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000301368 0247_ $$2ISSN$$a1541-4655
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000301368 1001_ $$00009-0000-4674-0838$$aTruong, Truong T$$b0
000301368 245__ $$aCoupling Immunoprecipitation with Multiplexed Digital PCR for Cell-Free DNA Methylation Detection in Small Plasma Volumes of Early-Onset Colorectal Cancer.
000301368 260__ $$aColumbus, Ohio$$bAmerican Chemical Society$$c2025
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000301368 520__ $$aColorectal 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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000301368 7001_ $$aMikloska, Klara$$b1
000301368 7001_ $$aSum, Judith$$b2
000301368 7001_ $$aOberländer, Martina$$b3
000301368 7001_ $$avon Bubnoff, Nikolas$$b4
000301368 7001_ $$aChristiansen, Lea$$b5
000301368 7001_ $$aTornow, Sebastian$$b6
000301368 7001_ $$aDerer, Stefanie$$b7
000301368 7001_ $$0P:(DE-He78)a8ba894a47d332308575c75acc0e80df$$aJanke, Florian$$b8$$udkfz
000301368 7001_ $$0P:(DE-He78)7483734fd8ab316391aa604c95f0e98a$$aSültmann, Holger$$b9$$udkfz
000301368 7001_ $$aZeissig, Sebastian$$b10
000301368 7001_ $$aLinnebacher, Michael$$b11
000301368 7001_ $$aSchafmayer, Clemens$$b12
000301368 7001_ $$aLehnert, Michael$$b13
000301368 7001_ $$aHutzenlaub, Tobias$$b14
000301368 7001_ $$aPaust, Nils$$b15
000301368 7001_ $$aJuelg, Peter$$b16
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