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

研究 PLWH 中的 EBV 甲基化组：血浆和唾液中新型 EBV 诊断的发现和开发

• 批准号: 10755171
• 负责人: RICHARD Frederick AMBINDER
• 金额: $ 71.37万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-08 至 2028-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10755171
• 关键词: AIDS-Related Lymphoma; Age; Autopsy; Baltimore; Biological Assay; Biological Markers; Burkitt Lymphoma; CD4 Lymphocyte Count; Cause of Death; Chicago; DNA; DNA Methylation; DNA analysis; Data; Development; Diagnosis; Diagnostic; Epigenetic Process; Epstein-Barr Virus latency; Epstein-Barr Virus-Related Lymphoma; Epstein-Barr Virus-Related Malignant Neoplasm; Evaluation; Future; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; HIV; Hodgkin Disease; Human Herpesvirus 4; Investigation; Large-Cell Immunoblastic Lymphoma; Lymphoma; Malignant Neoplasms; Malignant neoplasm of nasopharynx; Methylation; Modeling; Modification; Organ; Pathway interactions; Patients; Pattern; Persons; Plasma; Plasma Cells; Population; Prevalence; Saliva; Sampling; Screening for cancer; Source; Specificity; System; Testing; Training; Validation; Viral; Viral Genes; Viral Genome; Virus Latency; bisulfite sequencing; cell free DNA; cohort; cost; design; epigenome; health care availability; improved; large cell Diffuse non-Hodgkin' s lymphoma; latent infection; liquid biopsy; low and middle-income countries; lymphoblastoid cell line; methylation pattern; methylome; novel; point of care; primary effusion lymphoma; rapid test; sample collection; screening; sex; transcriptome; transcriptome sequencing; tumor

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

抽象的 EBV(+) 淋巴瘤是 HIV 感染者 (PLWH) 死亡的重要原因。病毒的不同模式 已发现细胞基因表达是 EBV(+) 淋巴瘤不同亚型的特征。 CPG EBV 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。现在， 我们对 PLWH EBV(+) 淋巴瘤中 CpG 甲基化的了解非常有限，以及这如何可能 确定病毒基因表达模式。尽管我们在治疗一些 EBV(+) 淋巴瘤方面非常成功 在艾滋病毒患者中，许多病例在器官功能受损后很晚才被诊断出来，或者直到 尸检。对于美国医疗保健机会有限的人群以及 艾滋病毒感染率高的低收入和中等收入国家 (LMIC)。游离 DNA (cfDNA) 的评估 人们越来越认识到血浆在早期癌症检测中的作用。血浆游离 EBV DNA 已被证明可用于筛查鼻咽癌。然而，一些感染者体内的 EBV DNA 水平较高 降低 EBV DNA 定量作为淋巴瘤诊断指标的特异性。越来越多的证据表明 EBV CpG 甲基化或甲基化模式可以准确识别 EBV(+) 恶性肿瘤。拟议的 研究应提高对 EBV 以及病毒和细胞基因表观遗传修饰的集体理解 表达，并能够发现用于 PLWH 早期癌症检测的新型 EBV 液体活检诊断方法。在 目标 1，我们将通过 RNA-Seq 表征淋巴瘤转录组，并通过高通量表征 EBV 甲基化组 亚硫酸氢盐测序 (bs-Seq) 用于研究细胞和病毒转录组之间的关系 EBV 甲基化。在目标 2 中，我们将系统地研究 PLWH 中的血浆 EBV DNA 甲基化组 EBV(+) 淋巴瘤和 PLWH 对照，以确定病毒基因组的差异甲基化区域 对于淋巴瘤来说信息最丰富。这些结果将指导甲基化特异性 PCR 的设计和评估 可以快速评估 EBV 甲基化状态的引物组。 qMSP 研究的结果将 用于开发和训练针对 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 中建立新的血浆（和唾液）样本采集中心，并且 匹配的控件。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 使用此 qMSP 分类器对唾液进行分类，以探索唾液 cfDNA 可能成为血浆有用替代品的可能性 cfDNA。在我们的研究结束时，我们期望对相互作用有更好的理解 淋巴瘤基因表达、EBV 基因表达和 EBV CpG 甲基化之间的关系。我们的结果将有助于 开发出第一个针对 PLWH 中 EBV(+) 淋巴瘤的血浆 EBV qMSP PCR 检测方法，并将使 探索唾液作为 cfDNA 的替代来源，用于未来 PLWH 液体活检应用。我们预计 我们的研究结果将为开发合适的即时多重 PCR 检测系统铺平道路 用于未来在美国和中低收入国家进行低成本筛查测定的研究。 1