PROJECT SUMMARY Background: Esophageal adenocarcinoma (EAC) arises from Barretts epithelium (BE) and colorectal cancer (CRC) arises from tubular and serrated adenomas, allowing for screening measures to prevent these cancers. The risk of CRC and EAC is variable and screening programs are most effective when optimized for high and low risk. However, our current methods for risk stratifying people are suboptimal, resulting in interval CRCs and EACs, which are preventable. In addition, current EAC screening tests are invasive and inconvenient, resulting in high social and economic burdens and low adherence. Epigenetic and genetic alterations are common in precursor lesions for CRC and EAC and can serve as early detection and as risk biomarkers for colon adenomas/CRC and BE/EAC, respectively. Hypothesis: For CRC, we hypothesis that a clinical testing program using methylated DNA biomarker assays in normal colonic mucosa will identify people at high risk of CRC who will benefit from more aggressive follow-up colonoscopies. For EAC, we hypothesize that clinical grade biomarkers assays based on epigenetic and genetic alterations for EAC risk prediction and early detection of high-grade dysplasia and early EAC in BE patients will improve EAC screening in BE patients and prevent EAC deaths. Proposal: We will clinically validate and develop GLP compliant assays, which can: 1) accurately detect high grade dysplasia (HGD) and early EAC; 2) can predict the risk of BE progression using DNA from either esophageal biopsies and ultimately brushings; and 3) can accurately identify people at high-risk for CRC, for whom aggressive CRC screening could be lifesaving. Assay design, protocols, controls, validation samples, and reference materials will be generated by the BDL and transferred to the BRL where the assays will undergo clinical validation. The BRL will develop harmonization and proficiency programs, along with GLP compliant kits in collaboration with industry partners to ensure other BCC BRLs can also conduct studies with these assays. BRL validated assays will support EDRN Phase 2/3 trials and will be based on a novel set of BDL discovered methylated DNA and genetic biomarkers that: 1) identify patients who are at increased risk of CRC from testing of normal colonic tissue biopsies; 2) improve risk stratification of patients with BE who are at increased risk of EAC and HGD using two classes of assays:one novel custom MPS assay and a smaller targeted MS-ddPCR assay, and 3) accurately detect HGD or early EAC in BE patients undergoing surveillance and that use esophageal brushing samples. Establishing proficiency testing programs with harmonized BRLs will ensure the biomarker tests are robust and reliable for Phase 2-4 clinical trials.