Abstract Removal of advanced adenomas during colonoscopy has been shown to significantly reduce colorectal cancer incidence and mortality. However, many patients recommended for colorectal screening remain unscreened due to the invasive nature of colonoscopy. Liquid biopsy cancer detection tests hold significant promise to improve patient outcomes by broadening access and enabling earlier intervention. However, the cost, complexity, and turnaround times of emerging sequencing-based assays present challenges for population-level adoption. To address these challenges, Harbinger Health applied a refined panel of methylation markers in a methylation-specific quantitative PCR (qMSP) assay. qMSP provides a rapid, low-cost, and scalable approach for advanced adenoma (AA) detection in cell-free DNA (cfDNA). Harbinger Health utilizes proprietary methylation biomarkers associated with the initiation of oncogenesis. Redundancy across these markers enabled the selection of six highly pan-cancer-informative methylation patterns that distinguish cancer from non-cancer cfDNA with a small genomic footprint. Harbinger Health developed a qMSP method that quantifies specific methylation patterns using probes that hybridize to highly methylated variants, combined with locked nucleic acid (LNA) blockers that suppress non-specific signal from partially methylated variants. This improved selectivity enables the detection of rare circulating DNA (ctDNA) variants within a heterogenous cfDNA background. The qMSP panel underwent analytical validation during development and demonstrated high sensitivity, specificity, and reproducibility. The qMSP panel was evaluated in a cohort comprised of 62 cfDNA samples collected from patients with no reported cancer in the CORE-HH clinical study (NCT05435066) and 38 commercially sourced cfDNA samples collected from patients with confirmed advanced colorectal adenomas. cfDNA samples were processed into bisulfite libraries and analyzed by qMSP at 5 ng of library per reaction. The cycle threshold (Ct) of each methylation marker was normalized to an internal reference assay and delta Ct values were used to measure methylation. Using target-specific detection thresholds established on 62 non-cancer samples to achieve an equivalent of 90% specificity, qMSP detected at least one positive target for 18 of 38 (47%) AA samples. This work demonstrates the use of qMSP to quantify highly informative methylation markers for AA detection in cfDNA. A six-marker panel effectively distinguished AA samples from non-cancer samples in a cohort of 100 cfDNA libraries. Notably, marker selection was not optimized for AA detection, and an expanded panel with AA-specific biomarkers is expected to further improve clinical performance. qMSP reactions in this study cost less than 5 per sample and exemplify a rapid, low-cost analytical method for the detection of advanced colorectal adenomas. Citation Format: Emily Neaga, Sarah Falotico, Mayur Gurnani, Miguel Williams, Anthony Shuber. . Low-cost detection of advanced adenomas in cfDNA using quantitative methylation-specific PCR abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts) ; 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86 (7 Suppl): Abstract nr 3861.
Neaga et al. (Fri,) studied this question.