It is now well established that epithelial cancers arise in a multi-decade process from precancerous lesions. Esophageal adenocarcinoma, a cancer whose incidence has risen 5-fold since 1950, initiates with precancerous lesions known as “Barrett's esophagus”, progresses to dysplasia, and finally emerges as malignant esophageal adenocarcinoma along a path that parallels the Correa Sequence for gastric adenocarcinoma. As the average life expectancy of patients diagnosed with esophageal adenocarcinoma is approximately one-year, considerable efforts are underway to define its Correa sequence, and especially its pre-malignant stages, as potential targets for interdiction ahead of the onset of malignant disease. Toward this end, we have applied novel stem cell cloning technologies originally developed in our laboratories for normal human gastrointestinal tract stem cells to reconstructing the Correa sequence in patients with early esophageal adenocarcinoma. Significantly, each of these patient-matched endoscopic biopsies of Barrett's, dysplasia, and esophageal adenocarcinoma yields discrete populations of stem cells that respectively yield intestinal metaplasia, dysplasia (but not tumors), and aggressive adenocarcinoma following transplantation to immunodeficient mice. From a detailed molecular genetics analysis of nearly 100 independent clones from across the Barrett's, dysplasia, and adenocarcinoma lesions of one patient, we have been able to reconstruct, at unprecedented resolution, both the direct phylogenetic sequence that led to this tumor as well as identify “dead-ends” at both the Barrett's and dysplasia stages that did not contribute to the final tumor. Moreover, each of the cloned stem cells of Barrett's, dysplasia, and adenocarcinoma lesions represent permanent lines that have enabled powerful approaches to drug discovery that has culminated in leads that selectively target the entire Correa sequence while sparing normal esophageal stem cells. In three specific aims, we will 1.) clonally reconstruct the Correa sequence from 10 patients with early esophageal adenocarcinoma; 2.) establish high-throughput screens involving co-cultures of normal esophageal and Correa sequence stem cells for lead discovery; and 3.) develop in vivo xenograft models of patient-matched normal esophageal and Correa sequence stem cells for validating lead combinations targeting these lesions. Based on extensive preliminary studies, we anticipate the analysis of patient-matched stem cells of these progressive lesions will provide fundamental insights into the evolution of esophageal adenocarcinoma and as well as epithelial cancers in general. From the standpoint of filling important gaps in patient care, the drug discovery enabled by these sets of lesional stem cells offers promising and novel interventions to prevent to onset of esophageal adenocarcinoma as well as ones to address disease that has already taken hold.