We integrated molecular data with available prognostic factors in patients undergoing allogeneic hematopoietic cell transplantation (alloHCT) for myelodysplastic syndrome (MDS) or secondary acute myeloid leukemia from MDS (sAML) to evaluate their impact on prognosis. 304 patients were sequenced for mutations in 54 genes. We used a Cox multivariate model and competing risk analysis with internal and cross validation to identify factors prognostic of overall survival (OS), cumulative incidence of relapse (CIR) and non-relapse mortality (NRM). In multivariate analysis, mutated NRAS, U2AF1, IDH2, TP53 and/or a complex karyotype were significant prognostic markers for OS besides age above 60 years, remission status, IPSS-R cytogenetic risk, HCT-CI > 2 and female donor sex. Mutated NRAS, IDH1, EZH2 and TP53 and/or a complex karyotype were
Introduction: The landscape of molecular aberrations in patients with myelodysplastic syndromes (MDS) has been well characterized and has identified ASXL1, BCOR, CUX1, IDH1, IDH2, SRSF2, RUNX1, U2AF1, TP53 and others as negative prognostic markers for overall survival (OS). We comprehensively investigated the prognostic impact of genetic aberrations in the context of allogeneic hematopoietic stem cell transplantation (alloHSCT) in a large cohort of patients with high risk MDS or secondary AML following MDS (sAML). Patients and Methods: 308 Patients with a diagnosis of MDS (47.4%) or sAML (52.6%) who received an alloHSCT at four German university medical centers and for whom genomic DNA was available at a time with active disease before transplantation were evaluated for the presence of mutations in 54 genes by Illumina high-throughput sequencing. Results: At least one mutation was identified in 82% of our patients with a median number of 2 mutations per patient. The most frequently mutated genes were ASXL1 (24.4%), DNMT3A, (23.1%), RUNX1 (16.9%), TET2 (16.9%), STAG2 (12%), TP53 (11.7%), and SRSF2 (11%) in agreement with previous reports. Mutation frequencies were similar between MDS and sAML patients for all mutated genes except SRSF2, TET2 and WT1, which were more frequently mutated in sAML. We grouped the mutated genes into functional classes and found that patients most frequently had mutations in modifiers of DNA methylation (42.2%), followed by chromatin modifiers (41.5%), splicing genes (31.1%), transcription factors (30.8%), signal transducers (28.8%) and tumor suppressors (14.6%). Mean variant allele frequencies were highest in modifiers of DNA methylation (33.2%), while signal transducers had the lowest allele frequency (20.4%). We next assessed the prognostic impact of gene classes and individual genes on outcome of patients after alloHSCT. Median follow up from transplantation was 4.15 years. Median patient age at time of HSCT was 58 years (range 19-75). 76 patients (25%) werein complete remission and 232 patients (75%) had active disease before transplantation. Low, intermediate, and high risk cytogenetics according to IPSS were found in 116 (38%), 59 (19%), and 115 (37%) patients, respectively. Matched and mismatched related donor HSCT was performed in 71 and 4 patients, respectively (23.1 and 1.3%), and matched and mismatched unrelated donor HSCT in 171 and 62 patients, respectively (55.5 and 20.1%). The six functional gene classes had no prognostic impact on survival, cumulative incidence of relapse and non-relapse mortality. We therefore evaluated the prognostic impact of individual gene mutations, of aberrations of chromosomes 3, 5, 7, 8, 17, 20 or a complex karyotype, and of transplant characteristics on OS. Parameters with significant impact on OS in univariate analysis were included in a multivariate cox proportional hazards model. Significant predictors of OS in multivariate analysis were mutations in PTPN11 (HR 3.1, present in 2.3% of pts.), IDH2 (HR 2.6, present in 4.2%), PHF6 (HR 2.2, present in 4.9%), NRAS (HR 1.8, present in 7.5%), presence of a complex karyotype (HR 1.8, present in 16.6%), transplantation from haploidentical donor (HR 5.5), RAEB/sAML not in complete remission before transplantation compared to untreated RA/RARS or RAEB/sAML and treated RAEB/sAML in remission (HR 2.0), GvHD prophylaxis other than calcineurin inhibitor with methotrexate or mycophenolate mofetil (HR 1.7) and female sex of the donor (HR 1.7). TP53 mutations lost their unfavorable prognostic impact when complex karyotype was added to the multivariate model. Competing risk analysis for cumulative incidence of relapse and non-relapse mortality showed that IDH2 and NRAS mutations and a complex karyotype were significantly associated with higher risk for relapse while PTPN11 and PHF6 mutations predicted for a higher incidence of non-relapse mortality. Importantly, a negative prognostic impact of ASXL1, BCOR, CUX1, IDH1, SRSF2, RUNX1 and U2AF1 seen previously in MDS patients not undergoing alloHSCT was not found in the transplant setting, suggesting that alloHSCT may overcome the unfavorable effect of these mutations. Conclusion: By extensive genetic characterisation of 308 MDS or sAML patients undergoing alloHSCT we identified mutations in IDH2, NRAS and complex karyotype as predictors of relapse and reduced OS and provide a matrix to refine risk prediction for allogeneic HSCT. Disclosures Heuser: Karyopharm: Research Funding. Platzbecker:Boehringer: Research Funding; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Thiede:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; AgenDix GmBH: Equity Ownership.
Introduction Aplastic anemia (AA) is a rare but life-threatening bone marrow failure syndrome, which is diagnosed based on cytopenias in peripheral blood and hypocellularity in the bone marrow. The distinction between AA and hypocellular myelodysplastic syndrome (MDS) is often difficult, and AA evolves into MDS at a 10-year cumulative incidence of 4-10%. As AA patients often respond to immunosuppressive therapy, an immune pathophysiology is widely assumed. However, the evolution of clonal cytogenetic aberrations in hematopoietic cells and the association with clonal paroxysmal nocturnal hemoglobinuria (PNH) suggest that at least some patients have a clonal hematopoietic disease. Walter et al. reported that 74% of MDS patients harbour a mutation in at least one of 94 genes (Walter et al. Leukemia 2013). We hypothesized that mutations that are found in MDS patients may also be present in AA patients. Aim To evaluate the mutation profile of 41 myelodysplasia-related genes in AA patients. Methods Bone marrow or peripheral blood was collected from 39 patients with moderate (n=11), severe (n=12), or very severe (n=16) AA before allogeneic transplantation (n=23) or when the patient was cytopenic in at least one blood lineage (non-transplanted patients, n=16, median time from diagnosis to sample harvest 2 years). The coding region of 33 genes was amplified by PCR and sequenced on the SOLiDTM sequencing system. The sequences were analyzed using the DNAnexus software and an in-house pipeline of bioinformatics software. All candidate SNPs were validated by Sanger sequencing and only those confirmed are reported. Eight additional genes were only sequenced by Sanger sequencing. Confirmed mutations were also sequenced using DNA from hair follicles as germline control. Telomere length was evaluated by monochrome multiplex quantitative PCR-based method in peripheral blood leukocytes of 13 AA patients and 20 healthy volunteers. Results The median age of AA patients at diagnosis was 30 years (range 9-79). Four patients (10%) had abnormal cytogenetics. In seven patients (18%), a GPI-deficient clone suggesting PNH/AA overlap syndrome was present. Twenty-three patients underwent allogeneic or syngeneic transplantation. The median follow-up from diagnosis of patients alive was 7.1 years. 36 of 39 patients were alive at last follow-up. Telomeres in peripheral blood leukocytes were significantly shorter in AA patients than in age matched healthy controls (P<.001). Next generation sequencing yielded an average coverage of 2015 reads per amplicon. In total, 6 mutations were identified in 5 patients (12.8%). One patient had a missense germline mutation in MYBL2, who developed trisomy 8 in the course of the disease; one patient had a missense germline mutation in TET2, another patient with very severe AA had a somatic missense TET2 mutation besides deletion of chromosome 5 (del5[q14q13]); one patient had a somatic missense mutation in SLIT1, and one patient with severe AA had two somatic mutations, i.e. one missense mutation in SETPB1 (D868N) and one frameshift mutation in ASXL1 (G646fs). This patient was diagnosed with severe AA at age 14 and received 4 courses of immunosuppressive therapy. Eleven years after diagnosis treatment with SCF and G-CSF was started, which induced a partial remission with signs of multilineage dysplasia. Two years later the patient received an allogeneic transplantation due to progressive thrombocytopenia. The current analysis was performed on cells harvested shortly before transplantation, and suggests that the patient had progressed to MDS. The patient with a SLIT1 mutation had very severe AA and responded well to the second course of anti-thymocyte globulin (ATG) and cyclosporine (CSA). The patient with a MYBL2 mutation is in remission after 2 courses of ATG/CSA for 16.8 years since diagnosis. The other two patients with mutations received an allogeneic or syngeneic transplant and are in remission 5.9 and 7.1 years after transplantation, respectively. Conclusions The frequency of MDS-related mutations is low in AA. We therefore suggest that mutation analysis of myelodysplasia-related genes may help to distinguish AA from MDS in ambiguous cases and may identify patients who are at risk for MDS-progression. Disclosures: No relevant conflicts of interest to declare.
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