Ultrasensitive multiplex ddPCR for integrated genomic-epigenomic ctDNA detection in colorectal cancer

Background: Liquid biopsy is rapidly emerging as a cornerstone of precision oncology, offering sensitive and accurate profiling of tumor-derived information in real-time. Although droplet digital PCR (ddPCR) delivers single-molecule sensitivity, its low-plex design has limited it to interrogating one or a few variants at a time. Here we introduce COMET plus, a 25-plex ddPCR assay that simultaneously detects both genetic and epigenetic changes in a single amplification reaction, enabling highly sensitive detection of circulating tumor DNA (ctDNA) in colorectal cancer (CRC). Methods: Following analysis of over 500 tumor tissue and more than 1,000 cfDNA specimens, we selected 18 methylation variants that simultaneously display (i) robust tumor-specific hyper-methylation, (ii) negligible signal in healthy donor plasma, and (iii) high recurrence across patients. These loci were co-amplified with somatic mutations and microsatellite instability loci (MSI) markers frequently altered in CRC. The assay runs on a 7- or 6-color ddPCR system (D3200 platform, Pilotgene; QX600 TM , Bio-Rad). A classifier was trained and subsequently locked to make sample-level calls of ctDNA positivity or negativity using optimized thresholds for each variant. Limit of detection (LoD) for each analyte was determined by Probit analysis of serially diluted contrived cfDNA and cell-line DNA. Limit of blank (LoB) was established from 10 independent replicates of two healthy donor cfDNA pools. Results: In 30 ng of contrived cfDNA or cell-line DNA we detected actionable KRAS/BRAF mutations at 0.08% VAF and MSI-H DNA at 0.1% in an MSS background. Individual methylation markers were called to 0.08-0.2%, while the aggregate 18-marker signature reached 0.01% (cell-line) or 0.02% (cfDNA); no false positives were observed across 20 blank replicates. Conclusions: We developed and validated a high-plex ddPCR assay that sensitively and simultaneously quantifies genetic and epigenetic alterations in colorectal cancer. The multiplexing strategy is compatible with most mainstream ddPCR instruments and readily extendable to other tumor types, offering a simple, streamlined workflow for cancer diagnosis, minimal residual disease (MRD) detection, and therapy selection.