We show that assessment of ctDNA is a non-invasive, exquisitely specific and highly sensitive approach for monitoring disease load, which has the potential to provide clinically relevant lead times compared with conventional methods. Furthermore, we provide a low-coverage protocol optimised for identifying SSVs with excellent correlation between SSVs identified in tumours and matched metastases. Application of ctDNA analysis has the potential to change clinical practice in the management of CRC.

We investigated whether detection of ctDNA after resection of colorectal cancer identifies the patients with the highest risk of relapse and, furthermore, whether longitudinal ctDNA analysis allows early detection of relapse and informs about response to intervention. In this longitudinal cohort study, we used massively parallel sequencing to identify somatic mutations and used these as ctDNA markers to detect minimal residual disease and to monitor changes in tumor burden during a 3-year follow-up period. A total of 45 patients and 371 plasma samples were included. Longitudinal samples from 27 patients revealed ctDNA postoperatively in all relapsing patients ( = 14), but not in any of the nonrelapsing patients. ctDNA detected relapse with an average lead time of 9.4 months compared with CT imaging. Of 21 patients treated for localized disease, six had ctDNA detected within 3 months after surgery. All six later relapsed compared with four of the remaining patients [HR, 37.7; 95% confidence interval (CI), 4.2-335.5; < 0.001]. The ability of a 3-month ctDNA analysis to predict relapse was confirmed in 23 liver metastasis patients (HR 4.9; 95% CI, 1.5-15.7; = 0.007). Changes in ctDNA levels induced by relapse intervention ( = 19) showed good agreement with changes in tumor volume (κ = 0.41; Spearman = 0.4). Postoperative ctDNA detection provides evidence of residual disease and identifies patients at very high risk of relapse. Longitudinal surveillance enables early detection of relapse and informs about response to intervention. These observations have implications for the postoperative management of colorectal cancer patients. .

<p>Comparison of serial ctDNA analysis performed using SSV and SPM ddPCR assays.</p>

<p>Supplementary Table 1 to 6</p>

<p>Comparison of serial ctDNA analysis performed using SSV and SPM ddPCR assays.</p>

<p>Changes in ctDNA levels after local or systemic interventions such as surgery or chemotherapy divided into response and non-response groups.</p>

<p>List of Supplementary Tables</p>

<p>Comparison of the time to relapse detection using CEA and standard-of-care CT imaging for fourteen relapse patients.</p>