Key Points• Mutations in genes of the cohesin complex are recurrent mutations in AML with a strong association with NPM1 mutations.• Cohesin gene mutations have no clear prognostic impact in AML patients.Mutations in the cohesin complex are novel, genetic lesions in acute myeloid leukemia (AML) that are not well characterized. In this study, we analyzed the frequency, clinical, and prognostic implications of mutations in STAG1, STAG2, SMC1A, SMC3, and RAD21, all members of the cohesin complex, in a cohort of 389 uniformly treated AML patients by next generation sequencing. We identified a total of 23 patients (5.9%) with somatic mutations in 1 of the cohesin genes. All gene mutations were mutually exclusive, and STAG1 (1.8%), STAG2 (1.3%), and SMC3 (1.3%) were most frequently mutated. Patients with any cohesin complex mutation had lower BAALC expression levels. We found a strong association between mutations affecting the cohesin complex and NPM1.

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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.

Background MicroRNAs are short (20-40 nucleotides) non-coding RNA molecules that are responsible for the post-transcriptional regulation of gene expression. Aberrant expression of MicroRNAs has been associated with various malignancies. Specifically, downregulation of MicroRNA-142 (miR-142) has been shown to occur in acute myeloid leukemia (AML). Interestingly, also gene mutations in miR-142 have been recently described in de novo AML. So far, little is known about mutations in miR-142 in myeloid malignancies. The aim of this study was to analyze mutations in the miR-142 in a large cohort of 944 patients with AML and myelodysplastic syndrome (MDS). Patients and Methods The patient group consisted of 425 de novo AML patients (excluding AML M3) who entered the multicenter treatment trials AML SHG 0199 or AML SHG 0295, 326 patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) for secondary acute myeloid leukemia after a prior diagnosis of MDS (sAML) (n=170) or primary MDS (n=156), and 193 primary MDS patients not undergoing intensive therapy or allogeneic HSCT. The genomic region of the miR-142 gene, containing miR-142-5p and miR-142-3p, was sequenced by Sanger sequencing. Patient samples were also assessed for other frequently mutated genes in AML and MDS. Results and Discussion We identified five patients with mutations in miR-142. All mutations were heterozygous point mutations affecting the seed region of miR-142-3p, thereby potentially changing the target specificity of miR-142. Mutations in miR-142 occurred in male and female patients. Of the five patients with mutations in miR-142, only one patient carried the diagnosis of de novo AML (0.2% in de novo AML), while two patients were diagnosed with sAML (1.2% in sAML) and two patients had MDS (0.56% in MDS, corresponding to 0.77% in MDS/AML from MDS). Apart from one patient who underwent allogeneic transplantation for sAML, all other patients with follow-up died of the disease in less than a year. 3 patients had normal cytogenetics, while one patient had a complex karyotype and one patient had a trisomy 8 with translocation t(1;4). No mutated patient showed aberrations typically associated with de novo AML (RUNX1/RUNX1T1, CBFB/MYH11, FLT3-ITD, NPM1 mutations or CEBPA mutations). However, myelodysplasia-related gene mutations such as mutations in the splicing genes or chromatin remodelling genes were found in two patients (one patient with mutated ASXL1 and SRSF2, one patient with mutated U2AF1). Furthermore, one patient had a concomitant mutation in NRAS and IDH1. Thus, the associated mutational profile suggests that miR-142 mutations play a role in the pathogenesis of MDS rather than de novo AML. Conclusion MicroRNA-142 is recurrently but infrequently mutated in MDS and secondary AML evolving from MDS, and some mutations co-occur with MDS-related gene aberrations. As miR-142 mutations affect the seed region of the miRNA the target specificity is likely changed, and the miRNA may lose its tumor suppressor function, which has been implicated from functional studies. Disclosures: Platzbecker: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.