphoma S. Van Steenweghen, J. Maertens, R. Schots, A. Van de Velde, AZ Stuivenberg, UZ Gasthuisberg, UZ VUB (Antwerp, Leuven, Brussels, B) Aim: In mantle cell lymhoma (expressing t(11;14) and unmutated IgVH genes), lasting remissions rarely occur. (ref1) High-dosesequential chemotherapy and in vivo immunotherapy (ref2) maximise first complete remission states.Although these constitute the optimal pretransplant situations, autologous transplantations face frequently delayed relapse (progression free survival 33% after 5 years). Given this timeframe and known graft-versus-lymphoma effect, after such autologous procedures, non myeloablative allogeneous stemcell transplantations theoretically can improve survival and lead to definite cure. Methods: We present seven patients, who received a non myeloablative conditioning with fludarabin 30 mg/m²/d for 4 consecutive days (days -6 till -3) , cyclophosphamide1000 mg/m²/d for 3 consecutive days (days -5 till -3) and antithymocyte-globulin (rabbitATG Fresenius 10 mg/kg/d on day -4 and day -3) with cyclosporin and mycophenolate mofetil graftversus-host prevention. These patients were highly pretreated. The aim of this study was to compare the outcome of allogeneic HSCT after myeloablative versus reduced intensity conditioning (RIC) for lymphoma. From January 1984 to August 2003, 45 patients underwent HSCT for lymphoma (including 11 patients with RIC, all grafted after June 2000). The diagnoses were NHL; 22 aggressive and 16 indolent (including 10 CLL) and 7 HD lymphoma. There were 34 males and 11 females with a median age of 40 (16-56) years. The donors were 27 HLA-identical siblings (one identical twin) and 18 unrelated donors (16 HLA-identical and 2 with 1-antigen mismatch). The myeloablative conditioning consisted of TBI/Cy (30), BuCy (3) and TLI/Cy (1) . The RIC consisted of fTBI/Cy/Flu (4), Flu/Cy (5), Flu/Bu (1) and Flu/Treosulfan (1) . As GVHD prohpylaxis, 39 patients received MTX and CsA, 3 MTX or CsA, 2 T-cell depletion and one patient had no prophylaxis. In the RIC group the median age was significantly higher 49 (34-56) than in the myeloablative group 38 (16-53) years (p=0.03). PBSC was more common used in the RIC group, 8 of 11 versus 9 of 34 in the myeloablative group. The diagnoses were aggressive lymphomas 5 (46%) and 17 (50%), indolent lymphomas 3 (27%) and 13 (38%), HD 3 (27%) and 4 (12%) in the RIC and myeloablative groups, respectively. The incidence of acute GVHD grades II-III was 11% and chronic GVHD mild to moderate 36%. The 2-years probability of TRM was 29% and 41% (ns) and relapse 29% and 17% (ns) in the RIC and myeloablative groups, respectively. The causes of deaths in the RIC group were 2 infections, 2 relapses and 1 EBV-related lymphoma (5/11, 45%) and in the myeloablative group; 6 infections, 1 GVHD, 7 relapses, 1 EBV-related lymphoma and 5 other causes (20/34, 59%). Results in the aggressive and indolent lymphomas will be presented. Conclusions: Long term survival was seen in more than 40% after myeloablative allogeneic HSCT for relapsing lymphoma. The outcomes were similar between RIC and myeloablative conditioning but each subgroup is small and the follow up short in the RIC group, therefore the results must be interpreted with care.

Allogenic haematopoetic stem cell transplantation after reduced intensity conditioning for patients with non-Hodkgin lymphoma H. Schieder, N. Kröger, T. Zabelina, F. Ayuk, A. Kratochwille, B. Fehse, N. Fehse, C. Wolschke, H. Renges, A. Zander, University Hospital Hamburg (Hamburg, D) Background: Allogenic transplantation for Non-Hodgkin Lymphoma ( NHL) is often associated with a high transplant related mortality.The aim of this retrospective single centre study was to explore feasibility and efficacy of reduced intensity conditioning (RIC) in patients with refractory or recurrent NHL. Patients: From 11/99 to 5/2003 19 patients with refractory or relapsed NHL (aggressive n=4, indolent n=15) were transplanted after dose reduced conditioning (11 Flu/Mel n=11, 5 Flu/BEAM n=5, 1 BEAM n=1, Flu/CY/TT450 n=1) with bone marrow ( n=3) or peripheral stem cells (n=16) from a related ( n=10) or an unrelated ( n=9) donor. Medium age was 46 y ( 30-60y).GVHD prophylaxis consisted of cyclosporin A/methotrexat ( n=18), cyclosporin A/MMF ( n=1) and anti-rabbit anti thymocyte globulin ( ATG Fresenius n=19). 8 Patients had an autologous transplantation before. Results:With a medium follow up of 536d ( 179-1281d) over all survival is 62 % and disease free survival 46%. One patient who relapsed achieved a complete remission after donor lymphocyte infusion. Transplant related mortality was 23%. Conclusion: RIC is associated with a low TRM and may lead to long lasting complete remission in patients with heavily pretreated lymphomas. Further follow-up is needed to determine long term toxicity and late relapse rate.

H. Kasparu, J. König, H. Hauser, O. Krieger, M. Girschikofsky, M. Bernhart, D. Lutz, Elisabethinen-Hospital, Hanusch Hospital (Linz, Vienna, A) Autologous stem cell transplantation (ASCT) is an established therpeutic option in the treatment of non-Hodgkin´s lymphoma. From 8/1984 to 9/2003 ASCT was performed in 102 patients (47 f, 55 m, age 15-65, median: 44,5 years) with either bone marrow (25), peripheral stem cells (73) or both (4). 29 patients were P921 Treosulfan/fludarabine as dose-reduced conditioning followed by allogeneic stem cell transplantation from related and unrelated donors in patients with AML and MDS/sAML N. Kröger, T. Zabelina, H. Schieder, K. Kratochwille, F. Ayuk, N. Stute, N. Fehse, J. Hassenpflug, J. Panse, J. Casper, M. Freund, A. Zander, University Hospital Eppendorf, University Hospital (Hamburg, Rostock, D) 15 patients with MDS (n=4; RA n=1; RAEB n=3), sAML (n=5) or AML (n=6; 1.CR n=4, 2.CR n =2) and a median age of 55 years (range: 20-69) who were not eligible for standard conditioning regimens were conditioned with treosulfan 10 g/m² i.v. (day -6 to day-4) and fludarabine 30 mg/m² (day -6 to day -2) followed by unrelated (n=9 ) or related (n=6) peripheral stem cell transplantation (PBSC). Five patients received grafts from donors with at least one locus HLA-mismatch. All but one patient received anti-rabbit anti thymocyte globulin (ATG-Fresenius 3 x10-20 mg/kg) as GvHD prophylaxis. Further GvHD prophylaxis consisted of cyclosporin A and short course MTX. One graft failure was observed in a patient with unrelated mismatched donor, but recovery was noted after reinfusion of autologous stem cell back-up. Median time to leucocyte (> 1 x 10 9 /L) engraftment was reached after 17 days (range 11-23). In general, toxicity was mild with skin toxicity (n=8), mucositis (n=12), and liver toxicity (n=4). So far, acute GvHD grade II was seen in 4 patients. Two patients died of treatment related causes (severe hepatitis, aspergillus infection). Three patients with AML experienced relapse 3, 4 and 6 months after transplantation. With a median follow-up of 5 months (range 2-8) the overall survival is 87% (95% CI 70-100%). Treosulfan/fludarabine provides rapid engraftment with low toxicity in patients with AML and MDS who are not eligible for standard myeloablative regimen. Further accrual and longer follow-up is needed to determine long term toxicity and late relapse-rate. The chemotherapy-based treatments of high-risk myelodysplastic syndrome patient's (MDS) or secondary acute leukemia (sAML) result in a few longterm survivors. We initiated a multicentric prospective study to assess the efficacy of intensive remission-induction and consolidation chemotherapy, followed by autologous (ASCT) or allogenic stem cell transplantation (allo-SCT) in this group of patients. Design and Methods: 74 patients (aged >15 years) diagnosed of high-risk MDS (International score prognostic factor >1 or Spanish prognostic factor >=3) or sAML in 10 Spanish hospitals from January 1999 to October 2003 were treated with an intensive protocol, consisting of Idarrubicin, Cytarabine and VP-16 (ICE) induction therapy followed by ASCT or allo-SCT. Only patients with hypoplastic marrow or patients with lower than 10% of blast cells in bone marrow and who underwent related donor, made allo-SCT without induction treatment. Analyses of response and overall survival (OS) were performed. Results: The median (range) age of the series was 53(16-69) years, 45 males and 29 females. Fifty-eight patients had MDS and sixteen sAML. Twelve patients had MDS or AML following treatment for previous malignancy. The median time between the first diagnosis of MDS and the start of treatment was 18 days. Seven patients received allo-SCT as initial treatment; all seven patients achieved complete response (CR). Three of these seven patients maintained the response at 10, 26 and 36 months respectively. Two relapsed at 22 and 38 months, and two died at 7 and 8 months due to infection related to transplant. Thirty-six of 64 evaluated patients (56%) achieved CR with ICE. Seventeen patients received ASCT, 5 miniallograft, 5 allo-SCT, 2 no treatment after, 1 died during consolidation, 5 no mobilized enough, and one is waiting for an ASCT. With a median followup of 13 months, 14 patients valuables are in continuous remission (3 allo-SCT direct, 6 allo-SCT consolidation and 4 ASCT). The median overall survival was 418 days.Patients who received allo-SCT for induction didn't yet achieve the median of survival. Conclusions: We conclude that this strategy of treatment is highly effective in obtained CR's. In 53% of the patients it was possible to make an intensive treatment. We need a longer follow-up to evaluate the impact of this protocol in the survival of these high-risk patients.