Using cell-free DNA tumor methylation score to detect and monitor cholangiocarcinoma

Cholangiocarcinoma (CCA) is characterized by late or unclear diagnosis, high recurrence rates, and limited options for noninvasive disease monitoring. The widely used serum biomarker CA 19-9 has suboptimal sensitivity and specificity, underscoring the need for more accurate and reliable quantitative measures of tumor burden. Cell-free DNA (cfDNA) methylation represents a promising approach for longitudinal disease monitoring.

In this single-center cohort study, we evaluated a cfDNA methylation-based Tumor Methylation Score (TMS) for detecting and monitoring CCA. Twenty-five patients with histologically confirmed CCA and twelve controls with benign hepatobiliary conditions were enrolled, yielding 100 plasma samples and 26 tissue samples. cfDNA methylation was quantified across 537 cancer-associated CpG loci by qPCR method, and TMS was calculated from the difference in methylation in paired plasma and buffy coat samples. For tissue samples, TMS was calculated by quantifying methylation in tumor and background liver separately. Response to treatment was assessed using blinded radiographic review. Tissue-based validation was performed using The Cancer Genome Atlas and 4 additional GEO cohorts. Finally, cellular pathways associated with the panel genes were explored using the Ingenuity Pathway Analysis (IPA).

TMS was significantly elevated in CCA samples compared with controls in both tissue (P = 0.007) and plasma (P < 0.0001). Plasma TMS distinguished CCA from controls with a sensitivity of 74.3% and specificity of 80% (AUC = 0.823), while CA 19-9 showed no discriminatory value. TMS decreased consistently following curative resection. Longitudinal changes in TMS (ΔTMS) robustly distinguished radiographic progression from non-progression during systemic therapy (AUC = 0.968), outperforming ΔCA 19-9. Tissue-based analyses across independent cohorts confirmed cancer specificity and association with adverse outcomes. IPA identified HOXA9 as a significantly altered gene implicated in CCA carcinogenesis.

Quantitative cfDNA methylation monitoring using TMS is feasible and accurately reflects dynamic tumor burden in CCA. TMS may serve as a complementary adjunct to imaging and CA 19-9 for diagnosis, prognosis, and longitudinal disease monitoring, warranting validation in larger prospective studies.