Investigating the EBV methylome in PLWH: Discovery and Development of Novel EBV Diagnostics in Plasma and Saliva

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ABSTRACT EBV(+) lymphomas are an important cause of death in people living with HIV (PLWH). Different patterns of viral and cellular gene expression have been found to characterize different subtypes of EBV(+) lymphomas. CpG methylation of EBV DNA is an important epigenetic regulator of viral and cellular gene expression. At present, we have a very limited understanding of CpG methylation in EBV(+) lymphomas in PLWH, and how this may determine patterns of viral gene expression. Although we are very successful in treating some EBV(+) lymphoma in HIV patients, many cases are diagnosed very late after organ function has been compromised, or only on post-mortem exam. This is especially true in populations with limited access to health care in the US, as well as in low- and middle-income countries (LMIC) with high HIV prevalence. Assessment of cell-free DNA (cfDNA) in plasma is increasingly recognized as useful in early cancer detection. Plasma cell-free EBV DNA has been shown to be useful in screening for nasopharyngeal cancer. However, high levels of EBV DNA in some PLWH reduce the specificity of EBV DNA quantitation as a diagnostic maker of lymphoma. Evidence is emerging that EBV CpG methylation, or patterns of methylation, could accurately identify EBV(+) malignancies. The proposed studies should improve the collective understanding of epigenetic modification of EBV and viral and cellular gene expression, and enable discovery of novel EBV liquid biopsy diagnostics for early cancer detection in PLWH. In aim 1, we will characterize lymphoma transcriptomes by RNA-Seq and EBV methylomes by high throughput bisulfite sequencing (bs-Seq) to investigate the relationship between the cellular and viral transcriptome and EBV methylation. In aim 2, we will systematically investigate the plasma EBV DNA methylome in PLWH with EBV(+) lymphoma and PLWH controls so as to identify differentially methylated regions of the viral genome that are most informative for lymphoma. These results will guide design and evaluation of methylation-specific PCR primer sets that can enable rapid assessment of EBV methylation states. The results from the qMSP studies will be used to develop and train an automated qMSP classifier for the presence of EBV(+) lymphoma. In aim 3, we propose to establish a new plasma (and saliva) specimen collection from PLWH with EBV(+) lymphoma and matched controls. We will validate the plasma classifier developed in this independent cohort. We will also apply this qMSP classifier to saliva to explore the possibility that saliva cfDNA may be a useful surrogate for plasma cfDNA. At the conclusion of our studies, we anticipate having an improved understanding of the interplay between lymphoma gene expression, EBV gene expression and EBV CpG methylation. Our results will aid in the development of the first plasma EBV qMSP PCR assay for EBV(+) lymphoma in PLWH, and will enable exploration of saliva as an alternate source for cfDNA for future liquid biopsy applications in PLWH. We anticipate that our findings will pave the way for the development of point of care multiplex PCR assay systems appropriate for future investigations of low-cost screening assays in the US and in LMICs. 1