key: cord-0842253-l0qke09d
authors: Ahmed, Ibrahim A.; Farooqi, Midhat S.; Vander Lugt, Mark T.; Boklan, Jessica; Rose, Melissa; Friehling, Erika D.; Triplett, Brandon; Lieuw, Kenneth; Saldana, Blachy Davila; Smith, Christine M.; Schwartz, Jason R.; Goyal, Rakesh K.
title: Outcomes of Hematopoietic Cell Transplantation in Patients with Germline SAMD9/SAMD9L Mutations
date: 2019-07-12
journal: Biol Blood Marrow Transplant
DOI: 10.1016/j.bbmt.2019.07.007
sha: 341a510819e8fa76bc13caf8b42d4c5fa1f86041
doc_id: 842253
cord_uid: l0qke09d

Germline mutations in SAMD9 and SAMD9L genes cause MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) (OMIM: *610456) and ataxia-pancytopenia (OMIM: *611170) syndromes, respectively, and are associated with chromosome 7 deletions, myelodysplastic syndrome (MDS), and bone marrow failure. In this retrospective series, we report outcomes of allogeneic hematopoietic cell transplantation (HCT) in patients with hematologic disorders associated with SAMD9/SAMD9L mutations. Twelve patients underwent allogeneic HCT for MDS (n = 10), congenital amegakaryocytic thrombocytopenia (n = 1), and dyskeratosis congenita (n = 1). Exome sequencing revealed heterozygous mutations in SAMD9 (n = 6) or SAMD9L (n = 6) genes. Four SAMD9 patients had features of MIRAGE syndrome. Median age at HCT was 2.8 years (range, 1.2 to 12.8 years). Conditioning was myeloablative in 9 cases and reduced intensity in 3 cases. Syndrome-related comorbidities (diarrhea, infections, adrenal insufficiency, malnutrition, and electrolyte imbalance) were present in MIRAGE syndrome cases. One patient with a familial SAMD9L mutation, MDS, and morbid obesity failed to engraft and died of refractory acute myeloid leukemia. The other 11 patients achieved neutrophil engraftment. Acute post-transplant course was complicated by syndrome-related comorbidities in MIRAGE cases. A patient with SAMD9L-associated MDS died of diffuse alveolar hemorrhage. The other 10 patients had resolution of hematologic disorder and sustained peripheral blood donor chimerism. Ten of 12 patients were alive with a median follow-up of 3.1 years (range, 0.1 to 14.7 years). More data are needed to refine transplant approaches in SAMD9/SAMD9L patients with significant comorbidities and to develop guidelines for their long-term follow-up.

In recent years, advances in genetic interrogation of patient samples have led to discovery of several novel genes that underlie inherited bone marrow failure and myelodysplastic syndrome (MDS) [1] . These include SAMD9 (sterile a-motif domain-containing protein 9) and SAMD9L (SAMD9-like) genes, located head to tail on chromosome 7q21.2 in a region that is frequently deleted in myeloid malignancies [2, 3] .

Germline mutations in SAMD9 and SAMD9L cause the multisystem disorders, MIRAGE (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy) and ataxia-pancytopenia syndromes, respectively [4] [5] [6] . Recent studies in children reported a rate of SAMD9 and SAMD9L mutations in 18.6% and 17% cases with suspected inherited bone marrow failure syndromes and those with primary MDS, respectively [7, 8] . Biology of Blood and Marrow Transplantation journal homepage : www.bbmt.org SAMD9 and SAMD9L proteins are involved in endosomal trafficking and negatively regulate cell proliferation [9] . Gain-of-function heterozygous mutations in these genes lead to cellular growth restriction and hypoplasia, resulting in cytopenias, bone marrow failure, and immunodeficiency. Interestingly, in many cases, there is a nonrandom loss of the mutated allele via full or partial deletion of chromosome 7 [4, [10] [11] [12] . The resultant monosomy 7 or deletion 7q can result in the development of MDS and acute myeloid leukemia (AML) [8, 11, 12] . Conversely, other "genetic correction" events such as in cis missense, nonsense, or loss of heterozygosity through uniparental disomy can result in normal hematopoiesis.

Since the initial report of MIRAGE syndrome in 2016, a series of studies has described clinical and genetic findings in patients and families with SAMD9/SAMD9L mutations [7, 11, 13] . Hematopoietic cell transplantation (HCT) therapy has been included in some reports, but transplantation details are lacking. A recent article by Sarthy et al. [14] documented 2 children with MIRAGE syndrome who succumbed to posttransplant complications due to syndrome-related comorbidities. We aimed to obtain a more complete assessment of transplant outcomes and the challenges and complications encountered in these patients.

After management of 2 cases with MIRAGE syndrome, additional cases were identified by literature search and peer consultations. For inclusion, patients were required to have a confirmed heterozygous mutation in the SAMD9 or SAMD9L gene and a minimum of 1-year follow-up post-transplant. Deidentified data for each case were collected by using a standardized questionnaire. All studies involving human subjects were performed in accordance with site-specific protocols approved by the institutional review board and in accordance with Declaration of Helsinki guidelines.

The primary study endpoints were overall survival and event-free survival. Safety and tolerability of HCT and impact of pretransplant comorbidities were evaluated by occurrence and severity of post-transplant complications, need for life support measures, and risk of transplant-related mortality. Transplant outcomes were defined using Center for International Blood and Marrow Transplant Research criteria [15] . Grading of acute graft-versus-host disease (GVHD) and diagnosis of chronic GVHD were based on standard criteria [16] . Surviving patients were censored at last follow-up. Continuous variables were summarized as median and range of values and analyzed using the Mann-Whitney test. Survival curves were generated using the Kaplan-Meier method and compared using the log-rank (Mantel-Cox) test using the GraphPad Prism 7 software (GraphPad Software, San Diego, CA).

Twelve patients underwent allogeneic HCT for hematologic disorders associated with germline SAMD9 (n = 6) or SAMD9L (n = 6) mutations (Table 1) . Patients 3, 4, 6, 11, and 12 (Table 1) were included in previous reports [11, 13, 17] . Indication for transplant was MDS in 10 of 12 (83%) cases. One SAMD9 patient with markedly reduced megakaryocytic precursors in marrow underwent transplantation for a presumed diagnosis of congenital amegakaryocytic thrombocytopenia, and 1 patient with SAMD9L mutation and shortened telomeres underwent transplantation on a presumed diagnosis of dyskeratosis congenita.

Median age at presentation for patients with SAMD9 mutations (1.65 years; range, 0.17 to 4.8 years) was similar to those with SAMD9L mutations (1.43 years; range, 0.67 to 12.6 years). Six patients had pancytopenia, including 5 with thrombocytopenia and 1 with anemia. Bone marrow was hypocellular in 11 (92%) cases and showed dysplasia most prominently in the megakaryocytic lineage in most cases. Chromosome 7 abnormalities, including monosomy 7 and chromosome 7q deletions, were present in all cases. All except 1 case showed somatic mosaicism for chromosome 7 abnormalities (ie, detection of a monosomy 7 or chromosome 7 deletion clone in only a fraction of hematopoietic cells in bone marrow).

Exome sequencing revealed 5 different missense heterozygous mutations in the 6 SAMD9 cases and 4 different missense mutations in the 6 SAMD9L cases. Their genomic details and pathogenicity assessment of variants are summarized in Table 2 and cross-referenced [5, 7, 8, 12, 13, [17] [18] [19] [20] . Six of 12 cases were familial. Four SAMD9 patients had phenotypic features of MIRAGE syndrome (patients 1, 2, 5, and 6; Tables 1 and 2); unique findings included panhypopituitarism, laryngeal cleft, and glomerulosclerosis. Two other cases with a SAMD9 mutation had milder phenotypes with growth restriction in 1 and hypospadias and a bifid scrotum in another. The remaining patients had no phenotypic abnormalities.

Transplant details of individual cases are summarized in Table 3 . Median age at HCT was 2.8 years (range, 1.16 to 12.8 years). Median age at HCT tended to be higher in SAMD9 patients versus SAMD9L patients at 4.15 years versus 2.2 years, respectively (P = .81). Median time from initial presentation to transplant was 0.45 years (range, 0.2 to 6.53 years). There was an interval of 5.5 and 6.53 years from initial diagnosis to HCT in 2 cases of MIRAGE syndrome because in these cases, blood counts seemed to show improvement before patients developed sustained marrow failure. Stem cell sources included bone marrow (matched unrelated, n = 7; HLA identical sibling, n = 2; and haploidentical parent, n = 1) and unrelated cord blood (n = 2). Nine patients received myeloablative conditioning (busulfan based, n = 7, or total-body irradiation based, n = 2). Three patients received reduced-intensity conditioning with fludarabine, cyclophosphamide, or melphalan, with rabbit antithymocyte globulin or alemtuzumab.

Clinically significant pretransplant comorbidities were present in SAMD9 cases with MIRAGE syndrome (Table 3 ). These included chronic diarrhea, electrolyte imbalance, infections, adrenal insufficiency, failure to thrive, lung disease, and renal dysfunction. One patient with SAMD9L mutation (patient 10, Table 2 and Table 3 ) had been treated for hemophagocytic lymphohistiocytosis, disseminated sepsis, invasive fungal infections before transplant.

Post-transplant complications included pericardial effusions (n = 3), veno-occlusive disease of liver (n = 3), thrombotic microangiopathy (n = 2), and diffuse alveolar hemorrhage (n = 1). Unique complications in several MIRAGE syndrome cases included large volume stool losses with dehydration and electrolyte imbalance, temperature and blood pressure instability, and hypoxia. Eight patients required transfer to intensive care for management of respiratory failure (n = 5), sepsis (n = 1), and severe hypertension (n = 1) and VOD of liver (n = 1).

One patient with a familial SAMD9L mutation, MDS, (patient 7, Table 3 ) and morbid obesity failed to engraft following reduced-intensity conditioning with double unrelated cord blood transplantation. All other patients achieved neutrophil and platelet engraftment at a median of 16 days (range, 12 to 19; n = 11) and 17 days (range, 12 to 40; n = 10) post-HCT, respectively. Two patients developed grade II to III acute GVHD, which resolved with treatment. Two patients developed mild skin chronic GVHD. Two patients have chronic lung disease, and 2 other patients have chronic kidney disease. One patient with SAMD9L mutation and MDS (patient 7, Table 3 ) with failed engraftment subsequently developed AML and died of its treatment complications. A second patient, with SAMD9L mutation and MDS (patient 10, Table 3 ), died of diffuse alveolar hemorrhage while receiving defibrotide for treatment of veno-occlusive disease of liver. Immune reconstitution data are summarized in Table 4 .

Ten of 12 patients were alive with a median follow-up of 3.1 years (range, 0.1 to 14.7 years). All surviving patients (n = 10) at time of last follow-up had resolution of hematologic disorder, had no chromosome 7 abnormalities, and sustained peripheral blood donor chimerism (90% to 100%). All patients were thriving. SAMD9 cases had varying degrees of developmental delays (n = 6) and chronic kidney disease (n = 3). All patients with clinical characteristics of MIRAGE syndrome (n = 4) were short for age, required supplemental feeds, and had persistent adrenal insufficiency. In SAMD9L cases (n = 4), no clinical neurologic manifestations have been observed so far.

In this report, we describe transplant details and outcomes in a series of patients with hematologic diseases associated with SAMD9/SAMD9L germline mutations. We found that most patients underwent transplantation for MDS with chromosome 7 abnormalities and received myeloablative conditioning with HCT from nonsibling donor graft sources. Allogeneic HCT led to successful resolution of MDS or marrow failure, with sustained donor chimerism and excellent survival.

On review of literature, we found 10 other cases with SAMD9 mutation who underwent HCT. A 4-year-old child with MIRAGE syndrome and monosomy 7 MDS underwent transplantation with active AML and died of Epstein-Barr virusrelated lymphoproliferative disorder a year later [4] . Wilson and colleagues [21] reported a patient with MIRAGE syndrome who underwent reduced-intensity conditioning and unrelated donor HCT that led to resolution of monosomy 7 MDS. Sarthy et al. [14] described a patient with marrow failure and another patient with MDS who had severe MIRAGE phenotypes and underwent HCT after reduced-intensity conditioning. Comorbidities, including enteropathy, electrolyte imbalances, adrenal crises, bacteremia, and lung disease, significantly led to a complicated transplant course and ultimately death in both cases. Although transplant details in 6 other cases are limited, 1 patient without the MIRAGE phenotype died of unknown cause, and 5 were surviving following HCT [7, 10] . There were 4 cases of MIRAGE syndrome in our series. Before transplant, 3 of 4 cases had chronic diarrhea, malnutrition, and adrenal insufficiency. Post-HCT, we observed severe gastrointestinal fluid losses, electrolyte imbalance, and acute dehydration in these 3 cases. Whether such dramatic stool losses without an infectious etiology were secretory and whether autonomic instability could have contributed are unknown. Patients also experienced temperature and blood pressure instability, respiratory distress, and acute renal dysfunction.

Several of these medical issues are similar to those reported in the report by Sarthy et al. [14] . Despite a complicated acute transplant course, all 4 patients with MIRAGE syndrome in our series survived.

We observed a high rate of ongoing medical issues in MIRAGE syndrome transplant survivors. These include adrenocortical insufficiency, diarrhea, need for supplemental nutrition, and developmental delays. Patients with pre-existing lung disease and nephropathy continue to have these issues following HCT. Most of these issues are related to pre-existing MIRAGE syndrome manifestations. The transplant survivor reported by Wilson et al. [21] had ongoing medical issues of adrenocortical insufficiency, growth and developmental delays, and chronic lung and chronic kidney diseases.

In this series, all 6 SAMD9L patients had cytopenias and MDS with chromosome 7 abnormalities. We did not observe ataxia, incoordination, or other neurologic manifestations before or following transplant. On review of the literature, we found 11 additional cases of patients with SAMD9L mutations who had undergone HCT [5, 7, 11] . Although transplant details are limited, 2 patients died of complications (cerebral [19] (continued) Tesi et al, [5] ; Bluteau et al, [7] ; Wong et al, [12] Abbreviations: WES indicates whole exome sequencing; WGS, whole genome sequencing; BMT, blood and marrow transplantation; ACMG, American College of Medical Genetics, and VUS, variant of unknown significance; NGS, Next generation sequencing. * Each pathogenic criterion was weighted as very strong (PVS1), strong (PS1-4); moderate (PM1-6) or supporting (PP1-5) and each benign criterion was weighted as stand-alone (BA1), strong (BS1-4) or supporting (BP1-6). From Richards et al, [20] .

y The SAMD9 variant c.3406G>C (p.E1136Q) was classified as a VUS using strict ACMG criteria. We believe this variant is pathogenic based on well-established functional data from two separate experimental studies showing that it has a deleterious effect on cells. The younger sibling of the patients above also carries the variant and had transient neonatal thrombocytopenia requiring transfusion. However, the mother of these patients carries the variant as well and presently lacks an apparent phenotype. Whether she was transiently affected in the past is unknown, but this is possible as somatic revertant mosaicism is a known associated phenomenon with SAMD9/SAMD9L variants. Other potential mechanisms that could account for the lack of phenotypic segregation include monoallelic gene expression, incomplete penetrance, or variable expressivity. We feel this is important to note for clinical reasons in case this variant is observed in another patient. Abbreviations: ATG (anti-thymocyte globulin); Ara-C (cytosine arabinoside); BU (busulfan); BMI (body mass index); Chr. 7 (chromosome 7); CKD (chronic kidney disease); Cy (cyclophosphamide); CsA (cyclosporine A); GI (gastrointestinal); Flu (fludarabine); HLH (hemophagocytic lymphohistiocytosis); MA (myeloablative); MDS (myelodysplastic syndrome); Mel (melphalan); MIRAGE syndrome (myelodysplasia, infection, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy); MMF (mycophenolate mofetil); Mtx (methotrexate); N.E. (not evaluable); RIC (reduced intensity conditioning); Tac (tacrolimus); TBI (total body irradiation); TMA (thrombotic microangiopathy); and VOD (veno-occlusive disease) hemorrhage and infection, 1 each), 1 had unknown survival status, and 8 were alive. Of the surviving patients, 1 had pulmonary fibrosis, and 3 had neurologic issues. Mutations in SAMD9 and SAMD9L add to a growing list of recently described heritable conditions associated with cytopenias, marrow failure, MDS, and AML [1, 7, 8] . Although these patients can be managed symptomatically with transfusions and treatment of infections, the only curative treatment is with allogeneic HCT.

Indications and timing of HCT in these patients are not straightforward because marrow cells can undergo somatic genetic correction events and spontaneous blood count recovery [4, 8, 12, 22] . In our series, there was an interval of several years from initial presentation to development of bone marrow failure or MDS in 2 cases. Most patients in our series underwent transplantation for MDS with transfusion dependence, and a diagnosis of SAMD9/SAMD9L was made retrospectively from archived specimens. Affected siblings of patients who underwent transplantation have been followed without transplant; however, these are anecdotal case reports, and long-term data are needed [8, 11] .

Patients who have relatively stable blood counts and do not show signs of development of MDS or AML may continue to be closely observed. However, in our view, patients who develop significant marrow failure (including if clinically symptomatic with infections, anemia, bleeding, and/or transfusion dependence), meet morphologic criteria of MDS, develop monosomy 7 or 7q-, or develop other cytogenetic abnormalities associated with myeloid malignancies should be evaluated for allogeneic HCT. Any potential family donors must undergo genetic evaluation for SAMD9/SAMD9L mutation as well.

In conclusion, in this small series of patients, we found that most patients with SAMD9/SAMD9L mutations tolerated transplant conditioning, with a high rate of engraftment and resolution of MDS or marrow failure. Clinically significant comorbidities were common in MIRAGE syndrome cases and contributed to unique adverse events in the acute posttransplant phase. These patients continue to require ongoing management and multispecialty care for syndromerelated nonhematologic manifestations. More data are needed to define timing of HCT in SAMD9/ SAMD9L patients and further refine conditioning regimens as well as management of patients with significant syndromerelated comorbidities. National and international transplant registries should be queried to examine reported outcomes in larger patient cohorts. Finally, long-term follow-up and care guidelines are needed for the survivors.

NK cells 480, CD19 740, all in per cumm. Patient 4 (SAMD9 without MIRAGE): Lymphocyte enumeration 3 years post-HCT, ALC 3700, CD3 2530, CD4 1090, CD8 1140, NK cells 400, CD19 770, all in per cumm. Patient 5 (SAMD9 with MIRAGE): IVIG infusions monthly until 1 year post-HCT. Patient 6 (SAMD9 with MIRAGE): IVIG infusions monthly until 6 months post-HCT

Introduction to acquired and inherited bone marrow failure

Recurrent genetic defects on chromosome 7q in myeloid neoplasms

The enigma of monosomy 7

SAMD9 mutations cause a novel multisystem disorder, MIRAGE syndrome, and are associated with loss of chromosome 7

Gain-of-function SAMD9L mutations cause a syndrome of cytopenia, immunodeficiency, MDS, and neurological symptoms

Ataxia-pancytopenia syndrome is caused by missense mutations in SAMD9L

A landscape of germ line mutations in a cohort of inherited bone marrow failure patients

The genomic landscape of pediatric myelodysplastic syndromes

Haploinsufficiency of SAMD9L, an endosome fusion facilitator, causes myeloid malignancies in mice mimicking human diseases with monosomy 7

Somatic mutations and progressive monosomy modify SAMD9-related phenotypes in humans

Constitutional SAMD9L mutations cause familial myelodysplastic syndrome and transient monosomy 7. Haematologica

Germline SAMD9 and SAMD9L mutations are associated with extensive genetic evolution and diverse hematologic outcomes

Germline SAMD9 mutation in siblings with monosomy 7 and myelodysplastic syndrome

Poor outcome with hematopoietic stem cell transplantation for bone marrow failure and MDS with severe MIRAGE syndrome phenotype

Forms Instruction Manual

Consensus Conference on Acute GVHD Grading

A novel SAMD9 variant identified in patient with MIRAGE syndrome: further defining syndromic phenotype and review of previous cases

A novel SAMD9 mutation causing MIRAGE syndrome: an expansion and review of phenotype, dysmorphology, and natural history

Novel V1551L mutation in SAMD9L inhibits cell cycle progression and results in pancytopenia that progresses to MDS with monosomy 7

Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology

Comment on: Acquired monosomy 7 myelodysplastic syndrome in a child with clinical features of dyskeratosis congenita and IMAGe association

SAMD9 and SAMD9L in inherited predisposition to ataxia, pancytopenia, and myeloid malignancies

The authors thank the patients and their families, as well as the clinical teams involved in their management.Disclaimer: The views expressed in this manuscript are those of the authors and do not reflect the official policy of the Department of Army/Navy/Air Force, Department of Defense, or US government.Financial disclosure: The authors have nothing to disclose. Conflict of interest statement: There are no conflicts of interest to report.