TY  - JOUR
AU  - Truong, Truong T
AU  - Mikloska, Klara
AU  - Sum, Judith
AU  - Oberländer, Martina
AU  - von Bubnoff, Nikolas
AU  - Christiansen, Lea
AU  - Tornow, Sebastian
AU  - Derer, Stefanie
AU  - Janke, Florian
AU  - Sültmann, Holger
AU  - Zeissig, Sebastian
AU  - Linnebacher, Michael
AU  - Schafmayer, Clemens
AU  - Lehnert, Michael
AU  - Hutzenlaub, Tobias
AU  - Paust, Nils
AU  - Juelg, Peter
TI  - Coupling Immunoprecipitation with Multiplexed Digital PCR for Cell-Free DNA Methylation Detection in Small Plasma Volumes of Early-Onset Colorectal Cancer.
JO  - Analytical chemistry
VL  - 97
IS  - 21
SN  - 0003-2700
CY  - Columbus, Ohio
PB  - American Chemical Society
M1  - DKFZ-2025-01006
SP  - 11259-11268
PY  - 2025
N1  - 2025 Jun 3;97(21):11259-11268
AB  - 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
LB  - PUB:(DE-HGF)16
C6  - pmid:40380352
DO  - DOI:10.1021/acs.analchem.5c01361
UR  - https://inrepo02.dkfz.de/record/301368
ER  -