Abstract 3242: DNA methylation profiling of Epstein-Barr virus (EBV) in lymphoproliferative disease reveals diagnostic and therapeutic applications

Abstract Introduction: Epstein-Barr Virus (EBV) is an oncogenic herpesvirus linked to hematologic malignancies. EBV enters latency within host cells and can undergo lytic activation, resulting in release of infectious virions. Latent EBV mediates malignant transformation, while lytic EBV is a frequent complication in immune suppression settings like post-transplant lymphoproliferative disease (PTLD). Current clinical assays detect only the presence of EBV via qPCR, limiting their diagnostic use. DNA methylation on EBV genomes is essential for interconversion of lytic/latent states, making it a promising virology biomarker. Methods: We have developed a high-throughput multiplex PCR profiling assay (iPLEX) to assess quantitative EBV DNA methylation at n=35 CpG dinucleotide sites in liquid biopsies (plasma/serum). We applied this assay to a cohort of n=246 plasma samples collected at the OSU Wexner Medical Center via the Leukemia Tissue Bank Shared Resource. Our cohort consisted of viremic patients with EBV-associated lymphoma or non-malignant EBV activation, including n=118 organ transplant recipients. We complemented our analysis with single molecule readout of EBV plasma methylomes (n=27) using Nanopore sequencing of native DNA methylation. Results: Low EBV methylation distinguished lytic from latent EBV (sensitivity: 93.3%, specificity: 97.2%), outperforming clinical EBV qPCR. We revealed distinct EBV methylation in lymphoma subtypes, ranging from intermediate (Hodgkin lymphoma, polymorphic PTLD) to high (Burkitt-, NK/T-cell lymphoma, monomorphic PTLD). Assessment of EBV methylation in treatment follow-up (n=55) highlighted malleable EBV methylation was associated with improved survival under treatment (p=0.0005). We further identified unique EBV methylation dynamics under epigenetic therapies, including DNA methyltransferase (DNMT) inhibitor decitabine. DNA methylation in the EBV protein kinase gene BGLF4 was further associated with response to antiviral ganciclovir (GCV) use. Lastly, assessment of overall methylation patterns revealed four EBV methylation groups. Single molecule methylome analysis via Nanopore sequencing validated our data and identified distinct states of molecular epigenetic heterogeneity in EBV lymphoma and PTLDs. Methylation groups showed highly significant association with overall survival in multiple disease entities, including Hodgkin- and diffuse large B-cell lymphoma (DLBCL). Conclusion: Our data reveal the epigenetic EBV landscape in lymphoma and non-malignant viremic conditions. EBV methylation can identify lymphoma through a minimally invasive plasma test for earlier and more reliable diagnosis of EBV-driven malignancy. EBV methylation could guide informed treatment decisions, especially with regard to epigenetic- and antiviral drug use, and aid in personalized care in the future. Citation Format: Christoph Weigel, Haley L. Klimaszewski, Cara Noel, Ada C. Sher, Kurtis M. Host, Yue-Zhong Wu, Sarah Y. Schlotter, Eric Brooks, Charles T. Gregory, Esko Kautto, Elshafa H. Ahmed, Rosemary Rochford, James S. Blachly, Mark Lustberg, Timothy Voorhees, Christopher C. Oakes, Robert A. Baiocchi. DNA methylation profiling of Epstein-Barr virus (EBV) in lymphoproliferative disease reveals diagnostic and therapeutic applications [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 3242.