Background: A high percentage of patients diagnosed with localized colon cancer (CC) will relapse after curative treatment. Although pathological staging currently guides our treatment decisions, there are no biomarkers determining minimal residual disease (MRD) and patients are at risk of being undertreated or even overtreated with chemotherapy in this setting. Circulatingtumor DNA (ctDNA) can to be a useful tool to better detect risk of relapse.Patients and methods: One hundred and fifty patients diagnosed with localized CC were prospectively enrolled in our study. Tumor tissue from those patients was sequenced by a custom-targeted next-generation sequencing (NGS) panel to characterize somatic mutations. A minimum variant allele frequency (VAF) of 5% was applied for variant filtering. Orthogonal droplet digital PCR (ddPCR) validation was carried out. We selected known variants with higher VAF to track ctDNA in the plasma samples by ddPCR.Results: NGS found known pathological mutations in 132 (88%) primary tumors. ddPCR showed high concordance with NGS (r ¼ 0.77) for VAF in primary tumors. Detection of ctDNA after surgery and in serial plasma samples during follow-up were associated with poorer disease-free survival (DFS) [hazard ratio (HR), 17.56; log-rank P ¼ 0.0014 and HR, 11.33; log-rank P ¼ 0.0001, respectively]. Tracking at least two variants in plasma increased the ability to identify MRD to 87.5%. ctDNA was the only significantly independent predictor of DFS in multivariable analysis. In patients treated with adjuvant chemotherapy, presence of ctDNA after therapy was associated with early relapse (HR 10.02; log-rank P < 0.0001). Detection of ctDNA at followup preceded radiological recurrence with a median lead time of 11.5 months.Conclusions: Plasma postoperative ctDNA detected MRD and identified patients at high risk of relapse in localized CC. Mutation tracking with more than one variant in serial plasma samples improved our accuracy in predicting MRD.

Statement of translational relevanceSensitive methods for recurrence risk stratification, monitoring therapeutic efficacy, and early recurrence detection may have a major impact on treatment decisions and outcomes for stage III colorectal cancer patients. Circulating tumor DNA assessments performed postoperative, postadjuvant, and serially during surveillance all allowed stratification of patients into high and low risk groups. CtDNA detected recurrence with a significant leadtime compared to CT-imaging and ctDNA growth rates were prognostic of survival.Treatment of ctDNA positive patients with standard adjuvant therapy prevented recurrence in only 20% of patients. Accordingly, further studies exploring the optimal treatment for ctDNA positive patients are needed, as well as interventional studies assessing the clinical utility of ctDNA-based risk-stratification. A promising opportunity is risk-stratified allocation of surveillance resources, which may improve both the cost-effectiveness and the overall clinical outcome of surveillance. Finally, ctDNA growth rates may identify patients who could benefit from immediate therapeutic intervention compared to awaiting recurrence.Research.

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