LAUSR

Immune checkpoint inhibitors (ICI) were recently approved as a first-line treatment for inoperable esophageal adenocarcinomas (EAC) in combination with chemotherapy (CTX). Unfortunately, even though EAC has one of the highest tumor mutation burdens among all cancer types, response to immunochemotherapy (ICI+CTX) is highly variable, and the underlying molecular basis is incompletely understood. While genomic features such as mutations and copy number alterations in EAC are highly variable across samples, DNA methylation array data from numerous studies suggested that EAC can be clustered into a few consistent subtypes. We thus hypothesize that epigenetic heterogeneity of EAC may contribute to or associate with patients’ heterogeneous response to ICI+CTX, possibly through modulation of key genes or neoantigens. To test this hypothesis, we took advantage of a unique ICI+CTX LUD2015-005 trial in which inoperable EAC patients received first-line ICI for four weeks (ICI-4W), followed by ICI+CTX. Instead of methylation array, we also used a new DNA methylation sequencing technology, TET-Assisted Pyridine-Borane Sequencing (TAPS), on 64 tumor and 15 adjacent normal tissue samples collected from 23 EAC patients before and throughout treatment. Unlike prior studies that used methylation arrays which only cover ~2.5% of all CpG sites in the genome, TAPS detects genome-wide, base-resolution DNA methylation information. Furthermore, many previous studies did not account for variability in tumor content between samples, which could impact downstream DNA methylation analyses. In view of this, we proposed an analytical framework that includes tumor content and local copy number as key parameters, which estimates the tumor and stromal methylation and tests for differentially methylated regions (DMR). We also performed unsupervised clustering based on large scale genome-wide methylation pattern and revealed 2 major tumor clusters. Using the whole genome data, we identified a large set of shared tumor-specific DMRs, revealing that hypomethylation across wide regions of the genome and hypermethylation of certain gene bodies are common features in EAC. We also identified a set of shared outcome-associated DMRs in pre-treatment samples, which predicts better progression-free survival at 12 months. We further performed subgroup analysis of the 2 tumor clusters. Interestingly, we found higher numbers of cluster-specific prognostic DMRs with stronger effect sizes. This suggests that tumor subtypes may respond differently to treatment, and should be considered separately in statistical analyses. Altogether, these results indicate that a detailed understanding of tumor epigenetic heterogeneity will improve patient stratification in immunochemotherapy. Citation Format: Phil F. Xie, Jaeho Chang, Paulina Siejka-Zielińska, Masato Inoue, Magdalena Drożdż, Joseph A. Chadwick, Thomas M. Carroll, Richard P. Owen, Michael J. White, Joseph Kaplinsky, Robert Amess, Mark Middleton, Skirmantas Kriaucionis, Chunxiao Song, Benjamin Schuster-Böckler, Xin Lu. Association between epigenetic heterogeneity of esophageal adenocarcinoma and response to first-line immunochemotherapy in LUD2015-005 Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB131.