Abstract
Adjuvant chemotherapy improves survival in early-stage colorectal cancer (CRC), yet accurate risk stratification remains an urgent clinical challenge. In this multi-center retrospective study, we evaluated the prognostic utility of circulating tumor DNA (ctDNA) methylation profiling in 438 patients with stage II and III CRC who underwent curative surgical resection followed by fluoropyrimidine-based adjuvant chemotherapy. Plasma samples collected postoperatively (prior to chemotherapy initiation) and following completion of adjuvant therapy were analyzed using a targeted bisulfite next-generation sequencing panel interrogating six CRC-specific hypermethylated gene loci (SEPT9, IKZF1, BCAT1, SDC2, VIM, and NPY). Postoperative ctDNA methylation positivity was detected in 18.9% of stage II and 34.0% of stage III patients, conferring a significantly elevated risk of disease recurrence compared to methylation-negative counterparts (hazard ratio [HR], 4.82; 95% CI, 3.24–7.18; P < 0.001). Persistent ctDNA methylation following adjuvant chemotherapy identified patients with exceptionally poor disease-free survival (3-year DFS: 22.4% vs. 86.7%; HR, 7.35; 95% CI, 4.89–11.05; P < 0.001), substantially outperforming conventional carcinoembryonic antigen (CEA) monitoring. Serial longitudinal clearance of methylated ctDNA during chemotherapy correlated with favorable clinical outcomes, mirroring the prognostic trajectory of baseline-negative individuals. Multivariate Cox regression confirmed post-treatment ctDNA methylation as an independent predictor of recurrence after adjusting for clinicopathological risk factors. These findings demonstrate that tumor-agnostic ctDNA methylation profiling reliably identifies minimal residual disease, gauges therapeutic response, and refines post-surgical prognostic assessment in early-stage CRC.
