The Effect of Epstein Barr Virus Latency on Cellular DNA Methylation Profile of DiffuseLarge B Cell Lymphoma

Epstein-Barr virus (EBV) is implicated in the pathogenesis of different B-cell lymphomas and lymphoproliferative disorders, including diffuse large B-cell lymphoma (DLBCL) arising in immunodeficiency settings. Despite its clinical significance, the mechanisms of EBV-mediated lymphomagenesis across different disease subtypes remains poorly understood. Global DNA methylation profiling can provide insight into tumor heterogeneity and disease mechanisms. To further characterize the underlying biology of EBV(+) DLBCL, we performed a global methylome analysis of a cohort of EBV(+)/(-) DLBCL. Illumina MethylationEPIC array data were generated from a curated set of DLBCL tissue samples (n=43) from a rural patient population with defined EBV status and immunodeficiency background. Differential methylation analyses were conducted using linear mixed models to identify significant methylation changes associated with EBV status. Principle component analysis (PCA) and probe-level comparisons revealed a distinct, globally hypermethylated DNA methylome in EBV(+) DLBCL compared to EBV(-) cases, and an overall hypomethylated profile in all DLBCL relative to control tissues. We identified a total of 117,334 differentially methylated probes mapping to 1,557 cancer-associated genes in EBV(+) versus EBV(-) DLBCL, and 330,872 probes mapping to 4,230 cancer-associated genes in all DLBCL versus controls. Pathway enrichment analysis highlighted distinct biological processes in EBV(+) DLBCL, including P53 feedback loops (hypermethylated genes) and MAPK signaling (hypomethylated genes). These findings demonstrated that EBV(+) DLBCL is epigenetically distinct from EBV(-) disease, with alterations that may contribute to clinical heterogeneity and potentially serve as biomarkers for disease classification and therapeutic targeting. We profiled the human cellular DNA methylation states of EBV-mediated diffuse large B-cell lymphomas (DLBCL) from our institution (UVM) utilizing the Illumina/Infinium MethylationEPIC array (v2.0), targeting ~935k CpG sites. Tumor cellular DNA was extracted from formalin-fixed, parrafin-embedded tissue blocks of patients samples of DLBCL. We analyzed a total of 43 patient samples following QC protocols (differential methylation values, p<0.05), which resulted in a total of 9 EBV(+) DLBCL, 22 EBV(-) DLBCL, and 12 control tissue (including EBV(+)/(-) reactive lymphoid proliferations and lymphoproliferative disorders of the lymph node and tonsil).