Barrett’s esophagus (BE) is the strongest known risk factor for, and obligate precursor of, esophageal high- grade dysplasia (HGD) and adenocarcinoma (EAC). However, determining future neoplastic progression risk is quite challenging in BE patients: current risk estimation is based solely on highly variable, subjective, observer-dependent histopathologic diagnoses (i.e., non-dysplastic (ND), Low-Grade Dysplasia (LGD), Indeterminate for Dysplasia (IFD), or High-Grade Dysplasia (HGD). Our ENVISAGE assay is a validated (CLIA # 21D2256153) laboratory-developed test (LDT) based on a 4-gene-plus-patient age DNA methylation-based PCR assay that was developed at Capsulomics built on foundational studies performed at Johns Hopkins University (JHU). Our preliminary studies yielded a ready-to-launch 1st-generation assay that accurately predicts the risk of future neoplastic progression in BE patients based on levels of molecular biomarkers. This prognostic assay risk-stratifies patients to vastly improve clinical decision-making, with a sensitivity of 71% and a specificity of 90%, outperforming any other clinically available tests. Notwithstanding this early success, we have identified a need for further improvement as well as a robust commercialization pathway. With funding of this direct-to-phase II proposal, we will make significant improvements to generate a more sensitive, more robustly validated second-generation product, while building a foundation for strategic commercialization and market adoption of both the current and future assays. Aim 1 will address assay sensitivity challenges due to using limited-quantity, low-quality fragmented DNA from biopsy specimens by engineering a new assay based on multiplex PCR. This advancement will also improve throughput, efficiency, cost, and processing speed. Aims 2 and 3 will augment our study population while incorporating the new multiplex protocol from Aim 1, retain and refine our algorithm to produce a 2nd-generation ENVISAGE assay, and more sharply delineate which patients will fall into the “Intermediate” risk category for improved clinical management. Aim 4 will establish early commercialization of the 1st-generation assay as an LDT while developing a platform for the future launch of our 2nd-generation product. Thus, this direct-to-phase II SBIR proposal will expand, refine, and validate the ENVISAGE assay, a novel LDT for stratifying future neoplastic progression risk in BE patients, thereby improving clinical decision-making.