Methylation signatures from liquid biopsies predict anti-EGFR therapy response in patients with colorectal cancer

Introduction: Anti-EGFR-based regimens are standard treatments for left-sided, RAS/BRAF wild-type metastatic colorectal cancer (mCRC), but current biomarkers, including RAS/BRAF mutation status and tumor sidedness, do not fully explain the variability in treatment response. Prior tissue-based studies suggest that genome-wide DNA methylation patterns define biologically distinct CRC subtypes. We aimed to develop and validate a circulating tumor DNA (ctDNA)-based methylation classifier to predict anti-EGFR efficacy in mCRC patients. Methods: Genome-wide methylation profiles from >3,000 CRC liquid biopsy samples were analyzed using Guardant360 Liquid (Guardant Health, Palo Alto, CA). After tumor fraction normalization, unsupervised clustering identified two reproducible subtypes: One cluster exhibited a globally high-methylation pattern consistent with the hypermethylated colorectal cancer (HMCC) state described in prior tissue-based studies, while the other represented a low-methylated colorectal cancer (LMCC) state. A random forest model trained on the top 50 principal components (PCs) of these methylation profiles was applied to a real-world cohort of patients receiving anti-EGFR or anti-VEGF therapies. Real-world survival, measured by time to treatment discontinuation (rwTTD), was assessed by treatment type, methylation status, and genotype ( RAS/BRAF wild-type vs mutant). Results: Methylation-based clustering successfully stratified mCRC into HMCC and LMCC groups in samples with evaluable tumor fractions (≥0.5%, N=162). HMCC tumors were enriched for BRAF mutations and exhibited significantly shorter PFS on anti-EGFR therapy, particularly among right- or unknown-sided patients. LMCC patients demonstrated improved outcomes on anti-EGFR therapy (HR = 1.76, p = 0.024), including in patients with right- or unknown-sided, RAS/BRAF wild-type disease, with survival comparable to patients with left-sided disease (HR = 0.94, p = 0.85). Combing methylation status with RAS/BRAF genotype and tumor sidedness expanded the anti-EGFR-eligible population by 18.8% (from 101 to 120 patients) while maintaining survival comparable to standard eligible patients. Conversely, no significant survival difference was observed between HMCC and LMCC patients treated with anti-VEGF therapy (HR = 1.08, p = 0.64), confirming methylation status as a predictive biomarker for treatment selection rather than a purely prognostic factor. Conclusions: ctDNA methylation profiling enables expanded prediction of anti-EGFR efficacy in mCRC beyond current selection criteria. Methylation-integrated eligibility captures additional patients who might benefit from anti-EGFR therapy that are missed by current sidedness-based guidelines. These findings support ctDNA methylation as a feasible and scalable predictive biomarker for precision therapy in colorectal cancer.