%0 Journal Article
%A Truong, Truong T
%A Mikloska, Klara
%A Sum, Judith
%A Oberländer, Martina
%A von Bubnoff, Nikolas
%A Christiansen, Lea
%A Tornow, Sebastian
%A Derer, Stefanie
%A Janke, Florian
%A Sültmann, Holger
%A Zeissig, Sebastian
%A Linnebacher, Michael
%A Schafmayer, Clemens
%A Lehnert, Michael
%A Hutzenlaub, Tobias
%A Paust, Nils
%A Juelg, Peter
%T Coupling Immunoprecipitation with Multiplexed Digital PCR for Cell-Free DNA Methylation Detection in Small Plasma Volumes of Early-Onset Colorectal Cancer.
%J Analytical chemistry
%V 97
%N 21
%@ 0003-2700
%C Columbus, Ohio
%I American Chemical Society
%M DKFZ-2025-01006
%P 11259-11268
%D 2025
%Z 2025 Jun 3;97(21):11259-11268
%X 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 ≤ 0.001) in the EO-CRC cohort. KCNQ5 demonstrated the highest diagnostic performance, achieving an 85
%F PUB:(DE-HGF)16
%9 Journal Article
%$ pmid:40380352
%R 10.1021/acs.analchem.5c01361
%U https://inrepo02.dkfz.de/record/301368