Recurrent DNMT3A Mutations In Patients with Myelodysplastic Syndrome

Abstract 608

Myelodysplastic syndrome (MDS) genomes are characterized by global DNA hypomethylation with concomitant hypermethylation of gene promoter regions compared to CD34+ cells from normal bone marrow samples. Currently, the underlying mechanism of altered DNA methylation in MDS genomes and the critical target genes affected by methylation remain largely unknown. The methylation of CpG dinucleotides in humans is mediated by DNA methyltransferases, including DNMT1, DNMT3A, and DNMT3B. DNMT3A and DNMT3B are the dominant DNA methyltransferases involved in de novo DNA methylation and act independent of replication, whereas DNMT1 acts predominantly during replication to maintain hemimethylated DNA. The function of these proteins in cancer cells is less well defined. Our group recently found that DNMT3A mutations are common in de novo acute myeloid leukemia (62/281 cases, 22%) and are associated with poor survival (Ley, et al, unpublished), providing a rationale for examining the mutation status of DNMT3A in MDS patients. MDS cases (n=150) were classified according to the French-American-British (FAB) system. The patients included refractory anemia (RA; n=67), RA with ringed sideroblasts (RARS; n=5), RA with excess blasts (RAEB; n=72), and RA with excess blasts in transformation (RAEB-T; n=6). The median International Prognostic Scoring System (IPSS) score was 1 (range 0–3), and the median myeloblast count was 4 (range 0–28%). We designed and validated 28 primer pairs covering the coding sequences and splice sites of all 23 exons for DNMT3A. Paired DNA samples were obtained from the bone marrow (tumor) and skin (normal) of each patient so that somatic mutations could be distinguished from inherited variants/polymorphisms. 17,120 reads were produced by capillary sequencing, providing at least 1X coverage for 82.6% of the target sequence (low/no coverage was obtained for 2 out of 28 amplicons). A semiautomated analysis pipeline was used to identify sequence variants and we restricted our analysis to nonsynonymous and splice site nucleotide changes. All mutations were confirmed by independent PCR and sequencing. We identified nonsynonymous DNMT3A mutations in 12/150 bone marrow samples (8% of cases). All the mutations were heterozygous (10 missense, 1 nonsense, 1 frameshift) and were computationally predicted (by SIFT and/or PolyPhen2) to have deleterious functional consequences. DNMT3A mRNA is expressed in normal CD34+ bone marrow cells and was expressed in all MDS patient samples tested (n=28), independent of mutation status. There was no difference in the expression level of total DNMT3A mRNA in CD34+ cells harvested from mutant (n=3) vs. non-mutant MDS samples (n=25). Amino acid R882, located in the methyltransferase domain of DNMT3A, was the most common mutation site, accounting for 4/12 mutations. The clinical characteristics of the 12 patients with DNMT3A mutations were similar to those of the 138 patients without mutations. Specifically, DNMT3A mutations were present in all MDS FAB subtypes (excluding CMML which was not tested) and in patients with IPSS scores ranging from 0–3. Mutations were not associated with a specific karyotype. In addition, there was no correlation between mutation detection and the myeloblast count of the banked bone marrow specimen, suggesting that mutations were not missed due to the cellular heterogeneity in the samples. We compared the overall (OS) and event-free survival (EFS) of the 12 patients with DNMT3A mutations vs. 138 patients without a mutation and observed a significantly worse OS in patients with mutations (p=0.02), with a median survival of 433 and 945 days, respectively. There was a trend towards worse EFS for patients with mutations (p=0.05). A multivariate analysis for outcomes could not be performed due to the small sample size of patients with mutations, indicating that a larger cohort from a clinical trial will be needed to properly address the affect of DNMT3A mutations on outcomes. The small sample size also precluded us from addressing whether the response to the hypomethylating agents 5-azacytidine or decitabine correlated with the mutation status of DNMT3A. If validated in larger cohort studies, we propose that DNMT3A mutation status could help risk stratify de novo MDS patients for more aggressive treatment early in their disease course.