Screening Pretest for HNPCC

HNPCC 筛查预测试

• 批准号: 7111331
• 负责人: JEREMY Z FIELDS
• 金额: $ 35.92万
• 依托单位: CA*TX, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7111331
• 关键词: cell line; clinical research; emotions; gene mutation; genes; lymphocyte; performance; proteins; western blottings

DESCRIPTION (provided by applicant): Our broad, long-term objective for Phase II is to develop an immunoassay to diagnose individuals carrying a hereditary nonpolyposis colon cancer (HNPCC) trait. There are >2.5 million HNPCC-carriers in the western world; they have >80% lifetime risk for cancer; if identified, cancer prevention strategies (eg, colonoscopy to remove premalignant lesions) would greatly increase their survival. Most (>95%) carriers have a germline mutation in one of two wild type alleles for a DNA mismatch repair (MMR) gene & they should have 50% reduction in a wild type MMR protein. This prediction was supported by our study of 42 lymphoblastoid & 11 control cell lines from colorectal cancer (CRC) patients in our familial CRC Registry. Controls showed western blot bands for full-length MSH2 and MLH1 of nearly equal signal intensity. In 7 of the 42 CRC patients we saw a clear imbalance in the MSH2/MLH1 ratio (p<0.0005); DNA sequencing showed that each of the 7 had an MMR mutation. We will now test the hypothesis that our immunoassay works using 1) fresh lymphocytes (WBC) and 2) either western blots (Aims 1-2) or a sandwich-type format (Aims 3-4). Aim 1. To do a retrospective study (on 25 trait carriers & 25 controls, all pre-confirmed by DNA sequencing), to establish a positivity criterion (MSH2/MLH1 ratio) for the western blot assay using fresh WBC. Aim 2. To do a prospective study (ratio & genotype for 120 individuals at high risk (approximately 40%) for the trait) to determine test performance characteristics (sensitivity, specificity) when fresh lymphocytes & western blots are used. Aim 3. To advance the immunoassay (using cell lines with known MMR mutations) into a manual prototype of an automated, magnetic bead-based, sandwich-type format that can eventually run on the widely available commercial platform of Bayer Corp. Aim 4. To conduct retrospective & prospective studies for the sandwich- type immunoassay using lymphocytes & methods from Aims 1-2. Aim 5. To study the feasibility of incorporating analyses for less frequent MMR mutations (MSH6). We predict: a) approximately 50% decrease in a wild type MMR protein in trait carriers, b) some ratio of wild type proteins (MSH2/MLH1) will accurately distinguish, with high sensitivity & specificity, between trait carriers & non-carriers. The fully developed, automated, sandwich-type assay (Phase III) will provide a practical method for early detection of HNPCC trait carriers, before they develop cancer, which should greatly reduce morbidity and mortality from hereditary CRC.

描述（由申请人提供）：我们第二阶段的广泛、长期目标是开发一种免疫测定法来诊断携带遗传性非息肉病结肠癌（HNPCC）特征的个体。西方世界有超过 250 万家 HNPCC 运营商；他们终生患癌症的风险>80%；如果确定，癌症预防策略（例如，结肠镜检查以去除癌前病变）将大大提高他们的生存率。大多数 (>95%) 携带者在 DNA 错配修复 (MMR) 基因的两个野生型等位基因之一中存在种系突变，并且他们的野生型 MMR 蛋白应减少 50%。这一预测得到了我们对家族 CRC 登记处结直肠癌 (CRC) 患者的 42 种淋巴母细胞和 11 种对照细胞系的研究的支持。对照显示全长 MSH2 和 MLH1 的蛋白质印迹带具有几乎相同的信号强度。在 42 名 CRC 患者中，有 7 名患者的 MSH2/MLH1 比率存在明显不平衡（p<0.0005）； DNA测序显示，这7人均存在错配修复突变。我们现在将测试以下假设：我们的免疫测定使用 1) 新鲜淋巴细胞 (WBC) 和 2) 蛋白质印迹（目标 1-2）或三明治型格式（目标 3-4）。目标 1. 进行回顾性研究（对 25 个性状携带者和 25 个对照，全部通过 DNA 测序预先确认），建立使用新鲜 WBC 进行蛋白质印迹测定的阳性标准（MSH2/MLH1 比率）。目标 2. 进行一项前瞻性研究（针对该性状高风险（约 40%）的 120 名个体的比率和基因型）以确定使用新鲜淋巴细胞和蛋白质印迹时的测试性能特征（敏感性、特异性）。目标 3. 将免疫测定（使用具有已知 MMR 突变的细胞系）推进为自动化、基于磁珠的三明治型格式的手动原型，最终可以在拜耳公司广泛使用的商业平台上运行。 目标 4. 使用淋巴细胞和目标 1-2 中的方法对三明治型免疫测定进行回顾性和前瞻性研究。目标 5. 研究纳入不太常见的 MMR 突变 (MSH6) 分析的可行性。我们预测：a) 性状携带者中野生型 MMR 蛋白减少约 50%，b) 一定比例的野生型蛋白 (MSH2/MLH1) 将以高灵敏度和特异性准确地区分性状携带者和非携带者。完全开发的自动化三明治型检测（第三阶段）将为 HNPCC 特征携带者在发展为癌症之前进行早期检测提供实用方法，这将大大降低遗传性 CRC 的发病率和死亡率。