Summary The incidence of esophageal adenocarcinoma (EAC) has increased 600-700% in North America since the 1980s. EAC only has 18-22% of an overall 5-year survival rate because most patients are discovered at an advanced stage. The single major risk factor for the development of EAC is Barrett’s esophagus (BE) where intestinal-like glandular epithelium replaces the normal squamous mucosa of the esophagus. BE affects approximately 2-5.4%of the adult population in the United States, with 0.12% to 1.6% of BE patients progressing to EAC annually. Most BE patients will not progress to EAC, but they will experience anxiety and fear about their uncertain future. It is urgent to find biomarkers to identify BE patients at high-risk progressing to EAC. DNA hypermethylation at specific genes is the best indicator and occurs early in tumorigenesis and typically increases with tumor progression. Recently, the role of epigenetic change in the pathogenesis of BE and EAC were extensively studied. Multiple methylation markers have been suggested to discriminate between high -risk and low risk BE. However, the sensitivity is relatively low for clinically predicting the patients at high-risk of progressing to EAC. Regardless of these methylation studies, the clinically useful methylation biomarkers for predicting BE patients to progress to EAC are still missing. Pre-Cancer Diagnosis is working to address this critical unmet need by developing a sensitive diagnostic test that will use a target DNA hypermethylation panel to identify those BE patients with the high-risk progressing to EAC. Recently, we found that several genes with DNA hypermethylation could identify high-risk BE patients that are progressing to EAC, which demonstrated an extremely high sensitivity (≈100%), and specificity (≈100%). With the DNA methylation test, we could triage the BE patients into high-risk and low-risk groups: the high-risk patients will receive early treatment including mucosal resection or radiofrequency ablation; the low-risk patients will have a longer follow-up interval, which will decrease the cost. The objective of this study is to validate our preliminary data and further develop a new methylation diagnostic kit to predict high-risk BE progression to EAC. In Aim 1, we will validate the hypermethylation genes and develop a methylation diagnostic kit that differentiate BE patients with and without a high risk of progressing to EAC by both DNA Methylation Epic array and PCR methylation tests. In Aim 2. We will use whole genome sequence to validate the methylation genes that can predict high -risk BE patients progressing to EAC and then we will develop a Target Methylation panel (a diagnostic kit) for the clinical trial. This test will identify high-risk BE patients before progressing to EAC, which is crucial for reducing the incidence of EAC and improving patient outcomes.