Sporadic Myelodysplastic Syndrome Research Articles

Prevalence and clinical outcomes of germline variants among patients with myeloid neoplasms

AimsMyeloid neoplasms (MNs) with germline predisposition have been recognised as a distinct entity. Emerging evidence suggests that sporadic myelodysplastic syndromes may also harbour undetected germline predispositions. We investigated germline alterations...

DNA Damage Response Constrains Cell Growth and Drives Clonal Hematopoiesis in Telomere Biology Disorders

Telomere biology disorders (TBD) are inherited diseases caused by genetic defects in telomere maintenance. Patients with TBD have critically shortened telomeres, leading to bone marrow failure (BMF) and an increased risk of myeloid neoplasms (MN). We conducted a study to understand the genetic changes driving clonal evolution in TBD patients and their distinct mechanisms compared to normal aging and sporadic myelodysplastic syndrome (MDS). We analyzed acquired genetic alterations in hematopoietic cells from 105 TBD patients. The diagnosis of TBD was made using a combination of clinical history, germline mutation status, and telomere length. Germline mutations were identified in 84% of patients, a family history consistent with TBD was seen in 46%, and 76% of patients had lymphocyte telomere lengths (TL) <1st %ile for their age, while 22% fell between the 1st and 10th %ile. Complications observed in our cohort included BMF (42%), myeloid neoplasms (17%), solid tumors (17%), cirrhosis (14%), and interstitial lung disease (34%). Clonal hematopoiesis was detected in 44 of 105 patients (42%), including 8 of 27 (30%) patients 18 years or younger. Somatic genetic alterations primarily involved the DNA damage response (DDR) pathway, with ATM and TP53 being the most frequently mutated genes (11 ATM mutations in 5 of 80 patients; 5 TP53 mutations in 4 of 84 patients; Fig. 1). Mutations typical of age-related clonal hematopoiesis were seen in 4 patients (3 ASXL1, 1 TET2). Somatic reversion of TBD germline lesions was found in 7 patients. Additionally, 11 somatic mutations were detected in genes involved in DDR, telomere maintenance, and cell senescence, including KAT6B, PALB2, TELO2, TOP1, and RTEL1. MDS-associated gene mutations were found in 15 patients (18%), 8 in the setting of MN. The most common mutations affected spliceosome genes with U2AF1 p.S34F and SF3B1 being most notable, including 1 U2AF1 S34F identified in a 15-year-old. Mutations in NPM1, RUNX1, WT1, SF3B1, and FLT3 were identified only after malignant transformation. Acquired cytogenetic changes were observed in 16 of 87 evaluable patients (18%), 8 in the setting of MN, and included gain of 1q in 5 patients (5.7%). Two patients with TP53 mutations developed a complex karyotype (Fig 1). To understand selective pressures contributing to clonal hematopoiesis development in TBD, we analyzed transcriptional changes in bone marrow (n=2) and peripheral blood (n=1) mononuclear cells (MNCs) of 3 TBD patients with different germline lesions using single cell RNA sequencing (scRNASeq). MNCs from TBD patients exhibited significant differences compared to healthy controls, showing downregulation of MYC targets, reduced translation, cell cycle arrest, and upregulation of ATM-dependent DDR pathway and FAS pathway (Fig 2). We hypothesized that mutations in DDR pathway genes, particularly somatic ATM loss, enable TBD hematopoietic stem and progenitor cells to overcome telomere-induced DDR induction and premature senescence. Consistent with our hypothesis, low dose ATM inhibition in TBD patients' fibroblasts significantly enhanced growth rate of TERC-mutated and DKC1-mutated cells, whereas control fibroblasts were unaffected. Somatic mutations in ATM in our cohort were typically observed in patients with TERC-mutated TBD (3 of 5, 3 of 34 with TERC-mutated TBD), very low TL, and extra-hematopoietic manifestations. Of the 11 ATM mutations, 3 were predicted loss of function and 4 were missense kinase domain mutations, known or predicted to result in loss of kinase activity. Somatic ATM loss occurred both as heterozygous or compound heterozygous mutations. The development of ATM mutations alone was not causative of malignancy; however, 2 patients with somatic ATM mutations developed myeloid neoplasms after acquisition of additional mutations (subclonal mutation in NPM1 in one patient and clonal replacement with a TP53-mutant clone in another patient). In conclusion, our findings highlight the role of telomere dysfunction-induced DDR and cellular senescence in bone marrow failure and clonal selection in TBD. ATM loss selectively enhances growth of TBD cells by inhibiting DDR activation which may ameliorate marrow failure but has the potential to increase the risk of myeloid neoplasms after acquisition of additional genetic events. Future studies should explore the possibility of a therapeutic window involving very low doses of ATM inhibition in TBD.

RUNX1 is required in granulocyte-monocyte progenitors to attenuate inflammatory cytokine production by neutrophils.

The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancy (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 was shown to regulate inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to epigenetically repress two inflammatory signaling pathways in neutrophils: Toll-like receptor 4 (TLR4) and type I interferon (IFN) signaling. RUNX1 loss in GMPs augments neutrophils' inflammatory response to the TLR4 ligand lipopolysaccharide through increased expression of the TLR4 coreceptor CD14. RUNX1 binds Cd14 and other genes encoding proteins in the TLR4 and type I IFN signaling pathways whose chromatin accessibility increases when RUNX1 is deleted. Transcription factor footprints for the effectors of type I IFN signaling-the signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRFs)-were enriched in chromatin that gained accessibility in both GMPs and neutrophils when RUNX1 was lost. STAT1::STAT2 and IRF motifs were also enriched in the chromatin of retrotransposons that were derepressed in RUNX1-deficient GMPs and neutrophils. We conclude that a major direct effect of RUNX1 loss in GMPs is the derepression of type I IFN and TLR4 signaling, resulting in a state of fixed maladaptive innate immunity.

Duplication on Chromosome 14q Identified in Familial Predisposition to Myeloid Malignancies and Myeloproliferative Neoplasms

Familial predispositions to myeloid malignancies are well recognized. Germline mutations in genes such as RUNX1, CEBPA, GATA2, TERT, TERC, ANKRD26, SRP72, ACD, ETV6, DDX41, SAMD9L and SAMD9, reported over the last 18 years, predispose predominantly to myeloid malignancies with or without cytopenias or other phenotypes. One study reported 4 families with predisposition to myeloid malignancies including myeloproliferative neoplasms (MPN) (Saliba et al. Nat Genet 47:1131, 2015). These French West Indies families harbored an identical 700 kb duplication within chromosome 14 encompassing the genes ATG2B, GSKIP and TCL1A . In functional assays, ATG2B and GSKIP were implicated in the predisposition and phenotype of the resultant malignancies.Here we report a 4 generational family with 6 affected individuals with myeloid malignancy (myelodysplastic syndrome (MDS), MPN and acute leukemia). Using copy number variant analyses directly from whole exome sequencing data, 3 affecteds were shown to harbor a ~700 kb germline duplication encompassing 9 genes including DICER1 and TCL1A . Only TCL1A and a long non-coding RNA (RP11-164H13.1) are located in the overlap duplicated region between this family and the West Indies families. T-cell leukemia/lymphoma 1A (TCL1A) gene is commonly over-expressed in T and B cell malignancies, sometimes due to chromosomal translocations that bring the strong TCR (A and B) and IGH enhancers adjacent to the TCL1A gene. To date, the TCL1A gene has not been implicated in predisposition to MPN.Mutational analysis of an MDS/MPN-U (RCMD-RS/MF) patient sample revealed that the germline duplicated region was further duplicated as part of a larger 18 Mb somatic duplication that also included ATG2B and GSKIP, likely contributing to the myelofibrosis phenotype. A somatic mosaic trisomy 14 acquisition further amplifying the duplicated region was also seen in one of the West Indies affecteds providing evidence that gene dosage of the predisposing gene or genes within the local Chr 14q32.2 region may be an important factor. Such trisomy 14 cases have been reported as rare events in MPN, MDS, CMML and aCML. In a cohort of 126 sporadic primary MDS samples, we have identified 2 cases with Chr 14q copy number neutral loss of heterozygosity encompassing the two 700 kb duplicated regions. Like the MDS/MPN-U proband (Fig.1A), these cases harbored deleterious SF3B1 mutations and displayed ring sideroblasts as a common feature. In summary, we have identified a novel germline duplication that segregates with MDS/MPN.Figure 1. MDS/MPN family and germline duplication on chromosome 14q. A. Four generation family with MDS/MPN.Myeloid phenotype (blue); age of diagnosis, dx; age of death, d; alive, a; in years. Proband (solid triangle) B. Copy number analysis of WES data from bone marrow (BM) of the RCMD-RS/MF proband. Ploidy calculation (dark blue) and z-score (light blue) for calculation of copy number variation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Germline SAMD9 and SAMD9L Alterations in Adult Myelodysplastic Syndromes

While familial leukemia and bone marrow failure syndromes present typically in children, it is possible that less damaging variants, such as heterozygous alterations of recessive genes, may be identified in older patients with seemingly spontaneously acquired disease. This may be particularly true for recessive variants with lower penetrance. Gain-of-function germline (GL) mutations in SAMD9 have been described in the context of MIRAGE syndrome, characterized by Myelodysplasia Infection, Restriction of growth, Adrenal hypoplasia,Genital phenotypes and Enteropathy. Recently, pediatric myelodysplastic syndromes (MDS) families affected by a somatically acquired -7/del(7q) were found to have GL mutations in SAMD9 and SAMD9L on 7q21.2. Here, we focused on sporadic adult myeloid neoplasm patients to assess how frequently mutations in SAMD9 and SAMD9L occur with or without -7/del(7q).We first sequenced a cohort of patients for the presence of variants in SAMD9/SAMD9L . Paired disease and normal GL DNAs were obtained from 312 MDS patients for whole exome sequencing (WES; n=220) and targeted deep sequencing (n=92). GL DNA was obtained from either buccal mucosa or paired CD3+ T cells. We used Broad Institute's Exome Aggregation Consortium (ExAC) data as ethnically matched healthy control (n=45,950).We defined “rare variants” as those present in <1% of ethnically matched healthy controls. Among 312 patients with myeloid neoplasms, we identified a total of 30 rare GL variants of SAMD9/SAMD9L in 29 patients (9.3%). Validation was performed by Sanger sequencing or PCR amplicon sequencing. None of these alterations were present in any of the previously described familial cases. All patients had a heterozygous configuration except for one. We used an algorithm that predicts the pathogenic significance of SAMD9/SAMD9L alterations by evaluating conserved amino acid residues and alterations of protein features. We identified 18 (10 in SAMD9 and 8 in SAMD9L) which were most likely to be functionally deleterious; 7/19 in SAMD9 and 9/11 in SAMD9L were not previously described, emphasizing their rarity. For example, I268T and D550V of SAMD9 which are present only in MDS patients, and E220G, G235S, W333C and Y705C SAMD9L alterations, were predicted to be highly deleterious based on structure/function analyses according to prediction algorithms. While those with known population frequencies were not over-represented individually, their combined frequency was higher in MDS vs . ethnically matched healthy controls, with an odds ratio = 1.54 (95% CI: 1.04-2.27, P=.03), and frequencies of 29/312 (9.3% in MDS) vs . 2,896/45,950 (6.3% in healthy controls). Two patients had a family history of MDS and unexplained anemia.We then focused on patients with -7/del(7q). In total, 82 out of 312 patients (26%) had -7/del(7q); 5 of these 82 patients (6.1%) had SAMD9 alterations. Variant allele frequencies (VAFs) were significantly lower in unpurified bone marrow fractions than in GL controls in 3 out of 5 patients. For example, the GL of one patient had the S86F alteration in SAMD9 in a heterozygous configuration, whereas the tumor of the patient with somatically acquired -7 showed lower VAFs. This observation suggests that the SAMD9/SAMD9L variant allele was selectively deleted at the onset of MDS, resulting in retention of only the wild-type allele during clonal selection (Figure 1). SAMD9/SAMD9L can maintain cell growth by attenuating proliferation. SAMD9/SAMD9L GL mutations identified in familial MDS cases strongly repressed cell proliferation by working as gain-of-function mutations. Somatic acquisition of -7/del(7q) can rescue the growth-repressing effects from GL gain-of-function alterations. However, cells which lost the GL gain-of-function alterations by -7/del(7q) to gain a survival advantage progressed to MDS due to haploinsufficiency of chr7 genes. This interpretation is further supported by the observation of a lower frequency of variants in those with vs . without -7/del(7q) (5/82 (6.1%) in -7/del(7q) carriers vs. 24/230 (10.4%) in diploid cells). Our results support a model wherein SAMD9/SAMD9L variants are selectively lost in MDS with -7/del(7q).In conclusion, while deletion of several important genes on chr7q are likely to be pathogenic in sporadic adult MDS, we show here that GL alterations in SAMD9/SAMD9L may also drive selection of -7/del(7q) clones in a small fraction of adult MDS. [Display omitted] DisclosuresMakishima:Yasuda Medical Foundation: Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

Human Germline HLTF E259K Mutation Identified in Familial MDS Patients Accumulates DNA Damage through Impaired PCNA Polyubiquitination

Although several causal genes of familial myelodysplastic syndromes (MDS) have been discovered, the genetic landscape and the molecular pathogenesis still remain unclear. To explore novel causal genetic alterations and their pathogenetic significance, we performed whole-exome sequencing analysis using four samples from a Japanese familial MDS pedigree, two affected and two unaffected as controls. In total, 16889 non-synonymous germline substitutions were obtained. Among them, twelve candidate single-nucleotide variations (ANK1, C9orf43, CYP7B1, EFHB, ENTPD7, FAM160B2, HELZ2, HLTF, INPP5J, ITPKB, RYK, TTN) were elucidated. Of the candidates, we elucidated HLTF, encoding a member of the SWItch/Sucrose Non-Fermenting (SWI/SNF) family, as a putative associated gene. HLTF works as an E3-ubiquitin ligase and binds with Methyl Methanesulfonate Sensitive 2 (MMS2)/Ubiquitin Conjugating Enzyme E2N (UBC13) complex. This complex is known to promote the Lys-63 linked polyubiquitination of proliferating cell nuclear antigen (PCNA), followed by an error-free postreplication DNA repair by arranging template switching (Mailand et al. Nat Rev Mol Cell Biol 2013). The obtained germline mutation of HLTF was p.Glu259Lys (E259K), heterozygous mutation.To study the functional role of HLTF, we firstly evaluated the effects of Hltf silencing on hematopoiesis by in vitro colony-formation assay with Hltf-knocked down mouse primary bone marrow cells, and found that these cells have enhanced in vitro colony-formation capacity. In agreement with the acquisition of an immature phenotype, the cells expressing c-kit, a hematopoietic stem/ progenitor cell marker, were increased in Hltf shRNA-expressing colonies after the first round. We then focused on DNA damage in HLTF knockdown HEL cells according to the function of HLTF post-replication DNA repair. We examined the phosphorylation of gamma H2AX foci by immunofluorescent staining and found that the number of foci was increased in HLTF knockdown cells compared with control cells, suggesting that DNA damage was accumulated in HLTF knockdown cells.To investigate the effects of exogenous expression of HLTF E259K mutant protein, we evaluated in vitro apoptosis status with HEL cells transduced with empty vector (EV), HLTF wild type (WT), and HLTF E259K mutant (MT), respectively. In apoptosis assay, annexin V positive cells were significantly increased in MT-overexpressed HEL cells compared with WT-overexpressed HEL cells. In addition, we found that the number of gamma H2AX foci was increased in HLTF MT-overexpressed HEL cells, suggesting that DNA double-strand breaks were accumulated in MT-overexpressed cells. To explore the mechanism of accumulated DNA double-strand breaks by mutant HLTF, we evaluated the binding ability of HLTF mutant protein with PCNA, and MMS2/UBC13, all of which play a critical role in the DNA double-strand break repair process. We found that binding of HLTF MT to MMS2 or UBC13 was significantly attenuated compared with HLTF WT although binding to PCNA remained unchanged, suggesting HLTF MT cannot efficiently form a complex that induces polyubiquitination of PCNA. In line with this, PCNA polyubiquitination was impaired in HLTF MT-overexpressed cells compared with HLTF WT-overexpressed cells.Finally, we searched somatic HLTF mutation in sporadic MDS bone marrow samples. Whole HLTF coding regions of 40 samples were sequenced. As a result, a novel HLTF missense heterozygous mutation (p.Thr50Ala, T50A) was identified in one sample (1/40 = 2.5%). These data suggested the possibility that HLTF mutation might contribute to the pathogenesis not only in familial MDS but also in sporadic MDS.In conclusion, our results indicated that HLTF E259K germ line mutation accumulates DNA damage through impaired PCNA polyubiquitination. The aberrant function of HLTF mutant might play a role in the pathogenesis of familial MDS. DisclosuresMasamoto:Astellas Pharma Inc.: Research Funding; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory; Pfizer Inc.: Research Funding. Arai:Bristol-Myers Squibb: Research Funding. Suzuki:Bristol-Myers K.K: Honoraria; Novarltis: Honoraria; Celgen: Honoraria; Ono: Honoraria; Fujimoto: Honoraria; Takeda: Honoraria. Harada:NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding; Celgene: Research Funding. Kurokawa:Bristol-Myers Squibb: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Other: Scholarship donations for the laboratory, Speakers Bureau; Astellas Pharma Inc.: Research Funding; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; Pfizer Inc.: Research Funding; Celgene Corporation: Speakers Bureau.

A novel RUNX1 mutation with ANKRD26 dysregulation is related to thrombocytopenia in a sporadic form of myelodysplastic syndrome.

Aging is associated with a higher risk of developing malignant diseases, including myelodysplastic syndromes, clonal disorders characterised by chronic cytopenias (anaemia, neutropenia and thrombocytopenia) and abnormal cellular maturation. Myelodysplastic syndromes arising in older subjects are influenced by combinations of acquired somatic genetic lesions driving evolution from clonal haematopoiesis to myelodysplastic syndromes and from myelodysplastic syndromes to acute leukaemia. A different pattern of mutations has been identified in a small subset of myelodysplastic syndromes arising in young patients with familial syndromes. In particular, dysregulation of ANKRD26, RUNX1 and ETV6 genes plays a role in familial thrombocytopenia with predisposition to myelodysplastic syndromes and acute leukaemia. Whether these genes affect thrombopoiesis in sporadic myelodysplastic syndrome with thrombocytopenia is still undefined. Thirty-one myelodysplastic syndromes subjects and 27 controls subjects were investigated. Genomic DNA was used for mutation screening (ETV6, RUNX1, 5'UTR ANKRD26 genes). Functional studies were performed in the MEG-01-akaryoblastic cell line. We found four novel variants of RUNX1 gene, all in elderly myelodysplastic syndromes subjects with thrombocytopenia. Functional studies of the variant p.Pro103Arg showed no changes in RUNX1 expression, but the variant was associated with deregulated high transcriptional activity of ANKRD26 in MEG-01 cells. RUNX1 variant p.Pro103Arg was also associated with increased viability and reduced apoptosis of MEG-01, as well as impaired platelet production. Our findings are consistent with dysregulation of ANKRD26 in RUNX1 haploinsufficiency. Lack of repression of ANKRD26 expression may contribute to thrombocytopenia of subjects with sporadic myelodysplastic syndromes.

Opposing Pathogenesis of Germline SAMD9/SAMD9L Variants in Adult Myelodysplastic Syndrome (MDS)

Familial myelodysplastic syndromes (MDS), leukemias, and bone marrow failure (BMF) syndromes, typically present in children and young adults. Such cases associate with germ line (GL) mutations in DDX41, RUNX1,ETV6, GATA2 and ANKRD26 and were recently designated by WHO to constitute a separate disease. Gain-of-function (GOF) GL mutations in SAMD9 and SAMD9L associate with familial pediatric myelodysplastic syndrome (MDS) cases that often have early onsets and -7/del(7q). It was not known if this was the case in sporadic adult MDS. With this question in mind, we compiled sequencing results on 799 adults with presumably acquired MDS and BMFs, and further enhanced these results through public WES data on 349 myeloid neoplasms. We also performed cell growth assays of exemplary SAMD9 and SAMD9L GL variants identified in adult MDS.We used Broad Institute's Genome Aggregation Database (gnomAD) as healthy controls (n = 138,632). After comparing ethnicities between gnomAD and our cohort, we matched healthy controls to patients by ethnicities. Defining rare variants as those present in <0.1% of ethnically-matched healthy controls, we found 26 rare SAMD9/SAMD9L GL variants in 3% (24/799) patients. Most were missense; 3 were truncating (nonsense or frameshift All variants were heterozygous except two patients with two variants. We further validated these data were validated in public WES databases. We identified 2% (7/349) of adult MDS patients in public database with GL SAMD9/SAMD9L variants. SAMD9/SAMD9L GL variants were located in the N-terminal significantly more in adult MDS than reported for pediatric MDS [SAMD9; 62% (8/13) vs. 17% (5/30), p=.044, SAMD9L; 62% (7/13) vs. 0% (0/30), p=.0026]. Compared to reported pediatric MDS patients, adult MDS patients had significantly less deletions of chromosome7/del(7q) [8% (2/24) vs. 69% (22/32), p=.0037] and more 5q deletions [33% (8/24) vs. 4% (1/24), p=.022].Conservative predictions for specific SAMD9 missense variants were made by using SAMD9 GL variants reported in previous papers or public databases of registered healthy donors, and comparing Area Under the Curve of 8 different prediction algorithms. It defined 11 missense variants as “pathogenic”. 57% (4/7) of the variants identified in public WES database were also likely pathogenic by our criteria. We evaluated the growth activity of these 11 unique pathogenic missense variants and 1 frameshift variant identified in a patient with -7. Compared to WT-SAMD9, Thr205Pro-SAMD9, Ile247Thr-SAMD9, and Leu574Pro-SAMD9 showed more proliferative cell growth, whereas, I268T-SAMD9 and D550V-SAMD9L had no significant differences compared to the WT. Compared to WT-SAMD9L, 6 variants (Glu220Gly, Leu1323fs, Cys228Tyr, Trp517Arg, Gly235Ser, and Trp333Cys), fused SAMD9L separately, also had higher proliferation rates than WT-SAMD9L. In total, 9 out of 13 variants (69%) cell growth was not suppressed compared to wild-type cells. Pediatric MDS variants showing less cell growth than the wild type cells suggests that they enhance the normal function of SAMD9/SAMD9L, which is to slow cell cycle progression. In this sense they are gain of function mutations (GOF). In contrast, the majority of variants identified in adult MDS were loss-of-function (LOF) mutations.Thus, given the different distribution of variants, lack of genetic reversion, and opposing functional results, we conclude that GL SAMD9/SAMD9L variants might have different consequences in familial childhood vs. adult disease. Sporadic adult MDS patients lacked early onset and genetic reversions following acquisition of -7/del(7q) or another mutations in cis characterized in pediatric MDS patients. SAMD9/SAMD9L variants could have two conflicting consequences; i) GOF mutations in pediatric patients that result in genetic reversion or MDS associated with aberrations of chromosome 7 with an early onset, or ii) LOF variants resulting in a late development of MDS due to haplo-insufficiency of SAMD9/SAMD9L. Our findings suggest that GL SAMD9/SAMD9L variants may be linked to the pathogenesis of a subset of adult sporadic MDS patients. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.

Telomere Length and Telomerase Complex Mutations Predict Fatal Treatment Toxicity after Stem Cell Transplantation in Patients with Myelodysplastic Syndrome

Introduction: Identifying patients at high risk of fatal treatment toxicity is a central challenge in hematopoietic stem cell transplantation (HSCT). Objective metrics that enable more accurate prediction of non-relapse mortality (NRM) could inform clinical decisions about timing and modality of HSCT. Short telomere length, mediated by inherited or acquired factors, impairs cellular response to genotoxic and replicative stress. We therefore evaluated the impact of recipient telomere length on clinical outcomes based on treatment intensity in patients with myelodysplastic syndrome (MDS) receiving HSCT.Methods: We used qPCR to measure relative telomere lengths in whole blood DNA samples from 1514 patients who received allogeneic HSCT for MDS and were enrolled in the Center for International Blood and Marrow Transplant Research Repository. Within the cohort, patients age 40 and older were grouped into those with short (<25th), intermediate (25-75th) or long (>75th percentile) telomeres. To evaluate germline determinants of telomere length, we sequenced 7 genes involved in telomere maintenance and mutated in dyskeratosis congenita: TERC, TERT, DKC1, TINF2, NHP2, WRAP53 and CTC1. Putative germline variants were classified as “rare” if the allele frequency was <0.001 in all Genome Aggregation Database (gnomAD) populations.Results: Among patients age 40 and older (n=1267), those with short (HR 1.52, 1.24-1.85, p<0.001) or intermediate (HR 1.35, 1.13-1.61, p<0.001) telomere length had poor overall survival compared with those having long telomeres (Fig 1A). In a competing risks regression model, the adverse effect of shorter telomeres was driven by a significantly higher risk of NRM among patients with short (HR 1.57, 1.20-2.06, p=0.001) and intermediate (HR 1.32, 1.03-1.69, p=0.03) telomere length. The association between telomere length and NRM was evident in patients receiving myeloablative (MAC, p=0.002) but not reduced-intensity conditioning (RIC, p=0.2) regimens (Fig 1B). We observed no association between telomere length and disease relapse in patients receiving MAC or RIC regimens. In a multivariable regression model, the prognostic significance of telomere length was independent of clinical and genetic factors, including age, Karnofsky performance status, hematologic parameters, IPSS-R risk category, donor mismatch, donor age, and TP53 mutation.We identified 40 patients (2.6% of the cohort) with rare germline TERT variants. Patients with rare TERT variants had significantly shorter telomeres than patients with common (p=0.001) or no (p<0.0001) TERT variants and were diagnosed with MDS at an earlier age than patients with common (52.2 vs. 58.4 years, p=0.01) or no (52.2 vs. 57.9 years, p=0.01) TERT variants. The domain distribution of rare TERT variants mirrored that of validated pathogenic germline TERT mutations, primarily affecting the reverse transcriptase and C-terminal extension domains. Rare variants in TERC (0.4%) and DKC1 (0.2%) were also associated with shorter telomeres (p=0.02 and p=0.04, respectively). In total, we identified germline telomerase complex mutations in 49 of 1514 MDS patients (3.2%), even though only 1 patient had a clinical diagnosis of dyskeratosis congenita. Together, patients with telomerase complex mutations had shorter overall survival than those without mutations (unadjusted p=0.008), attributable to a marked increase in the risk of early NRM among those receiving MAC (1 year cumulative incidence of NRM 48% vs. 26%, p=0.03). The impact of shorter telomeres on NRM was similar in patients with and without identified core telomerase complex mutations, suggesting that additional mechanisms of impaired telomere length maintenance may contribute to MDS pathogenesis and outcome.Conclusion: Recipient telomere length is independently associated with overall survival after allogeneic HSCT for MDS. Patients age 40 or older with shorter blood cell telomere length have a significantly elevated risk of early NRM with myeloablative conditioning regimens. Clinically unrecognized germline mutations in the telomerase genes TERT, TERC, and DKC1 define a distinct subset of adult patients with sporadic MDS and short telomeres who have poor transplant outcomes. Together, these results indicate that short telomere length in MDS patients mediates fatal treatment toxicity that may be attenuated by lower intensity conditioning approaches. DisclosuresAntin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Novel and Significant Impact of Germline Variants Predisposed to Pathogenic Somatic Mutations and Loss of Heterozygosity (LOH) in Myelodysplastic Syndromes (MDS) and Clonal Hematopoiesis of Indeterminate Potential (CHIP)

While germline predisposition to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) has long been recognized mainly through rare familial and pediatric cases, it has been drawing an increasing attention, on the basis of the recent discovery of novel risk alleles for MDS/AML through studies relying on revolutionized sequencing technologies; according to these studies, it suggest that more numbers of MDS/AML cases than expected might have germline predisposition. Moreover, it is suggested that germline variations may also confer predisposition to age-related clonal hematopoiesis or “CHIP”, which has been implicated in the development of MDS/AML.In this study, we explored germline predisposition to MDS and CHIP through intensive sequencing of blood samples from large cohorts of AML/MDS patients and ‘hematologically’ healthy individuals (HHIs), in which germline variants in 21 genes implicated in sporadic or familial MDS/AML or CHIP were interrogated among patients with MDS/AML from the Japan Marrow Donor Program (n=797) and HHIs aged >60 years from Biobank Japan (n=10,852). Germline variants were referred to NCBI dbSNP Build 151 database, excluding the entries in COSMIC ver.7 and in-house database, followed by manual curations. Somatic mutations and CHIP in the 21 genes were also analyzed for MDS/AML and HHIs, respectively.In total, 30,286 germline variants, including both synonymous and non-synonymous changes, were detected in 21 genes in the entire cohort. By comparing their frequencies between in MDS/AML and HHIs, we identified 6 germline variants in showing a significant enrichment in MDS/AML. Among these most frequently observed was variants in DDX41, for which a total of 3,721 variants were detected in 3,688 HHIs. Among these, 3 variants were significantly enriched in MDS/AML, including p.A500fs (OR=13.1 [6.6-25.9] (95%CI) (n=15), p.S363del (OR=41.0, [4.3-349.5]) (n=3), and p.Y259C (OR=34.2, [6.6-176.8]) (n=5). Of interest, 14 of 23 MDS patients with one of these alleles carried somatic DDX41 mutations, typically p.R525H, which were not found in any of HHIs, further supporting the relevance of these DDX41 risk alleles. Also including an additional 2 nonsense/splicing variants, 5 DDX41 alleles found in 25 MDS/AML patients were thought to represent germline predisposition to MDS/AML. Similarly, RUNX1 p.H85N (OR=9.10, [1.52-54.52]) (n=2), CBL p.P782L (OR=4.27, [1.56-11.70]) (n=5), and GNAS p.H69N (OR = 2.90, [1.28-6.59]) (n=7) showed a significant enrichment in MDS/AML. Combined, these putative risk alleles accounted for 4.6% (37/797) of sporadic MDS and sAML. None of these alleles were observed in the Caucasian population of Exome Aggregation Consortium dataset, suggesting Asian origins of these variants.We next evaluated the effects of germline variants on CHIP. CHIP mutations were detected in 929 HHIs, where DNMT3A mutations (n=290) were most prevalent, followed by TET2 (n=124) and ASXL1 (n=68) mutations. By comparing allele frequency of each of 1,276 germline variants between healthy donors with and without CHIP, we identified two haplotypes at the JAK2 and TET2 loci, defined by T/A at c.C489T/c.G2490A (JAK2) and G/G/T at c.G652A/c.G3117A/c.T4140C (TET2), which were significantly enriched in the cases carrying CHIP with the JAK2 (p.V617F) and TET2 mutations, respectively (T/A vs. C/G; OR=3.36, [1.41-8.01] for JAK2 and G/G/T vs. A/A/C; OR=1.85, [1.19-2.86] for TET2). Intriguingly, the JAK2 risk haplotype (C/G) were also enriched in MDS cases with JAK2 p.V617F mutations (T/A vs. C/G; OR=3.06, [1.26-7.60]). Similarly, the TET2 risk haplotype (G/G/T) tended to be enriched in MDS cases with TET2 mutations, although not statistically significant. Finally, variant allele frequency of JAK2 p.V617F mutations in CHIP exceeded 0.5 in 4 out of 26 JAK2 CHIP-positive patients (15%), suggesting the presence of loss of heterozygosity (LOH) in chromosome 9p.In conclusion, through a large-scale detection of germline variants in 21 common drivers of MDS/AML as well as CHIP, we identified multiple novel germline variants or haplotypes that showed a significant predisposition to the development of adult-onset MDS or CHIP, respectively. Our findings provide novel insights into the genetic basis of myeloid leukemogenesis and the development of CHIP. DisclosuresNakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Kanda:Otsuka: Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Nippon-Shinyaku: Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Pfizer: Research Funding; MSD: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Novartis: Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Taisho-Toyama: Research Funding; CSL Behring: Research Funding; Tanabe-Mitsubishi: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.

Universal genetic testing for inherited susceptibility in children and adults with myelodysplastic syndrome and acute myeloid leukemia: are we there yet?

Comprehensive genomic profiling of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) cases have enabled the detection and differentiation of driver and subclonal mutations, informed risk prognostication, and defined targeted therapies. These insights into disease biology, and management have made multigene-acquired mutation testing a critical part of the diagnostic assessment of patients with sporadic MDS and AML. More recently, our understanding of the role of an increasing number of inherited genetic factors on MDS/AML risk and management has rapidly progressed. In recognition of the growing impact of this field, clinical guidelines and disease classification systems for both MDS and AML have recently incorporated familial MDS/AML predisposition syndromes into their diagnostic algorithms. In this perspective piece, we contemplate the advantages, disadvantages, and barriers that would need to be overcome to incorporate inherited MDS/AML genetic testing into the upfront molecular diagnostic work-up of every MDS/AML patient. For centers already performing panel-based tumor-only testing, including genes associated with familial forms of MDS/AML (e.g., RUNX1, CEBPA, GATA2, TP53), we advocate optimizing these tests to detect all types of germline variants in these genes and moving toward upfront paired tumor/germline testing to maximize detection and streamline patient care.

RUNX1 Mutations in Inherited and Sporadic Leukemia.

RUNX1 is a recurrently mutated gene in sporadic myelodysplastic syndrome and leukemia. Inherited mutations in RUNX1 cause familial platelet disorder with predisposition to acute myeloid leukemia (FPD/AML). In sporadic AML, mutations in RUNX1 are usually secondary events, whereas in FPD/AML they are initiating events. Here we will describe mutations in RUNX1 in sporadic AML and in FPD/AML, discuss the mechanisms by which inherited mutations in RUNX1 could elevate the risk of AML in FPD/AML individuals, and speculate on why mutations in RUNX1 are rarely, if ever, the first event in sporadic AML.

Mutational Spectrum of Fanconi Anemia Associated Myeloid Neoplasms.

Individuals with Fanconi anemia (FA) have a high risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), yet the secondary somatic mutations lending to these malignancies remain to be further elucidated. We employed a next-generation sequencing myeloid neoplasia gene panel to determine the mutational spectrum of FA-related MDS/AML. Ten of 16 patients showed missense, nonsense, insertion or duplication mutations in 13 genes. In contrast to findings in MDS in the general population, mutations in genes involved in RNA splicing were rarely affected. Mutations in RUNX1 and genes of the RAS pathway appeared more instrumental in the pathogenesis of FA myeloid malignancies. RUNX1 mutations were associated with more advanced disease. Interestingly, one patient with refractory anemia with ring sideroblasts harbored the SF3B1 p.K700E mutation highlighting the mutation's causative role in MDS with ring sideroblasts even in the context of FA. On the whole, our findings implicate a different genetic architecture of FA MDS/AML from adult sporadic MDS. Notably, the genetic events resemble those described in pediatric MDS.

Overlap of Syndromes Associated with Myelodysplasia and Leukemia

Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are heterogeneous hematopoietic stem cell clonal disorders characterized by defective hematopoiesis and premature mortality in many patients. The majority of MDS and AML cases are sporadic but there are also rare cases where two or more affected individuals are found within the same family - “familial myelodysplasia and leukemia.” These familial cases represent a high-risk group of patients who require a unique management approach; they also provide an excellent opportunity for the identification of genetic changes that drive the disease. Over the last 15 years several genetic subtypes of familial MDS/AML have been identified and characterized. This includes families with constitutional heterozygous mutations in RUNX1, CEBPA, TERC, TERT, GATA2, and SRP72. The spectrum of mutations associated with familial MDS/AML implicates different biological pathways in the development of the disease, including transcription regulation (RUNX1, CEBPA and GATA2), telomere maintenance (TERC and TERT) and signal recognition (SRP72). It is notable that somatic mutations in RUNX1, CEBPA, and GATA2 are found in patients with sporadic MDS and AML. These discoveries demonstrate that familial MDS/AML is very heterogeneous and that the genetic characterization of the uncharacterized families will offer further insight into the primary pathophysiology of MDS/AML. Analysis of the MDS/AML families demonstrates considerable variability in the spectrum of features seen in these six genetic categories. There is heterogeneity even within each genetic subtype. The diverse range of different mutations in the disease gene in part explains the phenotypic difference between families. However, there are also differences amongst members of the same family (for example, in RUNX1 families some individuals only have a platelet abnormality and yet others have AML), suggesting that additional factors contribute to the phenotype. The mechanisms that underlie the development of progression to leukemia remain to be characterized. In families with CEBPA mutations there appears to be a near complete penetrance to developing AML. This is in contrast to the very diverse phenotypes associated with constitutional TERC and TERT mutations, ranging from dyskeratosis congenita (DC) to aplastic anemia, MDS, and AML. A similar situation has been observed with constitutional heterozygous GATA2 mutations. Here again affected individuals can display a wide range of phenotypes including MDS, AML, Emberger syndrome (primary lymphoedema with MDS/AML), MonoMAC syndrome (monocytopenia and Mycobacterium infection; the phenotype may also include Natural Killer cell and B-cell lymphopenia). It is notable that Natural Killer and B-cell deficiencies are also observed in DC. This highlights the overlap of features between the different genetic subtypes in addition to MDS/AML. Recognition of familial MDS/AML is important in guiding management. This includes appropriate genetic counselling of family members as well as careful selection of family donors regarding hematopoietic stem cell transplantation (HSCT). It also enables other specific modifications in the management. For example, patients with significant lymphopenia (be at secondary to TERC or GATA2 mutation) benefit from appropriate antimicrobial prophylaxis. Equally, patients with TERC and TERT mutations require low intensity Fludarabine-based HSCT (due to the abnormal/delayed healing of tissues as a consequence of the telomere deficiency) whereas patients with RUNX1 mutations appear to tolerate standard conditioning regimens. Further understanding of the biology and the associated phenotypic diversity will help to improve management of this group of patients. It is also likely to have relevance for sporadic MDS and AML. Disclosures No relevant conflicts of interest to declare.

GATA2 Mutations In Nonsyndromic Pediatric Myelodysplastic Syndrome

Myelodysplastic syndrome (MDS) is rare in children. Certain inherited bone marrow failure syndromes (IBMFS), such as Fanconi anemia, severe congenital neutropenia and Shwachman Diamond syndrome, markedly increase the risk of MDS during childhood. However, the genetic factor(s) underlying sporadic pediatric MDS are unknown. Germline mutations in GATA2, a hematopoietic transcription factor, explain four MDS-predisposing conditions: monocytopenia and mycobacterial infection (MonoMAC); dendritic cell, monocyte, B and NK cell lymphoid deficiency (DCML); primary lymphedema with myelodysplasia progressing to acute myeloid leukemia (Emberger syndrome); and a subset of familial MDS. Cases of pediatric MDS have been observed in some of the reported pedigrees. In addition, three individuals have been reported with large, de novo deletions encompassing GATA2 and surrounding genes and manifesting developmental delay, intellectual disability and dysmorphic features alongside their hematologic abnormalities. We identified a novel GATA2 splice site variant (c.1018-2A>C) in a teenager with MDS, WHO classification refractory cytopenia of childhood (RCC). Although he was found to have monocytopenia and B and NK cell deficiencies, he had no history of infections associated with MonoMAC or pertinent family history. We, therefore, hypothesized that mutations in GATA2 might be present in additional cases of pediatric MDS that were neither associated with an IBMFS nor relevant personal or family history.Two Baylor College of Medicine biology studies open to children with hematologic disease were queried for patients with the diagnosis of MDS. Exclusion criteria included treatment-related MDS, diagnosis of an IBMFS, prior diagnosis of severe aplastic anemia or infections suspicious for MonoMAC or DCML, and known or suspected family history of a GATA2-associated disorder. Cases lacking a pre-hematopoietic stem cell transplantation (HSCT) tissue sample available for study were also excluded. In addition to the patient described above, six children were identified who met eligibility criteria. DNA was isolated from banked peripheral blood or bone marrow cells and GATA2 sequencing performed, including upstream and intronic regulatory regions. Array comparative genomic hybridization was also performed on one sample that lacked GATA2 sequence variants, but was notable for complete absence of heterozygosity (AOH), including 6 polymorphic sites with minor allele frequencies of 0.20 or greater. Pertinent clinical and laboratory features were extracted by medical record review blinded to GATA2 status.We found heterozygous GATA2 mutations in three of the six additional patient samples. Thus, four of this seven patient, pediatric MDS cohort had mutated GATA2. Two of the newly identified mutations were splice site variants: a previously described c.1018-1G>A and a novel variant altering the exon 7 splice site acceptor (c.1114-1G>C). The third mutation was a de novo 3.1-3.3 Mb deletion encompassing the entire GATA2 locus and contiguous genes, and was established to be germline by analysis of skin fibroblasts. Notably, the patient had normal neurocognitive development and was without dysmorphic features. Their ages of presentation were 5, 9, 12 and 15 years. With the exception of the initial case, peripheral blood T and B cell phenotyping was not obtained prior to HSCT. Monocytopenia of less than 200/µL was present in five of seven patients, three of whom had a GATA2 mutation. All four GATA2 mutation cases had RCC and three of the four had monosomy 7 at diagnosis. In contrast, the three cases lacking GATA2 mutation presented with the MDS classification refractory anemia with excess blasts (RAEB-2), with either a normal karyotype, complex karyotypic changes or chromosome 13.q12q14 deletion.GATA2 mutation may explain a significant portion of sporadic, seemingly nonsyndromic pediatric MDS, particularly cases with monosomy 7. Evaluation of larger cohorts is warranted to ascertain the true prevalence. Although this cohort is small, we recommend GATA2 sequencing be performed as part of the initial evaluation of pediatric MDS as the identification of a germline mutation has critical implications for related donor selection and genetic counseling. AOH in GATA2 sequencing should prompt deletion analysis, even in cases without infections, dysmorphic features or neurocognitive impairment. Disclosures:No relevant conflicts of interest to declare.

Identification of Dido1 Mutation Associated with Familial Myelodysplastic Syndrome (MDS)/Acute Myeloid Leukemia (AML)

AbstractAbstract 169 IntroductionApproximately half of all patients with sporadic MDS will present with one of the known recurrent genetic alterations including additions/deletions of large chromosomal segments or mutations in single genes. Familial cases of MDS are rare, however, and the mutations that have been identified are not unique to MDS. Here we describe a family in which three of nine siblings were affected with early-onset MDS and a del(20) karyotype (Table 1). One of the brothers progressed to AML and died while the remaining 2 brothers are stable. Methods and ResultsWe performed whole exome sequencing on 2 affected brothers and 2 normal male family members followed by Sanger sequencing. Comparison of non-synonymous mutations common to the 2 brothers with MDS but not found in the normal controls revealed a mutated gene, Dido1, which is located at 20q13.33 upstream of the deleted region in the MDS brothers. The G to A mutation leads to substitution of a bulky basic, polar arginine in place of a small neutral non-polar glycine at amino acid 956 of the protein. This mutation, confirmed by PCR, was present in one allele in both the marrow and T-cells (not part of the MDS clone) of both brothers with MDS (Figure 1) showing that the mutation is not somatic but is, rather, a germline mutation. Since all three affected brothers harbored a del(20), it was of interest to investigate whether other patients with MDS or MDS/MPD and del(20)(q11.2) may also have this specific mutation. DNA from bone marrow mononuclear cells (BMMNC) of 10 del(20q) patients with either MDS or MDS/MPD was examined by PCR and Sanger sequencing. No mutation was found in any of the samples, suggesting that the mutation is unique to the affected family. BMMNC were available for 16 additional members of this extensive family. PCR followed by sequencing showed that 7/16 members of generation III have the identical heterozygous mutation. Interestingly, while no DNA was available from the brother who died of AML, analysis of his children revealed that 2 sons (III- Q and III-R) carry the mutation, confirming that the father also carried this mutation. Similarly, while DNA from sister II-G was not available for analysis, one of her daughters carries the mutation confirming the presence of mutation in the mother. DiscussionDeath inducer-obliterator 1 (Dido1) is a putative transcription factor originally identified in a screen for apoptosis related genes in a murine pre-B cell line. The gene encodes three alternate isoforms and gene targeting of all three isoforms in a murine model leads to development of a transplantable myeloid disorder with features similar to MDS/MPN. These studies also showed variable decreased Dido1 expression in a subset of patients with myeloid disorders when compared to healthy controls. Our results support the hypothesis that Dido1 aberrations in hematopoietic stem cells may contribute to MDS evolution. The fact that deletions in the long arm of chromosome 20 are one of the most frequent aberrations found in MDS and clearly was a secondary event in the 3 affected brothers of this family suggests that Dido1 may ontribute to this specific genomic instability. Generation III are still young, but those with the mutation may have an increased susceptibility to myeloid disease and should be clinically followed. ConclusionThe identification of the Dido1 mutation in this family suggests a novel pathogenic mechanism for the development of familial MDS.Table 1Clinical features of brothers with MDSBrothersAgeFABIPSSKaryotypeII-A66RAInt-146,XY,del(20)(q11.2q13.3)[17]/47,idem,+8[3]]II-E58RALow46,XY,del(20)(q11.2)[6]/46,XY[14]II-F52RAEB-2High6,XY,-4,-5,-7,-9,-10,-12,-16,-17,-18,-20[11]/ 36,XY,idem,add(2)(q33),add(11)(p14)[6]?46,XY[cp3] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Unexpected High Frequency of GATA2 Mutations in Children with Non-Familial MDS and Monosomy 7

AbstractAbstract 1699Haploinsufficiency of GATA2 results in three overlapping clinical entities unified by the predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML): i) familial MDS/AML, ii) Emberger syndrome, and iii) an immunodeficiency termed monocytopenia with mycobacterium avium infections (MonoMAC)/ dendritic cell, monocyte, B- and NK-lymphoid deficiency (DCML). All these conditions manifest with a wide heterogeneity of symptoms predominantly affecting the hematologic and immune systems.To investigate the frequency of GATA2 defects in children and adolescents with MDS registered in the retrospective and prospective studies of the European Working Group of MDS and JMML in Childhood (EWOG-MDS), we first identified children from families with MDS/AML affecting multiple individuals, or children with MDS and preexisting lymphedema or immunodeficiency. We measured telomere length in hematopoietic cells to rule out an underlying defect of the telomerase complex, performed SNP-Array analysis, and excluded cases with germline RUNX1 aberrations. We identified heterozygous GATA2 mutations in 11 offspring of 8 distinct pedigrees: 4 previously reported missense mutations affecting the 2nd zinc finger (ZF2) T357A, S447R and R396Q/W; a novel exon 5 skipping mutation c.1018-10_1037del; and one novel nonsense mutation S201X and two novel frameshift mutations S139CfsX45, and V70LfsX114, all resulting in a premature stop codon prior to ZF2. The median age at diagnosis of MDS was 13.6 (11.0–16.9) years. Karyotype abnormalities were detected in 10/11 (91%) children: in 9 at initial presentation and in one during disease progression. Most patients carried the aneuploidies monosomy 7 (−7; n=5), trisomy 8 (+8; n=4), or both (n=1). Additionally, the recurrent translocation +1,der(1;7)(q10;p10) was detected in three patients. Interestingly, in one patient, the initial karyotype 45,XY,-7 transformed at relapse after hematopoietic stem cell transplantation (HSCT) to 46,XY,-7,+8,+21. Two additional patients with hypocellular MDS since adolescence and GATA2 hotspot mutations referred to us carried the aneuploidies −7 or +8, respectively.We next extended our study to children registered to EWOG-MDS in Germany with non-familial MDS and monosomy 7. Unexpectedly 8/51 (16%) patients with −7 carried GATA2 aberrations: 7 missense mutations within ZF2 and a germline 3.6Mb deletion 3q21.2–21.3 encompassing GATA2. In depth review of patient’s history identified preexisting features of immunodeficiency as seen on laboratory tests and clinical exam (i.e. generalized verrucosis) in 5 of these 8 patients. The germline status was confirmed in cases where non-hematopoietic specimens were available.The combined analysis of all 21 patients identified additional comorbidities that, to our knowledge, were previously not reported in patients with GATA2-deficiency. These affected the urogenital system (nonselective glomerular proteinuria in one family, vesicoureteral reflux in a single case), neurodevelopmental delay and aggressive behavior in two cases, and ulcerative colitis in 2 families. At diagnosis of MDS, refractory cytopenia of childhood (RCC) was present in 43% of cases, while 33% presented with RAEB, and remaining 24% of patients with RAEBt and myelodysplasia related AML. Following HSCT as first line therapy, 10 of 12 children are alive, while all 4 children treated with AML-like therapy prior to HSCT succumbed. Five patients with RCC are followed closely with a watch and wait regimen.Finally, to study the genetic factors initiating clonal evolution, we subjected selected cases to targeted next generation sequencing of 103 cancer-associated genes. Based on preliminary data, some GATA2-deficient patients carry acquired mutations of genes previously reported in malignant processes (such as ASXL1, RUNX1, TET2, and 14 more genes).In summary, we identified a high frequency of GATA2 mutations among children and adolescents cases with sporadic MDS with monosomy 7 in children and adolescents. Some of these patients had a mild preexisting immunodeficiency. Outcome following HSCT without prior intensive chemotherapy was similar to what can be expected in children with MDS without GATA2 mutations. The genotype-phenotype correlation and mechanisms of clonal evolution are not understood and warrant further studies. Disclosures:No relevant conflicts of interest to declare.

Telomere Attrition and Chromosomal Instability During Long-Term Cultivation of Hematopoietic Stem Cells,

Abstract 4001By means of a large RNA interference screen, Ebert et al. identified RPS14 as the target of del(5q), since haploinsufficiency of RPS14 in human hematopoietic stem cells (HSC) induces an erythroid differentiation defect, a characteristic feature of myelodysplastic syndromes (MDS) with isolated deletion in 5q (Nature451, 335–339 (2008)). However, to the best of our knowledge, these cells have not been observed for more than two weeks in culture. We have shown that telomere shortening and chromosomal instability play a crucial role, particularly during progression of MDS with del(5q) into acute myeloid leukemia (AML) (K Lange et al, Genes Chromosomes Cancer49, 260–269 (2010) and G Göhring et al, Leukemia, 2011 (in press)). Because of the limited proliferation of primary MDS cells in culture and because there is no appropriate MDS cell line, a good human cell culture model is urgently needed to investigate the relationship of telomere attrition and chromosomal instability during the progression of MDS with del(5q).Therefore, it was our aim was to create a long-term culture (LTC) model to observe the effects of RPS14 haploinsufficiency on differentiation capacity, DNA repair, telomere shortening and chromosomal instability in human HSCs.CD34+ cells were isolated from umbilical cord blood via magnetic cell separation. Knockdown of RPS14 to a level of 40 – 50% was performed via lentiviral transduction of shRNA vectors. As controls, in addition to untransduced mock cells, CD34+ cells were transduced with scrambled shRNA. LTC was performed on murine feeder layers (M2-10B4).In short-term culture, as expected, knockdown of RPS14 led to an erythroid differentiation defect, decreased proliferative activity and an increased level of apoptosis. Cultivation on murine feeder layers enabled expansion of CD34+ cells for more than 6 weeks.Generally, within 6 weeks RPS14 -deficient CD34+ cells showed a poorer proliferation than the control cells with a 214-fold versus a 6080-fold multiplication, respectively. Median numbers of γH2AX foci, indicators of DNA double-strand breaks, did not differ between RPS14-deficient and control cells with a median number of 9.4 and 8.2 foci/cell, respectively. Induction of γH2AC foci via Mitomycin C, a DNA cross-linking agent inducing double-strand breaks, did not reveal an altered DNA repair capacity with 14.3 and 11.6 foci/cell.Colony-forming assays showed that, even though RPS14 haploinsufficiency immediately led to an erythroid differentiation defect, this differentiation capacity decreased even further within the weeks of follow-up. This however, although with a delay of about 4 weeks, could also be observed in the control cells (Figure 1). [Display omitted] Telomere length decreased in RPS14- deficient cells as well as in control cells. Initially, telomeres showed a median length of about 12.5 kb, decreasing to a median length of 10.3 kb (range 8.4 – 11.4 kb) and 5.8 kb (range 5.7 – 8.9 kb) after 2 and 4 weeks, respectively, which afterwards elongated to a level of 8.7 kb (range 8.5 –8.9 kb) after 6 weeks. The mechanism underlying this primary shortening and subsequent re-elongation might be due to an up-regulation of telomerase or of the alternative lengthening of telomeres (ALT) mechanism. Cytogenetic investigations demonstrated an increase in chromosomal breakage in all cultures, pointing towards induction during LTC rather than due to RPS14 deficiency. Remarkably, chromosomal breakage and chromosome aberrations in single cells within the first 4 weeks of cultivation of hematopoietic stem cells transduced with scrambled shRNA was followed by clonal dominance of monosomy 7 after 6 weeks. This may be an effect caused by insertional mutagenesis. LM-PCR is currently under way to obtain insights into the affected insertion site(s). Monosomy 7 is one of the most frequent chromosome aberrations in MDS and frequently occurs as an additional aberration in MDS/AML with inversion 3q generating an MDS-EVI1 fusion transcript. Thus, it will be interesting to see whether monosomy 7 in our LTC model cooperates with EVI1 activation as in sporadic MDS and AML.In conclusion, a LTC model on feeder layer cells seems to be an appropriate system to analyze hematopoietic stem cells and MDS in culture. However, cell culture artefacts inducing telomere shortening and chromosomal instability or insertional mutagenesis have to be taken into account and regular cytogenetic analyses should always be performed. Disclosures:No relevant conflicts of interest to declare.

Familial Myelodysplastic Syndromes with Centromere Instability of Chromosome 1 Origin.

Myelodysplastic syndrome (MDS) is a highly heterogeneous disease. The etiology involves both environmental and genetic factors. Identification of new pathogenetic mechanisms may have important impact on the choice of treatment as well as the development of new treatment options. Familial disease gives a unique opportunity to study those mechanisms. Familial occurrence of MDS is rare and mainly associated with autosomal recessive genetic syndromes such as Fanconi anaemia, Bloom syndrome, Diamond-Blackfan syndrome and megakaryocytic thrombocytopenia. Cytogenetic abnormalities are in general seen in 50% of MDS cases. Most frequently, abnormalities involving chromosome 5 or 7 are observed, which has also been described in rare cases of familial MDS. We here present a unique family in which a father and two of his three children were diagnosed with refractory anemia (RA) with cytogenetic aberrations resulting in three different translocations involving chromosome 1q and resulting in trisomy of 1q. Trisomy 1q is often seen in sporadic MDS, but the unbalanced translocations observed in this family may originate from inherited centromere instability of chromosome 1. The genetic background for the observed centromere instability could be a DNA methylation deficiency, which we plan to investigate further.

Predictive Markers of Myelodysplastic Syndrome in the Bone Marrow of Patients with Fanconi Anemia.

Fanconi Anemia (FA) patients are at increased risk of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Impaired cell cycle regulation may have a role in this predisposition. Over-expression of p53, Ki-67 and survivin proteins has been reported in association with MDS. We analyzed bone marrow samples from 138 individuals: 31 FA, 17 acquired aplastic anemia (AA), 40 sporadic low-grade MDS (MDS-1: 35 refractory cytopenia with multilineage dysplasia [RCMD], 5 RCMD with ring sideroblasts [RCMD-RS]), 11 high-grade MDS (MDS-2: refractory anemia with excess blasts [RAEB]), 12 AML, and 27 normal. To identify cell cycle regulation abnormalities that predict risk of MDS or AML in FA patients, we analyzed bone marrow morphology, cellularity, and immunohistochemical expression of cell cycle regulation proteins: p53 (cycle arrest and apoptosis), Ki-67 (cell proliferation), caspase-3 (apoptosis), and survivin (anti-apoptosis). Protein expression was classified as over-expressed if it was detected in a percentage of cells exceeding the highest values observed in normal marrow. Between group comparisons were made using the Fisher exact test. More FA marrows were hypocellular versus sporadic MDS-1, MDS-2 and AML (p <0.0001 in all). p53 was over-expressed at similar rates in FA, MDS-1, MDS-2 and AML patients (12/25 vs. 22/35, 4/11 and 8/12; p = 0.3, 0.7 and 0.3, respectively); p53 was not over-expressed in AA (0/13, p = 0.003). Ki-67 was also over-expressed at similar rates in FA, MDS-2 and AML patients (9/18 vs. 3/11 and 7/12, p = 0.3 and 0.7), in whom over-expression was higher than in AA or MDS-1 (0/12 and 4/35). Survivin expression was more common in FA than in MDS-1, MDS-2, AML or AA (12/16 vs. 2/32, 0/10, 2/12 or 1/13; p <0.01 for all comparisons). In contrast, the caspase-3 over-expression was higher in MDS-2 than in FA (4/11 vs. 0/18, p = 0.014). 7/31 FA patients had RCMD marrow changes; however, there was no correlation between morphologic features of myelodysplasia and the over-expression of cell cycle markers in these patients. p53 over-expression was frequent in FA, MDS-1, MDS-2, and AML; this distinguishes these disorders from AA. Previous studies found no p53 gene mutations in FA; thus, p53 over-expression observed here may represent upregulation of p53, or diminished p53 protein catabolism. Ki-67 over-expression may be a marker for higher grade MDS. The prevalence of over-expression of survivin was unique to FA, distinguishing it clearly from any of the acquired disorders (AA, MDS or AML), and may be a marker for any inherited bone marrow failure syndrome. Our cell cycle marker results suggest that patients in general in whom there is over-expression of Ki-67 and/or p53 may be at higher risk of evolution to MDS/AML; a longitudinal study is needed to address this prediction.