PROJECT SUMMARY/ABSTRACT The objective of this combined UH2/UH3 application is to develop a cost-effective, rapid, test that can be rapidly translated to the clinic and ultimately be used to re-purpose a class of FDA-approved colorectal cancer (CRC) EGFR inhibitor (EGFRi) therapeutics (cetuximab and panitumumab). To date, only negative genetic predictors (mutant KRAS/NRAS) of EGFRi response have been employed clinically, currently restricting EGFRi use to wild-type RAS/RAF subpopulations, and more recently, just to “left-sided” lesions. We recently reported a new prognostic role for APC that relates to the number of alleles mutated and to the association with other mutant genes such as KRAS and TP53 (Nat Commun, 2016). Further analysis also revealed that mutant APC (A) genotypes, in combination with mutant TP53 (P), are strongly correlated with a gene expression signature measuring cetuximab sensitivity (CTX-S). These data led to the provocative hypothesis that mutant APC + TP53 (AP) genotypes together---more so than either mutant gene alone (A_ / _P), or wild-type AP (_ _)---may have a new role in positively-predicting EGFRi outcomes (Nat Commun, Under Review, 2018). This hypothesis is based on 3 key observations we have recently made in our cohort and in TCGA data: (1) CTX-S scores are significantly higher in: AP > A_ / _P > _ _ tumors in both WT and MUT KRAS tumors; (2) AP mutations are more frequent in Left (cetuximab-sensitive) > Right (cetuximab-resistant) CRC; (3) AP mutations are almost non-existent in MSI tumors (highly cetuximab-resistant). These findings have two potentially high-impact clinical implications: (1) they re-define the patient selection strategy by further restricting EGFRi therapies to wild-type RAS/RAF patients harboring AP mutations, thereby increasing response rates; (2) they could expand the therapeutic opportunity to treat a substantial number of previously-excluded mutant KRAS/NRAS patients who have AP mutations in both left and right CRCs. If the test for these mutations is developed and clinically validated, the utilization of these drugs could be expanded to ~25% more patients, including more first-line patients. For the vast majority of CRC patients, AP mutations are not assessed in current practice. Unlike KRAS/NRAS oncogenes, both A and P are tumor suppressor genes that can have a multitude of inactivating mutations that must be detected. Thus, there is an opportunity to change clinical practice and standards of care to ultimately improve CRC outcomes with a new test. In the LabCorp CAP/CLIA environment, we plan to develop a new highly sensitive, specific and cost-effective targeted DNA-sequencing “assay” to detect mutations in the coding regions of the principal genes APC, TP53, KRAS, BRAF, NRAS from formalin fixed paraffin embedded (FFPE) tissue samples. In the UH2 Phase of this proposal, a new FFPE targeted DNA sequencing assay, with greater potential to accurately detect mutations at low allelic frequencie...