Development of Assays to Detect EBV in Breast Cancers

乳腺癌 EBV 检测方法的开发

• 批准号: 6771826
• 负责人: Margaret L Gulley
• 金额: $ 7.3万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6771826
• 关键词: Epstein Barr virus; actins; breast neoplasms; clinical research; diagnostic tests; gene expression; human tissue; laser capture microdissection; nasopharyngeal neoplasms; neoplasm /cancer genetics; neoplastic cell; neoplastic process; oncogenic virus; polymerase chain reaction; technology /technique development

DESCRIPTION (provided by applicant): Previous studies have suggested that Epstein-Barr virus (EBV) is present in some breast cancers. Although an association of this common virus with breast cancer has implications for disease etiology and treatment, it remains controversial, largely because of methodological problems such as: 1) detected EBV was not always localized to neoplastic cells, as opposed to bystander lymphocytes; and 2) the assays were often suspect with regard to their sensitivity and specificity for EBV. Newer technologies for detecting viral pathogens (quantitative real-time polymerase chain reaction (PCR) combined with laser capture microdissection technology for localizing the virus to certain cells), now make it feasible to both quantify viral DNA in archival tissues and determine which cell tractions harbor virus. In this study, we will combine and adapt these advanced technologies to look for EBV within malignant cells of breast tumor tissues. The Specific Aims are to: 1) develop a quantitative real-time PCR assay targeting the EBV LMP1 gene by designing TaqMan probe and primers, with attention to optimizing assay sensitivity, specificity, and linearity; 2) test the utility of this LMP1 assay, as well as predeveloped quantitative PCR assays for the BamHlW and EBNA1 portions of the EBV genome, by applying them to DNA extracted from 20 model tumors (EBV-related nasopharyngeal carcinomas (NPC); 3) test the ability to localize EBV in NPC tissues by separating tumor cells from reactive cells using laser capture microdissection, and measuring viral load in each fraction using the 3 PCR assays; 4) obtain archival specimens from 100 women with invasive breast cancer, randomly sampled from a population-based cancer registry to include epidemiologically relevant characteristics already recorded in the registry, such as younger and older age, white and nonwhite race, and less and more aggressive disease (according to clinical stage, tumor size and grade, lymph node involvement, hormone-receptor status, vital status); 5) apply the 3 PCR assays to quantitate EBV in the 100 breast cancer tissues and calculate the ratio of EBV DNA to cellular (actin) DNA for each specimen; 6) use microdissection and PCR in samples with the highest EBV levels to determine if the EBV DNA is in the tumor cells or in the normal cell fraction; and, 7) conduct exploratory descriptive analyses of EBV presence in breast tumors by patient demographic and clinical variables to characterize EBV-associated cancers epidemiologically. Study strengths include consideration of an understudied, potentially treatable factor (i.e., EBV) in breast cancer, development of a novel laboratory strategy to overcome prior methodological problems in studying this association, and the first use of a carefully sampled, representative case series in such research. The new assays should facilitate, and epidemiologic findings provide direction for, larger studies of breast cancer etiology and prognosis. The work should also be relevant to possible therapeutic and preventive strategies aimed at eliminating infected cells.

描述（由申请人提供）： 以前的研究表明，EB病毒（EBV）存在于一些乳腺癌中。尽管这种常见病毒与乳腺癌的关联对疾病病因学和治疗有影响，但它仍然存在争议，主要是因为方法学问题，例如：1）检测到的EBV并不总是定位于肿瘤细胞，而不是旁观者淋巴细胞; 2）检测方法对EBV的敏感性和特异性经常受到怀疑。检测病毒病原体的新技术（定量实时聚合酶链反应（PCR）结合激光捕获显微切割技术，用于将病毒定位到某些细胞）现在可以量化存档组织中的病毒DNA并确定哪些细胞片段携带病毒。 在本研究中，我们将联合收割机和适应这些先进的技术来寻找乳腺肿瘤组织的恶性细胞中的EBV。具体目标是：1）通过设计TaqMan探针和引物，建立了针对EBV LMP 1基因的实时荧光定量PCR检测方法，并优化了检测方法的灵敏度、特异性和线性; 2）测试该LMP 1测定法以及预先开发的用于EBV基因组的BamH 1 W和EBNA 1部分的定量PCR测定法的实用性，通过将它们应用于从20个模型肿瘤中提取的DNA，（EBV相关鼻咽癌（NPC）; 3）通过使用激光捕获显微切割将肿瘤细胞与反应性细胞分离来测试在NPC组织中定位EBV的能力，并使用3个PCR测定法测量每个级分中的病毒载量; 4）从100名患有浸润性乳腺癌的妇女获得存档标本，从基于人群的癌症登记处随机取样，以包括登记处中已经记录的流行病学相关特征，例如年轻和老年，白色和非白色种族，越来越少的侵袭性疾病（根据临床分期、肿瘤大小和分级、淋巴结受累、受体状态、生命状态）; 5）应用3种PCR检测方法对100例乳腺癌组织中的EBV进行定量，并计算每个样本的EBV DNA与细胞（肌动蛋白）DNA的比率; 6）在具有最高EBV水平的样品中使用显微切割和PCR以确定EBV DNA是在肿瘤细胞中还是在正常细胞部分中;和7）通过患者人口统计学和临床变量对乳腺肿瘤中的EBV存在进行探索性描述性分析以流行病学地表征EBV相关癌症。研究优势包括考虑一个未充分研究的、潜在的可治疗因素（即，EB病毒）在乳腺癌中的作用，开发一种新的实验室策略来克服先前研究这种关联的方法学问题，并在此类研究中首次使用仔细抽样的代表性病例系列。这些新的检测方法将促进乳腺癌病因学和预后的更大规模研究，流行病学研究结果将为这些研究提供方向。这项工作还应该与旨在消除受感染细胞的可能的治疗和预防策略有关。

项目成果

Real-time PCR measures Epstein-Barr Virus DNA in archival breast adenocarcinomas.

实时 PCR 测量存档乳腺癌腺癌中的 Epstein-Barr 病毒 DNA。

• DOI: 10.1097/01.pas.0000144448.23464.ab
• 发表时间: 2005
• 期刊: Diagnostic molecular pathology : the American journal of surgical pathology, part B.
• 作者: Thorne,LeighB;Ryan,JulieL;Elmore,SandraH;Glaser,SallyL;Gulley,MargaretL
• 通讯作者: Gulley,MargaretL