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）： 先前的研究表明，某些乳腺癌中存在 Epstein-Barr 病毒 (EBV)。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 并不总是局限于肿瘤细胞，而不是旁观者淋巴细胞； 2) 这些检测方法对 EBV 的敏感性和特异性经常受到怀疑。用于检测病毒病原体的新技术（实时定量聚合酶链式反应 (PCR) 与激光捕获显微切割技术相结合，可将病毒定位到某些细胞），现在可以量化档案组织中的病毒 DNA 并确定哪些细胞牵引力病毒。 在这项研究中，我们将结合并采用这些先进技术来寻找乳腺肿瘤组织恶性细胞内的 EBV。具体目标是：1）通过设计TaqMan探针和引物，开发针对EBV LMP1基因的定量实时PCR检测，并注意优化检测灵敏度、特异性和线性； 2) 通过将 LMP1 检测以及针对 EBV 基因组的 BamHlW 和 EBNA1 部分的预先开发的定量 PCR 检测应用于从 20 个模型肿瘤（EBV 相关鼻咽癌 (NPC)）中提取的 DNA 来测试其实用性；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，并使用 3 种 PCR 测定法测量每个部分中的病毒载量，测试在 NPC 组织中定位 EBV 的能力； 4) 从 100 名患有浸润性乳腺癌的女性中获取档案样本，这些样本是从基于人群的癌症登记处随机抽取的，包括登记处已记录的流行病学相关特征，例如年龄较小和较大、白人和非白人种族以及攻击性较低和较高等疾病（根据临床分期、肿瘤大小和分级、淋巴结受累、激素受体状态、生命状态）； 5) 应用3次PCR检测对100个乳腺癌组织中的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EBV水平最高的样本中使用显微切割和PCR来确定EBV DNA是否存在于肿瘤细胞中或正常细胞部分中； 7) 通过患者人口统计和临床变量对乳腺肿瘤中 EBV 的存在进行探索性描述性分析，以从流行病学角度描述 EBV 相关癌症的特征。研究优势包括考虑乳腺癌中尚未充分研究的潜在可治疗因素（即 EBV）、制定新的实验室策略以克服研究这种关联时先前的方法学问题，以及在此类研究中首次使用仔细采样的代表性病例系列。研究。新的检测方法应该有助于乳腺癌病因学和预后的更大规模研究，并为流行病学研究结果提供方向。这项工作还应该与旨在消除感染细胞的可能的治疗和预防策略相关。

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