Abstract 3311: Uncovering methylation signatures and pathways associated with resistance to anti-EGFR therapy in colorectal cancer

Abstract Background: Although anti-epidermal growth factor receptor (EGFR) therapy has established efficacy in RAS/BRAF wild type (WT) metastatic colorectal cancer (mCRC), a significant proportion (30-50%) of WT patients exhibit primary resistance to this treatment. Existing biomarker discovery has predominantly focused on tumor-specific DNA mutations, which suffer from variable mutation frequencies among patients. In contrast, DNA methylation represents a common and early event in carcinogenesis. For this reason, methylation markers hold great promise for detecting resistance mechanisms in primary tumors. Several studies have shown an association between DNA methylation patterns and resistance to anti-EGFR therapy in CRC. To validate and build upon these findings, we investigated the methylomes of primary tumors with resistant and responsive phenotypes. Material and Methods: We isolated DNA from FFPE tissue of 40 primary CRC tumors. DNA was subjected to bisulfite conversion and Infinium MethylationEPIC v2.0 BeadChip (Illumina). Subsequently, ChAMP was used to determine the methylation values (β values) and perform differential methylation analysis. The CpG sites were ranked based on the largest Δβ values and the significance of p-values across the patient groups. To ensure the reliability of our findings, we performed a false discovery rate (FDR) evaluation using the Benjamini-Hochberg method, which led to the selection of a subset of differentially methylated CpG sites (DMCs) with the highest likelihood of being genuine associations. Additionally, a gene set enrichment analysis (GSEA) was performed with methylGSA. Results: 48 DMCs were identified with adjusted p-values < 0.05. The affected genes are involved in cancer progression and invasiveness, suggesting a role in a yet-to-be discovered resistance mechanism. The GSEA revealed significant enrichment in 53 KEGG and 33 Reactome pathways, including MAPK-related and Akt signaling pathways. There were 470 significant gene ontology (GO) pathways: 347 in biological processes, 71 in cellular components and 52 in molecular functions. Conclusion and future work: We have found methylation sites associated with resistance to anti-EGFR therapy in CRC patients. In addition to identifying critical DMCs, the pathways uncovered hold potential as a rich source of predictive biomarkers. Next, we will include 24 additional patients. When increasing the sample size, we expect a higher statistical power and an enrichment of CpGs with significant p-values. Afterwards, we will independently validate the most significant CpG sites in a follow-up study using a highly sensitive multiplexed droplet digital PCR assay on additional tissue biopsies. Citation Format: Ana Regina de Abreu, Yann Heylen, Joe Ibrahim, Marc Peeters, Guy Van Camp, Ken Op de Beeck. Uncovering methylation signatures and pathways associated with resistance to anti-EGFR therapy in colorectal cancer [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 3311.