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| Home > Publications database > Coupling Immunoprecipitation with Multiplexed Digital PCR for Cell-Free DNA Methylation Detection in Small Plasma Volumes of Early-Onset Colorectal Cancer. > print |
| Home > Publications database > Coupling Immunoprecipitation with Multiplexed Digital PCR for Cell-Free DNA Methylation Detection in Small Plasma Volumes of Early-Onset Colorectal Cancer. > print |
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| 024 | 7 | _ | |a 10.1021/acs.analchem.5c01361 |2 doi |
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| 100 | 1 | _ | |a Truong, Truong T |0 0009-0000-4674-0838 |b 0 |
| 245 | _ | _ | |a Coupling Immunoprecipitation with Multiplexed Digital PCR for Cell-Free DNA Methylation Detection in Small Plasma Volumes of Early-Onset Colorectal Cancer. |
| 260 | _ | _ | |a Columbus, Ohio |c 2025 |b American Chemical Society |
| 336 | 7 | _ | |a article |2 DRIVER |
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| 500 | _ | _ | |a 2025 Jun 3;97(21):11259-11268 |
| 520 | _ | _ | |a 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% sensitivity at a 90% specificity, with methylation ratios correlating with the tumor stage. This study presents the first cfMeDIP-dPCR approach, demonstrating its potential as a sensitive liquid biopsy assay. Requiring only 0.5 mL of plasma, i.e., more than 20 times less than a sensitivity-matched bisulfite-based assay, cfMeDIP-dPCR facilitates clinical implementation for CRC and other diseases with epigenetic signatures. |
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| 700 | 1 | _ | |a Mikloska, Klara |b 1 |
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| 700 | 1 | _ | |a Tornow, Sebastian |b 6 |
| 700 | 1 | _ | |a Derer, Stefanie |b 7 |
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| 700 | 1 | _ | |a Zeissig, Sebastian |b 10 |
| 700 | 1 | _ | |a Linnebacher, Michael |b 11 |
| 700 | 1 | _ | |a Schafmayer, Clemens |b 12 |
| 700 | 1 | _ | |a Lehnert, Michael |b 13 |
| 700 | 1 | _ | |a Hutzenlaub, Tobias |b 14 |
| 700 | 1 | _ | |a Paust, Nils |b 15 |
| 700 | 1 | _ | |a Juelg, Peter |b 16 |
| 773 | _ | _ | |a 10.1021/acs.analchem.5c01361 |g p. acs.analchem.5c01361 |0 PERI:(DE-600)1483443-1 |n 21 |p 11259-11268 |t Analytical chemistry |v 97 |y 2025 |x 0003-2700 |
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