Abstract 6339: Methylation profiling of a novel donor-derived reference material using methylation-sensitive restriction enzyme digestion and droplet digital PCR

Abstract Aberrant DNA methylation, along with other epigenetic modifications, are important indicators in the development of cancer. Unique tissue-specific patterns of hyper-methylated genes have been linked to certain cancer types and are increasingly leveraged to identify tumor type and monitor disease progression including minimal residual disease (MRD) and ongoing cancer surveillance. Accurate DNA methylation analysis relies on robust reference standards to validate assay performance and ensure clinical relevance. Traditionally, methylation standards are produced by mixing fully methylated DNA and non-methylated DNA at defined ratios. While these mixtures provide a convenient benchmark for quantitative linearity, they fall short of capturing the complex methylation heterogeneity found in patient samples, which can diminish the utility of these standards for translational research and clinical diagnostics. To address this gap, our study evaluates a novel, differentially methylated DNA standard, specifically engineered to mimic physiologically relevant methylation patterns and better reflect the complexity observed in real-world patient samples. In this study, methylation-sensitive restriction enzymes (MSRE) are utilized in conjunction with droplet digital PCR (ddPCR) to establish a platform characterized by high sensitivity and precision for the quantitative analysis of DNA methylation. Compared to bisulfite conversion, MSRE digestion requires substantially less input material and eliminates the need for depurination, reducing the risk of DNA degradation; and when paired with ddPCR, it enables a streamlined, target-specific methylation detection in a single-tube workflow. We assessed methylation at seven colorectal cancer gene loci across three reference materials: enzymatically methylated DNA standard, non-methylated DNA standard, and the differentially methylated standard. Consistent with manufacturer’s stated performance, the enzymatically methylated standard showed consistently high methylation, while the non-methylated standard exhibited low background methylation. In contrast, the differentially methylated reference material displayed locus-specific methylation heterogeneity, with targets ranging from non-methylated to partially methylated, and none were found to be completely methylated. These results demonstrate that Bio-Rad’s ddPCR platform reliably detects methylation heterogeneity, supporting both research and assay development across both contrived and physiologically relevant control materials. (For Research Use Only) Citation Format: Daniel Wasik, Yves Konigshofer, Mona Shahbazian, Adam Corner, Eddy vanCollenburg, Jayanthi Ramprakash, Nish Kumar, Andrew Anfora, Mai Ho. Methylation profiling of a novel donor-derived reference material using methylation-sensitive restriction enzyme digestion and droplet digital PCR abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6339.