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 hi...
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| Hauptverfasser: | , , , , , , , , , , , , , , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
May 17, 2025
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| In: |
Analytical chemistry
Year: 2025, Jahrgang: 97, Heft: 21, Pages: 11259-11268 |
| ISSN: | 1520-6882 |
| DOI: | 10.1021/acs.analchem.5c01361 |
| Online-Zugang: | Verlag, kostenfrei, Volltext: https://doi.org/10.1021/acs.analchem.5c01361 Verlag, kostenfrei, Volltext: https://pubs.acs.org/doi/10.1021/acs.analchem.5c01361 
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| Verfasserangaben: | Truong T. Truong, Klara Mikloska, Judith Sum, Martina Oberländer, Nikolas von Bubnoff, Lea Christiansen, Sebastian Tornow, Stefanie Derer, Florian Janke, Holger Sültmann, Sebastian Zeissig, Michael Linnebacher, Clemens Schafmayer, Michael Lehnert, Tobias Hutzenlaub, Nils Paust, and Peter Juelg |
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| 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. | ||
| 700 | 1 | |a Mikloska, Klara |e VerfasserIn |4 aut | |
| 700 | 1 | |a Sum, Judith |e VerfasserIn |4 aut | |
| 700 | 1 | |a Oberländer, Martina |e VerfasserIn |4 aut | |
| 700 | 1 | |a von Bubnoff, Nikolas |e VerfasserIn |4 aut | |
| 700 | 1 | |a Christiansen, Lea |e VerfasserIn |4 aut | |
| 700 | 1 | |a Tornow, Sebastian |e VerfasserIn |4 aut | |
| 700 | 1 | |a Derer, Stefanie |e VerfasserIn |4 aut | |
| 700 | 1 | |a Janke, Florian |d 1991- |e VerfasserIn |0 (DE-588)1216211086 |0 (DE-627)1727474686 |4 aut | |
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| 700 | 1 | |a Zeissig, Sebastian |e VerfasserIn |4 aut | |
| 700 | 1 | |a Linnebacher, Michael |e VerfasserIn |4 aut | |
| 700 | 1 | |a Schafmayer, Clemens |e VerfasserIn |4 aut | |
| 700 | 1 | |a Lehnert, Michael |e VerfasserIn |4 aut | |
| 700 | 1 | |a Hutzenlaub, Tobias |e VerfasserIn |4 aut | |
| 700 | 1 | |a Paust, Nils |e VerfasserIn |4 aut | |
| 700 | 1 | |a Juelg, Peter |e VerfasserIn |4 aut | |
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