SBIR-STTR Award

Our goal is to develop a diagnostic assay to identify carriers of the hereditary colon cancer (HNPCC) trait using blood samples as the input specimen. In Phase 1 we will demonstrate the assay's feasibility, showing: 1) requisite predictive value, 2) practicality (easy, rapid, inexpensive), and 3) adaptability to automated technologies. Our assay should detect cellular changes in DNA mismatch repair (MMR) genes (MLH1 & MSH2), changes that occur in HNPCC individuals due to mutation of one of the two alleles of the MLH1 or MSH2 gene. We already demonstrated plausibility for this strategy in preliminary models in which the assay outcome correlated with wild-type gene status. Our AIMS are: 1) to develop a predictive assay for MMR mutations that 1a) accurately demonstrates that the assay outcome correlates with genotype; 1b) distinguishes between cells homozygous and those heterozygous for the wild-type allele and between individuals with and without the HNPCC trait; 2) to adapt our assay to work with automated systems. This will lead to our Phase 2 objective: to integrate our assay into quantitative, commercial, automated systems. PROPOSED COMMERCIAL APPLICATION: The commercial application of our assay involves a practical test for hereditary nonpolyposis colon cancer (HNPCC) affecting over 5 million people in the western world. Detecting HNPCC before cancer develops offers a clinical benefit since cancer prevention strategies reduce their mortality. If technically feasible (Phase 1), the likelihood of our assay becoming commercialized (Phase 2) is outsanding because of the strong commitment of this project by Bayer Diagnostics, which nationally distributes automated diagnostic systems

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 (~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) ~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.

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