Abstract Introduction: Radiation therapy is fundamental to the treatment of esophageal cancer. However, radioresistance is a significant clinical problem, with only ∼ 30% of patients achieving a complete pathological response to neoadjuvant chemoradiotherapy (CRT). The elucidation of biomarkers and molecular mechanisms underlying this radioresistance would be of substantial clinical benefit. Methods: An isogenic model of radioresistance in esophageal adenocarcinoma (EAC) was established by chronically irradiating OE33 cells with fractionated, clinically-relevant doses of 2 Gy X-ray radiation (cumulative dose, 50 Gy). Radiosensitivity was assessed by clonogenic assay. DNA damage and repair was assessed by immunofluorescence and high content analysis. Global microRNA (miRNA) expression was analyzed using miRNA arrays. Overexpression of miR-31 was performed using synthetic Pre-miRs. miR-31-regulated gene targets were investigated using qPCR-based gene arrays. miR-31 and gene target expression were measured in 37 and 29 esophageal diagnostic tumor biopsies, respectively, by qPCR. Expression levels were analyzed across tumor regression grade, lymph node metastasis and overall survival. Results: Chronic exposure of OE33 cells to fractionated doses of radiation resulted in a radioresistant subline, OE33 R. Characterization of this model revealed that, relative to the age- and passage-matched parent control (OE33 P), the radioresistant cells had a significantly enhanced DNA repair efficiency and a significantly altered miRNA profile, including a downregulation of miR-31. Overexpression of miR-31 re-sensitized the radioresistant esophageal cancer cells to radiation. miR-31 overexpression altered the mRNA expression levels of 13 DNA repair genes, suggesting miR-31-mediated regulation of DNA repair in this model. In vivo, decreased miR-31 expression was significantly associated with a poor response to neoadjuvant CRT. In parallel, increased expression of the miR-31 gene targets SMUG1, MLH1, RAD51L3 and MMS19 was significantly associated with a poor response to neoadjuvant CRT. Increased SMUG1 expression was also significantly associated with the presence of lymph node metastasis. Conclusion: To our knowledge we have developed the first isogenic model of radioresistance in EAC. Characterization of this model revealed alterations in DNA repair efficiency and miRNA expression. We demonstrate for the first time a functional role for miR-31 in modulating the cellular response to radiation. Reduced miR-31 expression and increased expression of gene targets SMUG1, MLH1, RAD51L3 and MMS19, in tumor tissue of poor responders suggests a mechanism for resistance to neoadjuvant CRT in vivo, possibly via enhanced DNA repair. Our data imply a role for miR-31 as a biomarker of response to neoadjuvant CRT in esophageal cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3991. doi:10.1158/1538-7445.AM2011-3991