Abstract A006: Assessment of EBV DNA methylation to guide antiviral use in EBV-associated lymphoma

Abstract Introduction/Objective: Epstein-Barr Virus (EBV) is an oncogenic herpesvirus driving development of human lymphomas. EBV uses DNA methylation to silence its genome and switch from a lytic (actively replicating) to a latent (dormant) state. Clinical treatment decisions for EBV-associated (EBV+) lymphomas are limited by current diagnostics which provide no information on EBV’s activation state, despite this playing an important role in lymphoma pathogenesis. The antiviral ganciclovir (GCV) is effective specifically against lytic EBV due to viral BGLF4 expression, a kinase that activates GCV. The Baiocchi group discovered that BGLF4 expression in otherwise latent EBV+CNS lymphomas was associated with favorable outcomes when patients received GCV-containing treatment regimen GARD. This indicated improved EBV+ lymphoma outcomes with GCV, yet we cannot determine which patients will benefit. We hypothesized that DNA methylation loss at BGLF4 indicates its expression and is associated with GCV response, serving as a potential DNA biomarker to determine which patients are suitable GCV candidates. Methods: We established a high-throughput PCR and mass spectrometry assay quantifying methylation of n=12 CpG sites at the BGLF4 promoter. The assay was validated against the EpiTYPER methylation assay (R^2=0.766), methylation standards, and the limit of detection was determined to be 625 copies/reaction. We assessed n=136 EBV+ patient plasma samples from a cross-sectional study cohort including EBV-reactivations, post-transplant lymphoproliferation (PTLD), B-, T- and NK-cell lymphomas. Methylation was analyzed with unsupervised clustering and overlaid with in vitro luciferase reporter data for respective BGLF4 DNA elements. Retrospective chart review was performed to determine GCV use and response association with BGLF4 methylation. Results: We identified n=4 CpG sites within BLGF4 that are heterogeneously methylated among samples and highlight the BGLF4 core promoter. Preliminary analysis showed that across disease groups, patients who responded to GCV had a significantly lower BGLF4 methylation (p=0.0007) than patients who did not respond to GCV. Comparatively, we saw no significant difference in BGLF4 methylation of rituximab responders versus non-responders (p=0.343). These results suggest BGLF4 demethylation may indicate lytic EBV activity and thus response to GCV. Conclusions/Significance: Site specific BGLF4 methylation loss is widespread in EBV+ lymphoma. Administration of GCV, a drug that has already been well studied and is FDA-approved, may be guided by EBV methylation, serving as a biomarker to improve patient outcomes in EBV-driven lymphomas. Citation Format: Cara M. Noel, Christoph Weigel, Haley LT. Klimaszewski, Ada C. Sher, Kurits M. Host, Yue-Zhong Wu, Sarah Y. Schlotter, Elshafa H. Ahmed, Eric Brooks, Christopher C. Oakes, James S. Blachly, Mark Lustberg, Timothy Voorhees, Robert A. Baiocchi. Assessment of EBV DNA methylation to guide antiviral use in EBV-associated lymphoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Mechanisms of Cancer Immunity and Cancer-related Autoimmunity; 2025 Sep 24-27; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(9 Suppl):Abstract nr A006.