Identification of differential epigenetic landscapes in subtypes of appendiceal cancer.

4199 Background: Appendiceal cancers (AC) represent a rare and heterogeneous group of malignancies, often managed with strategies adapted from colorectal cancer (CRC) due to their anatomical proximity. However, marked differences in biological behavior, treatment response, and molecular profiles between AC and CRC necessitate independent study. While genomic data on AC has been explored, epigenetic landscapes remain uncharted. This study establishes the first comprehensive epigenetic profile of appendiceal cancer subtypes, aiming to identify novel biomarkers, therapeutic targets, and prognostic indicators. Methods: Tissue specimens from 20 patients with histologically confirmed appendiceal neoplasms and 4 age-matched non-neoplastic controls were analyzed. Malignant subtypes were categorized into three groups: Normal, Low-Grade Appendiceal Mucinous Neoplasms (LAMN, with and without pseudomyxoma peritonei), and Advanced Neoplasia (mucinous adenocarcinoma, non-mucinous adenocarcinoma, and goblet cell adenocarcinoma). Whole-genome methylome profiling was performed using enzymatic methyl-seq for single-base resolution of DNA methylation. Differentially methylated regions (DMRs) were identified with a q-value cutoff of < 0.05 and analyzed using the Whole Genome R package with the methylKit analysis pipeline. Results: Epigenetic clustering revealed progressive dysregulation from normal tissue to LAMN and further to advanced neoplasia, supporting a continuum of malignancy. Our preliminary analysis of CpG islands identified 2,621 DMRs between LAMN and normal tissues and 395 DMRs between Advanced Neoplasia and LAMN. In promoter regions, 1,852 DMRs differentiated LAMN from normal, and 283 distinguished Advanced Neoplasia from LAMN. LAMN samples exhibited predominantly hypomethylated regions relative to normal tissues (2,299 vs. 322 for CpG islands; 1,579 vs. 273 for promoters). Conversely, advanced neoplasia demonstrated more hypermethylated regions than LAMN (243 vs. 152 for CpG islands; 195 vs. 88 for promoters). Most DMRs were localized to intronic, distal intergenic, and promoter regions. Key overlapping DMRs included 8 hypomethylated CpG islands, 41 hypermethylated CpG islands, 3 hypomethylated promoters, and 14 hypermethylated promoters. These regions implicate pivotal genes in the progression from LAMN to advanced neoplasia. Conclusions: This study pioneers the epigenetic characterization of appendiceal cancers, uncovering unique methylation signatures that differentiate malignant subtypes and normal tissue. Integrating these findings with genomic data highlights critical targets for the detection and molecular classification of appendiceal neoplasms. These insights pave the way for improved diagnostic and therapeutic strategies tailored to this rare malignancy.