Abstract 934: Defining the role of Epstein-Barr virus (EBV) in diffuse large B-cell lymphoma (DLBCL) pathogenesis

Abstract Background: Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive lymphoma. DLBCL is not a single disease, but a group of histologically similar yet genetically distinct cancers with unique prognostic and therapeutic features. The recently defined genetic landscape of DLBCL includes at least seven subtypes, but does not consider Epstein-Barr virus (EBV) status, despite up to 14% of DLBCL being EBV-positive and EBV’s known role in B cell lymphomagenesis. We performed a reanalysis of published DLBCL genetic data to molecularly define EBV-positive DLBCL, and characterized its cell line models. Methods: Published whole exome (WES) and RNA sequencing data were analyzed in silico to identify EBV-positive (EBV+) DLBCL tumors. Differential analyses of the mutation and transcriptomic profiles of the viral and cellular genes of EBV+ and EBV-negative tumors were performed. WES, RNAseq, and immunoblotting were performed on EBV+ DLBCL cell lines Farage, BCKN1, IBL1, and Val to assess the extent to which they accurately reflect profiles from EBV+ DLBCL tumors. Results: We identified 20 EBV+ DLBCLs in the NCI cohort (n=481). EBV+ DLBCL is significantly enriched within the BN2 subtype (p=0.0051, Barnard’s test) and comprised ∼1/3 of EBV+ DLBCLs. The remaining ∼2/3 are distinct from currently defined subtypes. Several differentially mutated genes within the BN2 tumors were found to correlate with EBV status. These include subtype-defining genes, genes involved in B cell receptor signaling and cell cycle regulation, and those known to be downstream of EBV. This finding supports our hypothesis that EBV oncogenes can functionally compensate for specific cellular mutations in DLBCL. Preliminary transcriptomic analyses found EBV+ DLBCL to have increased inflammatory and immune signatures, and JAK/STAT and NF-kB signaling. By RNAseq and immunoblot we determined the cell lines Farage, BCKN1, and IBL1 to have EBV Latency III, while Val has Latency II. All lines are infected with EBV Type 1 strain, except IBL1 which harbors EBV Type 2. Genes mutated in BCKN1, IBL1, and Val are consistent with ‘Unclassified’ subtype, whereas Farage is ST2 subtype. Conclusion: EBV+ DLBCL, like DLBCL itself, is not a single disease. A subset of EBV+ DLBCL is enriched within, and likely have a shared pathogenesis with, BN2 DLBCL. The remaining EBV+, ‘Unclassified’ DLBCL may constitute a novel subtype(s). EBV status correlates with distinct mutation and gene expression profiles, including subtype-defining features. Thus, integrating EBV status into the classification of DLBCL is likely to better inform tumor subtyping and molecular studies, and resolve the specific role(s) of EBV in DLBCL. Characterization of EBV+ DLBCL cell lines revealed limited correspondence with EBV+ tumors, underscoring the need for additional cell line models to support in vitro studies of EBV+ DLBCL. Citation Format: Quincy Rosemarie, Mitch Hayes, Eric C. Johannsen. Defining the role of Epstein-Barr virus (EBV) in diffuse large B-cell lymphoma (DLBCL) pathogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 934.