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

摘要 EB病毒（+）淋巴瘤是艾滋病毒感染者（PLWH）死亡的一个重要原因。不同的病毒模式 和细胞基因表达已被发现是不同亚型EB病毒（+）淋巴瘤的特征。CPG EBV DNA的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， 确定病毒基因表达的模式。虽然我们在治疗某些EBV（+）淋巴瘤方面非常成功， 在艾滋病患者中，许多病例在器官功能受损后很晚才被诊断出来，或仅在 在美国，这在获得医疗保健有限的人群中尤其如此， 在艾滋病毒流行率高的低收入和中等收入国家。评估细胞游离DNA（cfDNA）， 血浆越来越被认为在早期癌症检测中有用。血浆无细胞EBV DNA已被 显示其可用于筛查鼻咽癌。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。有证据表明， EBV CpG甲基化或甲基化模式可以准确识别EBV（+）恶性肿瘤。拟议 研究应该提高对EBV和病毒及细胞基因的表观遗传修饰的集体理解 表达，并且能够发现用于PLWH中的早期癌症检测的新型EBV液体活检诊断。在 目的1，我们将通过RNA-Seq和高通量EBV甲基化来表征淋巴瘤转录组 亚硫酸氢盐测序（bs-Seq）研究细胞和病毒转录组之间的关系， EBV甲基化。在目的2中，我们将系统地研究PLWH中血浆EBV DNA甲基化组， EBV（+）淋巴瘤和PLWH对照，以鉴定病毒基因组的差异甲基化区域， 对淋巴瘤的诊断最有帮助这些结果将指导甲基化特异性PCR的设计和评价 可以快速评估EBV甲基化状态的引物组。qMSP研究的结果将 用于开发和训练用于EBV（+）淋巴瘤存在的自动qMSP分类器。在目标3中，我们 建议建立一个新的血浆（和唾液）标本收集PLWH与EBV（+）淋巴瘤， 匹配的控制。我们将验证在该独立队列中开发的血浆分类器。我们还将应用 该qMSP分类器用于唾液，以探索唾液cfDNA可能是血浆cfDNA的有用替代物的可能性。 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 开发了第一个用于PLWH中EBV（+）淋巴瘤的血浆EBV qMSP PCR检测方法， 探索唾液作为cfDNA的替代来源用于PLWH中未来的液体活检应用。我们预计 我们的研究结果将为开发适合的护理点多重PCR检测系统铺平道路 用于在美国和中低收入国家进行低成本筛查试验的未来研究。 1