Clinical utility of longitudinal ctDNA monitoring by multiplex MS-ddPCR for risk stratification and follow-up in rectal cancer.

INTRODUCTION: Personalized treatment strategies in rectal cancer aim to balance escalation and de-escalation based on recurrence risk. Accurately identifying which patients will benefit from each approach is essential for optimizing outcomes and guiding follow-up. However, current clinical methods lack the precision needed to reliably predict response and long-term prognosis. METHODS: In this feasibility study, we evaluated the prognostic utility of a novel methylation-specific droplet digital PCR (MS-ddPCR) multiplex assay in 56 patients with locally advanced rectal cancer (LARC) undergoing neoadjuvant treatment (nT)  and surgery. Circulating tumor DNA (ctDNA) was analyzed at four time points (baseline, during nT, preoperatively, 6 months post-surgery). Associations between ctDNA status and dynamics with tumor regression grade (TRG), disease recurrence, and overall survival (OS) were assessed using receiver operating characteristics (ROC) analyses and survival statistics. RESULTS: ctDNA was detected in 59% of the patients at baseline. Preoperative ctDNA had limited discriminative value for pathologic response, AUC 0.60 (95% CI 0.45-0.76). In contrast, ctDNA positivity 6 months postoperatively was strongly associated with recurrence within 2 years following surgery, AUC 0.96 (95% CI, 0.91-1.00). CtDNA positivity 6 months post-surgery was associated with inferior 2-year DFS (38% vs 94%, p for log-rank < 0.001) and 3-year OS (63% vs 100%, p for log-rank < 0.001). CONCLUSION: With this MS-ddPCR assay, preoperative ctDNA showed limited prognostic value, whereas ctDNA 6 months postoperatively was strongly associated with recurrence and overall survival. The absence of an immediate postoperative sample limited assessment of early molecular response-a time point critical for guiding treatment decisions and follow-up strategies-underscoring the need for earlier sampling in future studies to optimize ctDNA-guided management. Given the small cohort and exploratory design, these findings are hypothesis-generating and support further validation of the assay in larger, prospective trials.