Coupling Immunoprecipitation with Multiplexed Digital PCR for Cell-Free DNA Methylation Detection in Small Plasma Volumes of Early-Onset Colorectal Cancer

Colorectal cancer (CRC) remains a major global health challenge, with an increasing incidence of early-onset cases among young adults. Targeted analysis of cell-free DNA (cfDNA) methylation in blood has emerged as a promising minimally invasive diagnostic approach. While digital PCR (dPCR) offers high sensitivity and low turnaround times, conventional bisulfite-based dPCR assays require large plasma volumes due to cfDNA degradation, limiting clinical feasibility. To overcome this limitation, we developed a bisulfite-free, low-plasma-volume assay by coupling cell-free methylated DNA immunoprecipitation (cfMeDIP) with multiplexed dPCR for methylation detection. Assays were designed for CRC targets based on publicly available bisulfite-based plasma data and optimized for native, bisulfite-untreated cfDNA. The cfMeDIP-dPCR assays were first developed and optimized on circulating tumor DNA surrogates derived from HCT116 cells and subsequently validated in a pilot study, including 32 early-onset CRC (EO-CRC) patients and 29 non-CRC individuals. Methylation ratios, defined as the proportion of methylated to total cfDNA copies per marker, served as a diagnostic indicator. Three out of four selected markers (SEPT9, KCNQ5, and C9orf50) were successfully adapted, with significantly higher methylation ratios (p

• Truong, T. T.
• Mikloska, K.
• Sum, J.
• Oberländer, M.
• von Bubnoff, N.
• Christiansen, L.
• Tornow, S.
• Derer, S.
• Janke, F.
• Sültmann, H.
• Zeissig, S.
• Linnebacher, M.
• Schafmayer, C.
• Lehnert, M.
• Hutzenlaub, T.
• Paust, N.
• Juelg, P.