Abstract 5203: Patient derived organoids capture heterogeneity and uncover therapeutic opportunities in esophageal adenocarcinoma

Abstract Introduction: Esophageal adenocarcinoma (EAC) is a hard-to-treat cancer that contributes to considerable morbidity and mortality. A major treatment challenge is the inherent disease heterogeneity. We sought to recapitulate this in vitro using a comprehensive organoid platform to allow correlation with genomic driver events, including extrachromosomal circular DNA (ecDNA), and response to systemic therapies and radiation. Methods: A total of 48 patient-derived organoids (PDOs) and their matched parental tissue were characterized using clinical annotations, whole-genome sequencing (WGS), and RNA sequencing. Additional phenotypic characterization was undertaken via immunohistochemistry (IHC) for key protein markers. Ten EAC organoids with distinct genomic driver events were selected for in-depth analysis, including evaluation of ecDNA events using IHC, Fluorescence in situ hybridization (FISH), and AmpliconArchitect tool, as well as characterization of sensitivity to chemotherapeutics and radiation. PDO cultures were treated with five anticancer compounds as single agents based on genomic drivers and GI50 values were calculated. Radiation sensitivity was assessed by exposure to a single fraction of 0-8Gy x-ray irradiation and subsequent evaluation of organoid forming efficiency (OFE) using an IncuCyte. Results: The organoid platform successfully captured the heterogeneity of EAC, replicating key histological and molecular features observed in matched patient samples. WGS analysis demonstrated that EAC organoids reflect the heterogeneous genomic landscape of their primary tumor including point mutations, copy number alterations, structural variations, mutational signatures, and driver alterations. FISH analysis and AmpliconArchitect confirmed the presence of ecDNA events leading to focal amplification of up to 41 copy numbers of functionally relevant genes with corresponding RNA expression. HER2 amplified organoids were confirmed through IHC and FISH analysis. In addition, IHC staining for CDK6 demonstrated biomarker potential of ecDNA events. Therapeutic sensitivities were observed to CDK4/6 (GI50 < 10 μM) CDK2 (GI50 < 0.03 μM), HER2 (GI50 1-10 μM), MEK (GI50 0.3 - 10 μM) and mTOR (GI50 0.3-10 μM) inhibitors. The most sensitive of responses were seen in the CDK2 inhibitor, with most organoids responding (n = 6). Putative ecDNA events were found to confer sensitivity to predicted targeted compounds and, in some cases, showed a greater response than nuclear amplification of the same oncogenic driver. Significant variation in OFE was seen in response to x-ray irradiation, highlighting novel molecular determinants of radiation response. Conclusion: Our organoid platform captures the molecular and phenotypic heterogeneity of EAC and provides a platform in which to identify novel molecular determinants of therapeutic response. Citation Format: Dylan Peter McClurg, Kayli Bolton, Christopher M. Jones, Daniel Jacobson, Ahmad Miremadi, Ginny Devonshire, Xiaodun Li, Sriganesh Jammula, Adrienn Blasko, Rebecca C. Fitzgerald. Patient derived organoids capture heterogeneity and uncover therapeutic opportunities in esophageal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5203.