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

基本信息

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
项目类别：
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&apos 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

项目摘要

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（+）淋巴瘤是艾滋病毒（PLWH）患者的重要原因。病毒的不同模式已经发现细胞基因表达表征了EBV（+）淋巴瘤的不同亚型。 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剂。现在，我们对PLWH中EBV（+）淋巴瘤中CpG甲基化的了解非常有限，这可能如何确定病毒基因表达的模式。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，在器官功能被妥协后，许多病例被诊断出很晚，或仅在验尸考试。在美国获得医疗保健的人口中，尤其如此在艾滋病毒率高的低收入和中等收入国家（LMIC）中。评估无细胞DNA（CFDNA）血浆越来越多地被认为在早期癌症检测中有用。不含等离子体细胞的EBV DNA已经显示可用于筛查鼻咽癌。但是，某些PLWH中的高水平EBV DNA 降低EBV DNA定量的特异性，作为淋巴瘤的诊断制造商。有证据表明 EBV CpG甲基化或甲基化模式可以准确鉴定EBV（+）恶性肿瘤。提议研究应提高对EBV以及病毒和细胞基因表观遗传修饰的集体理解表达，并能够发现新型的EBV液体活检诊断，以用于PLWH中的早期癌症检测。在 AIM 1，我们将通过RNA-SEQ和EBV甲基甲基淋巴瘤转录组来表征高吞吐量的淋巴瘤转录组亚硫酸盐测序（BS-Seq），以研究细胞和病毒转录组与病毒转录组之间的关系 EBV甲基化。在AIM 2中，我们将系统地研究PLWH中的等离子体EBV DNA甲基甲基 EBV（+）淋巴瘤和PLWH控制，以鉴定病毒基因组的差异甲基化区域最有用的淋巴瘤。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甲基化状态的底漆集。 QMSP研究的结果将用于开发和训练自动QMSP分类器，以实现EBV（+）淋巴瘤的存在。在AIM 3中，我们建议建立来自PLWH的新等离子体（和唾液），并用EBV（+）淋巴瘤和匹配的控件。我们将验证该独立队列中开发的等离子分类器。我们还将申请唾液的QMSP分类器探索唾液CFDNA可能是血浆有用的替代的可能性 cfDNA。在我们的研究结束时，我们预计对相互作用有了改进的了解在淋巴瘤基因表达，EBV基因表达和EBV CpG甲基化之间。我们的结果将有助于 PLWH中EBV（+）淋巴瘤的第一个等离子体EBV QMSP PCR分析的开发，并将启用将唾液作为CFDNA的替代来源，用于将来的液体活检应用中的PLWH。我们期待我们的发现将为开发护理点多重PCR测定系统的发展铺平道路为了将来对美国和LMIC的低成本筛查测定法进行调查。 1