The EBV-Positive Tumor Methylome Is Distinct from EBV-Negative in Diffuse Large B-Cell Lymphoma

<b>Backgrounds</b>: 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 remain poorly understood. Global DNA methylation profiling can provide insight into tumor heterogeneity and disease mechanisms. <b>Methods</b>: 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 (<i>n</i> = 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. <b>Results</b>: 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 1557 cancer-associated genes in EBV(+) versus EBV(-) DLBCL, and 330,872 probes mapping to 4230 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). <b>Conclusions</b>: These findings demonstrate 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.