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

摘要 EBV(+)淋巴瘤是艾滋病毒携带者(PLWH)死亡的重要原因。不同的病毒模式 细胞基因表达是EBV(+)淋巴瘤不同亚型的特征。CPG EB病毒DNA甲基化是病毒和细胞基因表达的重要表观遗传调节因子。目前， 我们对PLWH中EBV(+)淋巴瘤中CpG甲基化的了解非常有限，以及这种甲基化是如何发生的 确定病毒基因表达的模式。虽然我们在治疗一些EBV(+)淋巴瘤方面非常成功 在艾滋病毒患者中，许多病例是在器官功能受损后很晚才被诊断出来的，或者只是在 验尸报告。在美国，获得医疗保健的机会有限的人群尤其如此， 在艾滋病毒流行率较高的低收入和中等收入国家。人体内游离DNA(CfDNA)的检测 血浆在癌症早期检测中的作用日益得到认可。无浆细胞EBV DNA已经被 这对鼻咽癌的筛查很有帮助。然而，在一些PLWH中，高水平的EBV DNA 降低EBV DNA定量作为淋巴瘤诊断标志物的特异性。越来越多的证据表明 EBV CpG甲基化，或甲基化模式，可以准确地识别EBV(+)恶性肿瘤。建议数 研究应提高对EBV、病毒和细胞基因表观遗传修饰的集体理解 表达，并使发现新的EBV液体活检诊断用于PLWH的早期癌症检测。在……里面 目的1.我们将通过高通量的RNA-Seq和EBV甲基组来鉴定淋巴瘤的转录本 亚硫酸氢盐测序(bs-Seq)研究细胞和病毒转录组与 EB病毒甲基化。在目标2中，我们将系统地研究PLWH患者的血浆EBV DNA甲基组 EBV(+)淋巴瘤和PLWH对照，以确定病毒基因组的差异甲基化区域 对淋巴瘤来说是最有价值的。这些结果将指导甲基化特异性聚合酶链式反应的设计和评价 能够快速评估EBV甲基化状态的引物集。QMSP研究的结果将 用于开发和培训用于EBV(+)淋巴瘤存在的自动qMSP分类器。在目标3中，我们 建议从患有EBV(+)淋巴瘤的PLWH中建立一种新的血浆(和唾液)样本采集方法 配对的对照组。我们将验证在这个独立队列中开发的血浆分类器。我们也会申请 这种qMSP分类器可以用来探索唾液cfdna作为血浆替代品的可能性。 CfDNA。在我们的研究结束时，我们预计会对相互作用有更好的理解 淋巴瘤基因表达、EBV基因表达与EBV CpG甲基化之间的关系我们的结果将有助于 首次建立血浆EBV qMSP聚合酶链式反应检测PLWH中EBV(+)淋巴瘤，并将使 探索唾液作为cfDNA的替代来源，用于未来PLWH的液体活检应用。我们期待着 我们的发现将为开发适当的护理点多重聚合酶链式反应检测系统铺平道路 用于未来在美国和低成本集成电路中进行低成本筛查分析的研究。 1

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