Screening Pretest for HNPCC

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分析方法来诊断携带遗传性非息肉病性结肠癌(HNPCC)特征的个体。在西方世界有250万HNPCC携带者；他们有80%的终生癌症风险；如果被发现，癌症预防策略(例如，通过结肠镜检查移除癌前病变)将极大地提高他们的存活率。大多数(&gt；95%)携带者在DNA错配修复(MMR)基因的两个野生型等位基因中的一个有胚系突变，他们的野生型MMR蛋白应该减少50%。这一预测得到了我们家族性结直肠癌登记中42个淋巴母细胞系和11个对照细胞系的研究的支持。对照显示全长MSH2和MLH1的蛋白质印迹带信号强度几乎相同。在42名结直肠癌患者中，有7名患者的msh2/mlh1比例明显失衡(p&lt；0.0005)；DNA测序显示，这7名患者中的每一人都有mmr突变。我们现在将测试的假设是，我们的免疫分析使用1)新鲜淋巴细胞(WBC)和2)蛋白质印迹法(目标1-2)或三明治形式(目标3-4)。目的1.对25名性状携带者和25名对照(均经DNA测序证实)进行回顾性研究，建立用新鲜WBC进行免疫印迹检测的阳性标准(MSH2/MLH1比值)。目的2.进行一项前瞻性研究(120名高危个体(约40%)的比率和基因型)，以确定使用新鲜淋巴细胞和免疫印迹法时的测试表现特征(敏感性、特异性)。目标3.将免疫分析(使用具有已知MMR突变的细胞系)推进到最终可在拜耳公司广泛可用的商业平台上运行的自动化、磁珠为基础的夹心型格式的手动原型。目标4.对使用淋巴细胞的夹心型免疫分析进行回顾和前瞻性研究，目标1-2中的方法。目的5.研究纳入较低频率MMR突变(MSH6)分析的可行性。我们预测：a)野生型MMR蛋白在性状携带者中大约减少50%，b)某些野生型蛋白(MSH2/MLH1)将以高度的敏感性和特异性准确地区分性状携带者和非携带者。这种完全开发的自动化夹心式检测(第三阶段)将提供一种实用的方法，在HNPCC特征携带者发展成癌症之前及早发现他们，这将大大降低遗传性CRC的发病率和死亡率。

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.