Summary At least 500,000 people in the United States are at high-risk for Lynch syndrome, based on inheritance of a genetic mutation in the mismatch repair (MMR) or double strand break (DSB) repair pathway. More than half of them are unaware of their risk, because their family history is uninformative or unknown. Genetic testing is important for identifying mutations in this pathway, but in a large number of cases no mutation or a variant of uncertain significance will be identified, leading to ambiguous, unsatisfactory results. As more people are seeking testing to identify their risk of Lynch syndrome, accurate alternatives to sequencing are needed to predict the molecular phenotypic effects of mutations in genes in colon cancer and endometrial cancer- predisposing pathways. Risk classification scores based on flow variant assays (FVAs) are a new technology that can accurately identify people with heterozygous germline mutations in these pathways. In response to treatment with chemical agents, FVAs identify decreased nuclear localization of repair proteins and decreased phosphorylation of damage-sensing proteins in cells that bear mutations in these genes. FVAs are rapid, inexpensive and highly reproducible and can be performed on circulating and cultured human blood cells, thus lending themselves to becoming a Next Generation, non-sequencing, standalone test for assessing cancer risks. The goal of this STTR project is to develop a, simple, rapid and inexpensive clinical test that will accurately identify those at high risk for Lynch syndrome. Phase I hypothesis. The standalone FVA test using whole blood samples will identify those at high-risk with 95% accuracy. Specific aim 1. Achieve MMR pathway risk classification score for 99% of subjects with at least 95% accuracy on 180 subjects from well-characterized risk groups. Specific Aim 2. Achieve risk classification score results for all subjects from Aim 1 with comparable accuracy using an automated gating and analysis protocol and a newly created commercial kit. Having demonstrated analytical validity in Phase I, MMG will demonstrate clinical utility in Phase II by calculating and validating 10-year hazard ratios for colon cancer by age decade for 1,800 people followed by up to 20 years by the NCI?s Colon Cancer Family Registry. This product will be sold to clinical laboratories in collaboration with a designated good manufacturing practices facility commercial partner, initially as a laboratory developed test and then as an FDA approved test. Several factors will drive this commercialization into the $1B market cancer risk assessment market: 1. low entry and performance costs, 2. greater accuracy than sequencing, and 3. application to understanding risks for endometrial, gastric, ovarian, small bowel, pancreatic, urinary tract, kidney, bile duct and brain cancers. The creation of simplified, commercial FVA kits will change risk assessment for Lynch syndrome.