Epigenetic Profiling of Primary DLBCLs Reveals Novel DNA Methylation-Based Clusters and New Underlying Mechanisms of Lymphomagenesis
Authors: Chambwe Nyasha , Kormaksson Matthias , De Subhajyoti , Michor Franziska , Johnson Nathalie , Scott David W , Gascoyne Randy D. , Melnick Ari , Campagne Fabien , Shaknovich Rita 
Published: October 2019

Abstract
Abstract 556
Epigenetic profiling of primary DLBCLs reveals novel DNA methylation-based clusters and new underlying mechanisms of lymphomagenesis. Nyasha Chambwe, Matthias Kormaksson, Subhajyoti De, Franziska Michor, Nathalie Johnson, David W. Scott, Randy D. Gascoyne, Ari Melnick, Fabien Campagne and Rita Shaknovich. Diffuse Large B Cell Lymphomas (DLBCLs) are a heterogeneous group of diseases from the clinical, molecular and genetic standpoints. While gene expression profiling has identified clinically and biologically relevant DLBCL subtypes, there is still considerable heterogeneity beyond what has been resolved through transcriptional and genetic profiling. It is increasingly clear that lesions in epigenetic regulatory proteins and transcription factors are a hallmark of DLBCL, which suggests that aberrant epigenetic programming is likely to be a significant factor in these tumors. Our recent preliminary data suggests that aberrant DNA methylation is also widespread in DLBCL and contributes to the abnormal expression patterns of GCB and ABC DLBCLs. Moreover recent data in the setting of AML show that DNA methylation profiles delineate disease subtypes not captured through transcriptional or genetic profiling. We hypothesized that DNA methylation profiles would allow us to identify new, biologically significant DLBCL subtypes. We therefore examined the DNA methylation of over 140 patients with DLBCL using the HELP assay covering multiple CpGs at over 14,000 gene loci. We next performed an unbiased (unsupervised) analysis of probesets that display significant variability (n=3,005), using K-means consensus clustering. This procedure identified four robust DLBCL subtypes based on epigenetic profiles. To identify the genes that define these four clusters we next performed supervised analysis of the DLBCL subtypes including the normal counterpart germinal center B-cells as a normal control, using three independent statistical methods. 46 genes defined cluster A, 236 genes defined cluster B, 376 genes defined cluster C and 1271 genes defined cluster D (selected genes displayed change in methylation of at least 30% at BH corrected p-value < 0.05). Each of these epigenetically defined DLBCL subtypes featured aberrant DNA methylation of genes and pathways with potential relevance to pathogenesis. For example cluster A was notable for aberrant DNA methylation of REL, STAT3, CD30; cluster B for aberrant methylation of the TNFa and IFN1 networks; cluster C of IDH2, FOXG1 genes; and cluster D of CDKN2A, ATF3, FOXL3 genes. Other defining characteristics of Cluster D was enrichment for ABC DLBCLs (Fisher exact test, p=0.007) and most remarkably, marked intra-tumor and inter-individual heterogeneity of DNA methylation patterning. This latter feature is suggestive of potential epigenetic clonal complexity and failure to properly control the boundaries of methylated regions of the genome in these tumors. Clusters A and B revealed enrichment for GC features and cluster B had increased expression of MUM1 (Fisher exact test, p=0.007 and p=0.002). Furthermore, we noted that higher expression of DNMT3B and DNMT3L were associated with hypermethylation in DLBCL samples, while higher expression level of AICDA was associated with aberrant hypomethylation in DLBCLs as compared to normal GC B-cells. Higher expression levels of epigenetic modifiers EZH2 and MBD4 was associated with greater heterogeneity of DNA methylation patterning compared to the normal methylation pattern in germinal center B cells. Collectively, the data indicate that DLBCLs are composed of entities defined by specific DNA methylation profiles that only partially overlap with the ABC and GCB classification. The DLBCL subtypes display perturbation of genes likely to play significant biological roles, and aberrant methylation patterning can be traced in part to aberrant expression of epigenetic regulators including DNA methyltransferases, AICDA and EZH2.

Disclosures:
No relevant conflicts of interest to declare.