Development of a BRCA Breast Cancer Diagnostic Test

描述（由申请人提供）：这是第一阶段STTR提案的第一次修订。本提案旨在使用基于组织的IHC方法开发BRCA2遗传性乳腺癌的基于组织的基因检测。了解哪些乳腺癌患者患有BRCA2遗传性乳腺癌是很重要的，因为BRCA2患者患乳腺癌的风险增加，他们的亲属有突变的乳腺癌和卵巢癌的风险增加。如果能够进行广泛的基因筛查，这些随后发生的癌症将被及早发现或预防。我们开发了一种基于抗体的组织截断方法来识别BRCA2遗传性癌症，并证明了该技术的概念证明。该方法通过免疫组织化学（IHC）显示n端存在而c端缺失，从而可视化蛋白质截断。初步数据显示，3例BRCA2截断突变乳腺癌通过IHC检测到截断蛋白，20例散发性乳腺癌通过N端和c端抗体检测到全长蛋白。该项目将资助研究和开发，最终将导致适当规模的II期大型临床研究，以说服FDA和医学界，该测试可以有效地识别遗传性BRCA2乳腺癌患者。该产品（测试）将是一种基于抗体的免疫组化诊断试剂盒，用于癌症组织样本，医院实验室将使用它来识别遗传性癌症。I期提案的里程碑是：1)确定具有n端和c端BRCA2抗体的IHC的最佳组织制备方案。2)建立卵巢组织N端和c端BRCA2免疫染色定量评分系统，测定内变异性为10%，测定间变异性为15%或更低。3)证明蛋白质截断的c端与n端比值（BRCA2 truncation ratio）可以区分20例遗传性BRCA2乳腺癌和50例散发性乳腺癌，灵敏度和特异性大于90%（比较不同评分系统）。4)开发基于组织的pcr测序方法，使用测序基准验证IHC研究结果。该I期提案的目标是为II期提案开发技术，以确定BRCA2截断测试是否可以在500例乳腺癌样本（样本量为提供30例BRCA2遗传性癌症）的临床试验中达到90%的灵敏度和特异性。乳腺癌相关性：这项I期研究的成功完成将导致II期临床试验，该临床试验可能建立一种新的基于组织的BRCA2遗传性乳腺癌基因检测，该检测基于IHC方法来可视化蛋白质截断。这种更简单、更广泛适用的方法将识别出更多患有遗传性乳腺癌和卵巢癌的家庭，从而帮助患者及其亲属识别出可能通过他莫昔芬和/或筛查检查和x射线预防的后续乳腺癌或卵巢癌的个体。

DESCRIPTION (provided by applicant): This is the first revision of a Phase I STTR proposal. This proposal aims to develop a tissue-based genetic test for BRCA2 hereditary breast cancer using a tissue-based IHC method. It is important to know which breast cancer patients have BRCA2 hereditary breast cancer because BRCA2 patients have an increased risk of breast cancer and their relatives with mutations have an increased risk of breast and ovarian cancer. These subsequent cancers would be discovered early or prevented if widespread genetic screening was available. We have developed an antibodybased tissue truncation method to identify BRCA2 hereditary cancers and have demonstrated the proof of concept for this technology. This approach visualizes protein truncation by showing with immunohistochemistry (IHC) that the N-terminus is present but the C-terminus is absent. Preliminary Data shows that 3 breast cancers with BRCA2 truncating mutations have truncated proteins by IHC while 20 sporadic breast cancers have full-length proteins detected by both N- and C-terminal antibodies. This project will fund research and development which will ultimately lead to a Phase II large clinical study of appropriate size to convince the FDA and the medical community that this test can efficiently and effectively identify patients with hereditary BRCA2 breast cancer. The product (test) will be an antibody-based IHC diagnostic kit for cancer tissue samples which hospital labs will use to identify hereditary cancers. The milestones for the Phase I proposal are: 1) Determine optimal tissue preparation protocols for IHC with both N-terminal and C-terminal BRCA2 antibodies. 2) Develop a quantitative scoring system for N- terminal and C-terminal BRCA2 immunostaining on ovarian tissues with intra-assay variability of 10% and inter-assay variability of 15% or less. 3) Demonstrate that a C-terminal to N-terminal ratio for protein truncation (BRCA2 Truncation Ratio) can distinguish 20 hereditary BRCA2 breast cancers from 50 sporadic breast cases with greater than 90% sensitivity and specificity (comparing different scoring systems). 4) Develop tissue based methods for PCR-sequencing to validate results of IHC studies using the sequencing benchmark. The goal of this Phase I proposal is to develop technology for a Phase II proposal to determine if the BRCA2 truncation test can achieve 90% sensitivity and specificity in a clinical trial of 500 breast cancer samples (sample size to provide 30 BRCA2 hereditary cancers). Breast Cancer relevance: Successful completion of this Phase I research would lead to a Phase II clinical trial which could establish a new tissue-based genetic test for BRCA2 hereditary breast cancer based on an IHC method to visualize protein truncation. This simpler more widely applicable approach will identify many more families with hereditary breast and ovarian cancer and consequently help patients and their relatives by identifying individuals likely to develop subsequent breast or ovarian cancer which might be prevented by tamoxifen and/or screening exams and X-rays.