Early Detection of Molecular Residual Disease and Risk Stratification for Stage I to III Colorectal Cancer via Circulating Tumor DNA Methylation

Abstract

Importance: Detection of molecular residual disease and risk stratification as early as possible may improve the treatment of patients with cancer. Efficient pragmatic tests are therefore required.

Objective: To measure circulating tumor DNA (ctDNA) with 6 DNA methylation markers in blood samples and to evaluate the association of the presence of ctDNA with colorectal cancer (CRC) recurrence throughout the disease course.

Design, setting, and participants: In this multicenter prospective longitudinal cohort study performed from December 12, 2019, to February 28, 2022, 350 patients with stage I to III CRC were recruited from 2 hospitals for collection of blood samples before and after surgery, during and after adjuvant chemotherapy, and every 3 months for up to 2 years. A multiplex, ctDNA methylation, quantitative polymerase chain reaction assay was used to detect ctDNA in plasma samples.

Results: A total of 299 patients with stage I to III CRC were evaluated. Of 296 patients with preoperative samples, 232 (78.4%) tested positive for any of the 6 ctDNA methylation markers. A total of 186 patients (62.2%) were male, and the mean (SD) age was 60.1 (10.3) years. At postoperative month 1, ctDNA-positive patients were 17.5 times more likely to relapse than were ctDNA-negative patients (hazard ratio [HR], 17.5; 95% CI, 8.9-34.4; P < .001). The integration of ctDNA and carcinoembryonic antigen tests showed risk stratification for recurrence with an HR of 19.0 (95% CI, 8.9-40.7; P < .001). Furthermore, ctDNA status at postoperative month 1 was strongly associated with prognosis in patients treated with adjuvant chemotherapy of different durations and intensities. After adjuvant chemotherapy, ctDNA-positive patients had a significantly shorter recurrence-free survival than did the ctDNA-negative patients (HR, 13.8; 95% CI, 5.9-32.1; P < .001). Longitudinal ctDNA analysis after the postdefinitive treatment showed a discriminating effect in that ctDNA-positive patients had poorer recurrence-free survival than did the ctDNA-negative patients (HR, 20.6; 95% CI, 9.5-44.9; P < .001). The discriminating effect was enhanced (HR, 68.8; 95% CI, 18.4-257.7; P < .001) when ctDNA status was maintained longitudinally. Postdefinitive treatment analysis detected CRC recurrence earlier than radiologically confirmed recurrence, with a median lead time of 3.3 months (IQR, 0.5-6.5 months).

Conclusions and relevance: The findings of this cohort study suggest that longitudinal assessment of ctDNA methylation may enable the early detection of recurrence, potentially optimizing risk stratification and postoperative treatment of patients with CRC.

Figure 1.. Patient Enrollment and Definition of the Patient Subgroups

ACT indicates adjuvant chemotherapy; CEA, carcinoembryonic antigen; CRC, colorectal cancer; CT, computed tomography; and ctDNA, circulating tumor DNA.

Figure 2.. Circulating Tumor DNA (ctDNA) Analysis of Plasma at Postoperative Month 1 for Detecting Colorectal Cancer Recurrence

A, Kaplan-Meier estimates of recurrence-free survival, stratified by ctDNA status at postoperative month 1. The recurrence risk for ctDNA-positive patients was significantly higher than that of ctDNA-negative patients (hazard ratio, 17.5; 95% CI, 8.9-34.4; P < .001). P value was determined by the log-rank test. Shaded areas indicate 95% CIs. B, Network map of 6 targets of the ColonAiQ assay in patients with recurrence. The size of the nodes is proportional to the number of patients who tested positive, and the thickness of lines is proportional to the number of patients with recurrence who tested positive for 2 targets. Positive rates of 6 targets are shown. BCAN indicates brevican; BCAT1, branched-chain amino acid transaminase 1; IKZF1, ikaros family zinc finger 1; Septin9_1, septin 9 locus 1; Septin9_2, septin 9 locus 2; and VAV3, vav guanine nucleotide exchange factor 3.

Figure 3.. Longitudinal Circulating Tumor DNA (ctDNA) Analysis for Detecting Colorectal Cancer Recurrence

A, Kaplan-Meier estimates of recurrence-free survival (RFS) for patients who completed definitive treatment (surgery and adjuvant chemotherapy [ACT]), stratified by the first time point at postdefinitive treatment ctDNA status. After curative treatment, ctDNA positivity was associated with a strikingly reduced RFS compared with ctDNA negativity (hazard ratio [HR], 20.6; 95% CI, 9.5-44.9; P < .001). P value was determined by the log-rank test. B, Comparison of time to relapse by ctDNA status at postdefinitive-treatment and standard-of-care computed tomography (CT). ID indicates identification. C, Kaplan-Meier estimates of RFS for patients during surveillance, stratified by the longitudinal ctDNA status. A patient was classified as testing positive if there was at least 1 positive ctDNA test result. An inferior prognosis was associated with patients with ctDNA-positive results at any point (HR, 13.5; 95% CI, 5.4-33.8; P < .001). P value was determined by the log-rank test. D, Kaplan-Meier estimates of RFS stratified by ctDNA status in blood samples obtained after surgery. A patient was classified as testing positive (or negative) if all plasma samples were ctDNA positive (or negative). The ctDNA status maintenance indicated that ctDNA-positive patients had a significantly shorter RFS than ctDNA-negative patients (HR, 68.8; 95% CI, 18.4-257.7; P < .001), indicating a worse prognosis when the residual ctDNA was retained. P value was determined by the log-rank test. Shaded areas indicate 95% CIs.