Screening Pretest for HNPCC

描述（由申请人提供）：我们II期的广泛长期目标是开发一种免疫测定法，以诊断携带遗传性非息肉病性结肠癌（HNPCC）性状的个体。在西方世界有> 250万HNPCC携带者;他们有>80%的癌症终生风险;如果被发现，癌症预防策略（例如，结肠镜检查以去除癌前病变）将大大增加他们的生存率。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具有种系突变&他们应该在野生型MMR蛋白中具有50%的减少。这一预测得到了我们对来自我们家族性结直肠癌登记处的结直肠癌（CRC）患者的42个淋巴母细胞和11个对照细胞系的研究的支持。对照显示全长MSH 2和MLH 1的蛋白质印迹条带几乎相等的信号强度。在42名CRC患者中，我们发现7名患者的MSH 2/MLH 1比例明显失衡（p<0.0005）; DNA测序显示，这7名患者均存在MMR突变。我们现在将测试我们的免疫测定使用1）新鲜淋巴细胞（WBC）和2）蛋白质印迹（目的1-2）或免疫印迹型形式（目的3-4）起作用的假设。目标1.对25例遗传性状携带者和25例对照者进行回顾性研究，以建立新鲜白细胞免疫印迹试验的阳性标准（MSH 2/MLH 1比值）。目标二。进行前瞻性研究（120例高风险个体（约40%）的比例和基因型），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（灵敏度，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、可在Bayer Corp. Aim 4广泛使用的商业平台上运行的自动化、磁珠型格式的手动原型。目的1-2：对淋巴细胞夹心免疫分析方法进行回顾性和前瞻性研究.目标5。研究合并分析不常见MMR突变（MSH 6）的可行性。我们预测：a）在性状携带者中野生型MMR蛋白减少约50%，B）野生型蛋白的一定比例（MSH 2/MLH 1）将以高灵敏度和特异性准确区分性状携带者和非携带者。完全开发的，自动化的，自动化的检测方法（III期）将提供一种实用的方法，用于早期检测HNPCC性状携带者，在他们发展成癌症之前，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.