DNA methylation-based immune profiling reveals distinct subtypes in esophageal adenocarcinoma

Background Only a subset of patients with esophageal adenocarcinoma (EAC) achieve complete or partial histopathological response to immunotherapy, highlighting the need for better understanding of the tumor microenvironment (TME). Despite efforts to characterize the EAC TME at the molecular level, its epigenetic landscape remains understudied. Methods We combined bulk DNA methylation data from a large Norwegian multicenter EAC cohort ( n = 232) with immune gene annotations to identify immune-related CpG sites differentially methylated between tumor and normal tissue. Hierarchical consensus clustering of EAC samples based on these sites was performed, and the identified subtypes were characterized according to their immune cell type composition, association with clinical features and potential prognostic relevance. Results We identified four immune subtypes (IS1-IS4) with distinct DNA methylation patterns and tumor-infiltrating immune cell composition. The highest DNA methylation levels at immune-related CpG sites were found in IS3, which showed the lowest relative fractions of CD8 + T cells and the highest fractions of regulatory T cells (Tregs), consistent with an immunosuppressed phenotype. Conversely, IS1, with the lowest DNA methylation levels at immune-related CpG sites, displayed the highest relative fractions of CD8 + T cells and the lowest fractions of Tregs, aligning with an "inflamed" immune profile. These findings were validated in an independent multicenter cohort ( n = 285). No significant differences in 5-year overall survival were observed across immune subtypes, possibly reflecting the influence of variable response to neoadjuvant treatment. Conclusion The identified immune subtypes offer new insights into the epigenetic landscape of the EAC TME and reveal differences in its cellular composition.