Abstract

Introduction : DNA methyltransferases catalyze the addition of a methyl group in CpG islands. New sequencing technologies have enabled the identification of mutations in DNA methyltransferase-3-alpha ( DNMT3A ), which predict a worse prognosis in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). DNMT3A mutations are considered as initiating events and are believed to result in clonal expansion of hematopoietic stem/progenitor cells (HSPCs) and clonal hematopoiesis of indeterminate potential (CHIP). These mutations can also cooperate with other mutations in the context of myeloid malignancies including MDS and AML. Although DNMT3A mutations have been thoroughly investigated in myeloid diseases, DNMT3A mRNA expression and its clinical impact has been poorly explored. A previous study using competitive PCR analysis reported that DNMT3A is highly expressed in a cohort of thirty-three patients with AML compared to five normal bone marrow samples. Aims : We herein aimed to investigate and to compare the DNMT3A expression in bone marrow cells from healthy donors, MDS and AML patients, as well as between AML patients with wild-type and mutant DNMT3A. Methods : Twenty-one healthy donors and 153 patients with diagnosis of de novo MDS (according to WHO 2008 classifcation; n=54) or AML (n=99) were included in the study. DNMT3A mutational status was determined for 49 AML patients by next generation sequencing as part of the GeneTrails® AML/MDS Genotyping Panel or by Sanger sequencing. All healthy controls and patients provided informed written consent and the study was approved by the Ethics Committees of participating institutions. All patients were untreated at the time of sample collection. Gene expression was evaluated by qPCR. Statistical analyses were performed using GraphPad Prism 5 or SAS System for Windows 9.2. Mann-Whitney test (two groups) or Kruskal-Wallis test and Dunn post-hoc test (more than two groups) were used for measured factors. Due to data availability, survival analysis was performed for the cohort of MDS patients. Log-rank (Mantel-Cox) was used to estimate overall survival (OS) and event-free survival (EFS). For MDS patients, OS was defined from time of sampling to date of death or last seen; EFS was defined as time of sampling to date of progression to high-risk MDS or AML with myelodysplasia-related changes, or date of death. The level of significance was set at p < 0.05. Results : Higher DNMT3A expression was observed in bone marrow cells from AML patients (median: 1.39 [range: 0.01 - 7.54]) compared to healthy donors (0.74 [0.22 - 1.53]; p <0.001) and MDS patients (0.59 [0.01 - 3.46]; p <0.0001). The pattern of DNMT3A mRNA levels was similar in bone marrow cells from healthy donors compared to MDS. No difference was observed in DNMT3A expression when MDS patients were stratified by WHO 2008 classification into RA/RARS/RCMD versus RAEB-1/RAEB-2 subgroups (0.56 [0.01 - 2.76] versus 0.66 [0.01 - 3.46], p >0.05). For the 35 and 14 AML patients with wild-type or mutated DNMT3A , respectively,mRNA levels did not differ significantly (1.92 [0.51 - 7.54] versus 1.54 [0.89 - 5.80], p >0.05). Survival analysis among MDS patients revealed that gender and WHO 2008 classification were independent factors for EFS and OS. Conclusions : DNMT3A is highly expressed in bone marrow cells from AML but not MDS patients, and its expression does not differ according to DNMT3A mutation status.

Disclosures

Druker: Baxalta US Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; GRAIL: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cylene: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; CTI Biopharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; McGraw Hill: Patents & Royalties; Henry Stewart Talks: Patents & Royalties; Third Coast Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties: Royalties from Dana-Farber Cancer Institute, which has an exclusive commercial license with Millipore for monoclonal antiphosphotyrosine antibody 4G10, which I developed while employed at DFCI.; MED-C: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; The Leukemia & Lymphoma Society: Other: Joint Steering Committee of AML Master Protocol, Research Funding; Bristol-Myers Squibb: Research Funding; Oregon Health & Science University: Patents & Royalties: #843 Mutated ABL Kinase Domains (licensed to various companies); #0996 Detection of Gleevec Resistant Mutations (licensed to various companies, including MolecularMD); #0606 Treatment of Gastrointestinal Stromal Tumors (exclusively licensed to Novartis); Beta Cat: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees; ARIAD: Research Funding; Roche TCRC: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Author notes

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Asterisk with author names denotes non-ASH members.