Variability in DNA methylation defines novel epigenetic subgroups of DLBCL associated with different clinical outcomes

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive form of non-Hodgkin lymphoma with variable biology and clinical behavior. The current classification does not fully explain the biological and clinical heterogeneity of DLBCLs. In this study, we carried out genomewide DNA methylation profiling of 140 DLBCL samples and 10 normal germinal center B cells using the HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction assay and hybridization to a custom Roche NimbleGen promoter array. We defined methylation disruption as a main epigenetic event in DLBCLs and designed a method for measuring the methylation variability of individual cases. We then used a novel approach for unsupervised hierarchical clustering based on the extent of DNA methylation variability. This approach identified 6 clusters (A-F). The extent of methylation variability was associated with survival outcomes, with significant differences in overall and progression-free survival. The novel clusters are characterized by disruption of specific biological pathways such as cytokine-mediated signaling, ephrin signaling, and pathways associated with apoptosis and cell-cycle regulation. In a subset of patients, we profiled gene expression and genomic variation to investigate their interplay with methylation changes. This study is the first to identify novel epigenetic clusters of DLBCLs and their aberrantly methylated genes, molecular associations, and survival.