PROJECT SUMMARY: Esophageal adenocarcinoma (EAC) remain among the most lethal cancers, with a 5- year survival rate of ~20%. Present in 1-6% of the general population, Barrett’s Esophagus (BE) has been identified as the definitive precursor lesion, but confers a risk of progression at an astoundingly low rate of 0.12% per year. Despite identification of multiple clinical risk factors for progression, such factors alone are not sufficient to explain why so few individuals subsequently develop high-grade dysplasia and adenocarcinoma. Extensive somatic genomic characterization of non-dysplastic Barrett’s Esophagus and EAC have demonstrated that early acquisition of TP53 mutations (prevalence 70-83%) and subsequent genome doubling, aneuploidy, and/or chromothriptic events as genomic hallmarks of BE progression. However, these somatic alterations have not yielded insight to why so few individuals with BE progress to dysplasia, or account for the 25-30% of tumors with wildtype TP53. Our robust preliminary data from 742 BE progressors reveal that individuals who progress are enriched with pathogenic germline mutations in cancer-predisposing genes associated with multicancer susceptibility, and that these inheritable mutations can shape the somatic mutanome in Barrett’s esophagus. Moreover, these germline mutations are enriched in TP53 wildtype tumors. In Aim #1, we seek to provide more precise risk estimates of germline alterations occurring in two prevalent genes associated with TP53-wildtype tumor development, ATM and BRCA2. In Aim #2, we assess if introduction of heterozygosity of ATM and BRCA2, the most commonly mutated genes that demonstrate mutual exclusivity with TP53, into nondysplastic BE organoids accelerate accumulation of copy number alterations. In Aim #3, we perform single-cell copy number analysis of BE from BRCA2 and ATM mutation carriers to see if such individuals demonstrate increased subchromosomal aneuploidy compared to matched controls. Understanding how enriched germline driver mutations, particularly those that remain heterozygous in Barrett’s esophagus and retain wildtype TP53 acquire high-risk genomic features is critical for comprehensive risk assessment, biomarker design, and chemoprevention efforts.