Development of Assays to Detect EBV in Breast Cancers

描述(由申请人提供)： 以前的研究表明，一些乳腺癌中存在EB病毒(EBV)。尽管这种常见病毒与乳腺癌之间的联系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：1)检测到的EBV并不总是局限于肿瘤细胞，而不是旁观者淋巴细胞；2)对于EBV的敏感性和特异性，检测往往是可疑的。检测病毒病原体的新技术(定量实时聚合酶链式反应(PCR)与激光捕获显微切割技术相结合，将病毒定位于某些细胞)现在使量化档案组织中的病毒DNA和确定哪些细胞痕迹中含有病毒成为可能。 在这项研究中，我们将结合和适应这些先进的技术来寻找乳腺癌组织中的恶性细胞内的EBV。其具体目的是：1)通过设计TaqMan探针和引物，建立针对EBV LMP1基因的实时定量聚合酶链式反应方法，重点是优化检测灵敏度、特异性和线性；2)测试这种LMP1方法的实用性，以及预先建立的EBV基因组BamHlW和EBNA1部分的定量PCR方法，通过应用于从20个模型肿瘤(EBV相关鼻咽癌)提取的DNA，测试EBV在鼻咽癌组织中的定位能力；3)通过激光捕获显微切割将肿瘤细胞从反应细胞中分离出来，并使用3种PCR方法测量每个部分的病毒载量，来测试EBV在鼻咽癌组织中的定位能力；4)从基于人群的癌症登记中随机抽取100名浸润性乳腺癌妇女的档案样本，以包括已在登记中记录的流行病学相关特征，例如年轻和老年、白人和非白人种族，以及更少和更具侵袭性的疾病(根据临床分期、肿瘤大小和分级、淋巴转移、激素受体状况、生命状况)；5)应用3种聚合酶链式反应方法对100例乳腺癌组织中的EBV进行定量，并计算每个样本的EBVDNA与细胞(肌动蛋白)DNA的比率；6)在EBV水平最高的样本中使用显微解剖和PCR来确定EBV DNA是在肿瘤细胞中还是在正常细胞部分中；以及，7)根据患者人口统计和临床变量对乳腺癌中EBV的存在进行探索性描述性分析，以确定EBV相关癌症的流行病学特征。研究的优势包括考虑乳腺癌中未被充分研究的潜在可治疗因素(即EBV)，开发新的实验室策略以克服研究这种联系时先前的方法学问题，以及首次在此类研究中使用仔细采样的代表性病例系列。新的分析方法应该有助于更大规模的乳腺癌病因和预后研究，并为流行病学研究提供方向。这项工作还应与旨在消除受感染细胞的可能的治疗和预防战略有关。

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.