Abstract 1943: Promoter methylation as a cancer biomarker: insights from ctDNA-based targeted methylation data

Abstract Introduction: Promoter methylation is a hallmark of cancer and holds promise as a biomarker for early cancer detection, prognostication, treatment guidance, and response monitoring. This study explores the utility of GRAIL’s ctDNA-based targeted methylation assay in detecting gene-specific promoter methylation signals and assessing their potential as cancer biomarkers. Methods: A comprehensive literature review was conducted to identify genes with promoter methylation proposed as a cancer biomarker, focusing on those covered by GRAIL's ctDNA targeted methylation assay. We then prioritized genes implicated in multiple cancer types across several studies. As a proof-of-concept, four genes (EYA4, SFRP1, BNIP3, ASCL1) were evaluated using the ctDNA targeted methylation data from the Circulating Cell-free Genome Atlas (CCGA; NCT02889978) study. Contrived samples with known methylation fractions were utilized to establish the limit of detection (LoD) and linearity. Our analysis included 6127 CCGA cancer samples, spanning 21 cancer types, and 2323 non-cancer samples. We estimated the fraction of methylated fragments in each promoter region for all samples and the prevalence of promoter methylation for each cancer type. A Cox proportional hazard model was employed to evaluate the association of promoter methylation for a given gene with 5-year overall survival, adjusting for cancer stage. Results: EYA4 promoter methylation was prevalent in colorectal (39%,108/279, 95% CI: 32%-44%) and liver/bile duct cancer (68%,47/69, 95% CI: 56%-79%). Significant association of EYA4 promoter methylation with survival was observed in colorectal cancer (HR=2.79, p<0.001) and in the pan-cancer setting (HR=1.71, p<0.001). SFRP1 promoter methylation was significantly associated with poorer survival in lung (HR=1.62, p=0.006), colorectal (HR=1.96,p=0.036) and stomach cancers (HR=2.79, p=0.048). BNIP3 promoter methylation, reportedly associated with chemoresistance, was prevalent in colorectal (37%, 104/279, 95% CI: 32%-43%) and pancreatic (34%, 70/205, 95% CI: 23%-41%) cancer, with significant association with worse survival (pancreatic: HR=2.72, p<0.001; colorectal: HR=1.98, p=0.009). ASCL1 promoter methylation correlated with clinical HPV status of HPV-associated cancers, with higher fraction of methylated fragments in HPV-positive head and neck and cervical cancers (p=0.015). Conclusion: This proof-of-concept study underscores the potential of GRAIL’s ctDNA-based targeted methylation assay in detecting clinically informative promoter methylation signals. It also demonstrates the value of the CCGA cohort in facilitating biomarker discovery in plasma samples across multiple cancer types. Further validation and investigation in therapeutic settings are necessary to fully leverage promoter methylation as a biomarker in precision oncology. Citation Format: Margaret Antonio, Eran Mick, Roham Razaghi, Chun Zhang. Promoter methylation as a cancer biomarker: insights from ctDNA-based targeted methylation data [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 1943.