DNMT3A‐2 EXPRESSION LEVELS CHARACTERISE DIFFUSE LARGE B ‐CELL LYMPHOMA WITH DISTINCT METHYLATION PATTERNS AND OUTCOME

Abstract

Background: DNA methylation changes have been implicated in the pathogenesis of diffuse large B-cell lymphoma (DLBCL), but molecular mechanisms driving aberrant methylation remain largely unknown. Alterations of the DNA methyltransferase DNMT3A are known pathogenetic events in acute leukaemia and T-cell lymphoma, with the short isoform 2 (DNMT3A-2) probably being of particular relevance. The role of DNMT3A in DLBCL has not been investigated yet. Method(s): DNMT3A-2 mRNA expression was derived from Illumina DASL microarray data (ILMN-1654945) of 175 newly diagnosed DLBCL patients from the UK R-CHOP14v21 trial. DNA methylation was analysed in 144/175 cases with Illumina Infinium 450 k arrays using the RnBeads package in R 3.2.3. Differentially methylated regions (DMRs) were analysed on RnBeads tiling level, comparing the highest and lowest DNMT3A-2 expression quartiles. Result(s): DNMT3A-2 expression showed significant impact on global DNA methylation, with 27 344 DMRs between the high and low expression groups (false discovery rate < 0.05). In contrast, only 50 DMRs were associated with expression of DNMT3A isoform 3 and 6485 DMRs with cell-of-origin subgroups. We further analysed DNMT3A-2-associated DMRs with at least 15% methylation difference (n = 3298), 2806 being hypomethylated (predominantly gene bodies) and 492 hypermethylated (predominantly promoters). Hypomethylated genes were enriched for KEGG cancer- and MAPKsignalling pathways (P < .0001), including MDM2, BCL2L1, LEF1, HDAC1, and JAK1, whereas WNT7A, BMP4, DKK3, ETV1, and WT1 were among hypermethylated genes. DNMT3A-2 expression was significantly lower in patients with extranodal involvement of >1 sites (P = .03) and in cases with BCL2-rearrangement (P = .03). There was a trend towards lower DNMT3A-2 expression in bulky disease (P = .09) and GCB subtypes (P = .10). DNMT3A-2 expression correlated with expression of CREBBP, DOT1L, and MLL2 (all P < .05). No association was observed between DNMT3A-2 expression and MYC- and BCL6-rearrangements, double-hit-lymphoma, or with patients' age. Patients with higher DNMT3A-2 expression (median) had worse progression-free survival (HR = 2.01; 95% CI, 1.21-3.35; P = .01) and a trend towards worse overall survival (HR = 1.72; 95% CI, 0.96-3.10; P = .07).We found methylation of 5 sites within the DNMT3A gene to be inversely correlated with DNMT3A-2 expression as a potential mechanism of isoform-specific regulation. Copy number gain of DNMT3A was seen in 9 cases, copy number loss in 3 cases, with no obvious impact on expression of isoform 2. DNMT3A mutational analyses will be provided at the meeting. Conclusion(s): DNMT3A-2 expression is associated with global DNA methylation changes and outcome in DLBCL, suggesting a potential role in disease pathogenesis. DNMT3A-2 expression levels might identify clinically meaningful subgroups of DLBCL suitable for epigenetic therapies.