Screening Pretest for HNPCC

HNPCC 筛查预测试

• 批准号: 7294901
• 负责人: JEREMY Z FIELDS
• 金额: $ 36.06万
• 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 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7294901
• 关键词: Abbreviations; Affect; Alleles; Am 80; Americas; Area; Biological; Biological Assay; Blood specimen; Bos taurus; CHGA gene; Cancer Center; Cancer Etiology; Cancer Patient; Cattle; Cell Line; Cells; Centrifugation; Characteristics; Chromogranin A; Clinic; Clinical; Collaborations; Colonoscopy; Colorectal Cancer; DNA; DNA Mismatch Repair Protein MLH1; DNA Sequence; Data; Databases; Detection; Development; Diagnosis; Diagnostic; Doctor of Philosophy; Early Diagnosis; Electrophoresis; Enrollment; European; Feasibility Studies; Fluorescein; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorouracil; Gel; Gene Dosage; Genes; Genetic Counseling; Genotype; Germ-Line Mutation; Goals; Grant; HCT116 Cells; Health Professional; Healthcare; Hereditary Malignant Neoplasm; Hereditary Nonpolyposis Colorectal Neoplasms; Immunoassay; Immunofluorescence Immunologic; Immunohistochemistry; Immunoprecipitation; Individual; Inherited; Introns; Isothiocyanates; Laboratories; Legal patent; Length; Lesion; Leukocytes; Lymphocyte; MLH1 gene; MSH2 gene; MSH6 gene; Malignant Neoplasms; Manuals; Marketing; Measures; Methods; Microsatellite Instability; Mismatch Repair; Morbidity - disease rate; Mutation; Names; PMS1 gene; PMS2 gene; Patients; Phase; Phase I Clinical Trials; Phycoerythrin; Polymerase Chain Reaction; Population; Predictive Value; Premalignant; Preparation; Prevention strategy; Prospective Studies; Proteins; ROC Curve; Radioimmunoassay; Range; Rate; Receiver Operating Characteristics; Recruitment Activity; Retrospective Studies; Risk; Running; Sampling; Screening procedure; Sensitivity and Specificity; Serum; Signal Transduction; Small Business Funding Mechanisms; Small Business Innovation Research Grant; System; Target Populations; Technology; Test Result; Testing; Tube; Universities; Western Blotting; Western World; Work; base; cancer prevention; cancer risk; commercialization; denaturing gradient gel electrophoresis; design; experience; fetal; human SGTA protein; immortalized cell; immunocytochemistry; lifetime risk; lymphoblastoid cell line; magnetic beads; mortality; mutant; neoplasm registry; particle; performance tests; polyposis; protein expression; prototype; repaired; success; trait

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）：我们广泛的、长期的II期目标是开发一种免疫分析法来诊断携带遗传性非息肉性结肠癌（HNPCC）特征的个体。在西方世界有大约250万hnpcc携带者；他们一生中患癌症的风险是80%；如果确诊，癌症预防策略（如结肠镜检查切除癌前病变）将大大提高患者的生存率。大多数（约95%）携带者在DNA错配修复（MMR）基因的两个野生型等位基因中有一个发生种系突变，他们的野生型MMR蛋白应该减少50%。我们对结直肠癌（CRC）患者的42个淋巴母细胞系和11个对照细胞系的研究支持了这一预测。对照显示全长MSH2和MLH1的western blot条带信号强度几乎相等。在42例结直肠癌患者中，我们发现7例MSH2/MLH1比例明显失衡（p<0.0005）；DNA测序显示，这7个人都有一个MMR突变。现在，我们将使用1)新鲜淋巴细胞（WBC）和2)western blots （Aims 1-2）或三明治型格式（Aims 3-4）来测试我们的免疫测定是否有效的假设。目的1。进行回顾性研究（25名性状携带者和25名对照组，全部通过DNA测序预先确认），为使用新鲜白细胞进行western blot检测建立阳性标准（MSH2/MLH1比率）。目标2。做一项前瞻性研究（120名高危个体（约40%）的比例和基因型），以确定使用新鲜淋巴细胞和western blots时的测试性能特征（敏感性、特异性）。目标3。将免疫测定（使用已知MMR突变的细胞系）推进到自动化的手动原型，基于磁珠的三明治式格式，最终可以在Bayer公司的广泛可用的商业平台上运行。利用淋巴细胞和Aims 1-2的方法进行三明治型免疫分析的回顾性和前瞻性研究。目标5。目的：研究将分析纳入低频率MMR突变（MSH6）的可行性。我们预测：a)野生型MMR蛋白在性状携带者中减少约50%，b)野生型蛋白（MSH2/MLH1）的一定比例将以高灵敏度和特异性准确区分性状携带者和非携带者。完全开发的自动化三明治式检测（III期）将提供一种实用的方法，在HNPCC特征携带者发展为癌症之前就进行早期检测，这将大大降低遗传性CRC的发病率和死亡率。

项目成果

Detection of DNA mismatch repair proteins in fresh human blood lymphocytes--towards a novel method for hereditary non-polyposis colorectal cancer (Lynch syndrome) screening.

• DOI: 10.1186/1756-9966-30-100
• 发表时间: 2011-10-21
• 期刊: Journal of experimental & clinical cancer research : CR
• 作者: Hassen S;Boman BM;Ali N;Parker M;Somerman C;Ali-Khan Catts ZJ;Ali AA;Fields JZ
• 通讯作者: Fields JZ