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Purpose: Sensitive methods for risk stratification, monitoring therapeutic efficacy, and early relapse detection may have a major impact on treatment decisions and patient management for stage III colorectal cancer patients. Beyond assessing the predictive power of postoperative ctDNA detection, we explored the added benefits of serial analysis: assessing adjuvant chemotherapy (ACT) efficacy, early relapse detection, and ctDNA growth rates. Experimental Design: We recruited 168 patients with stage III colorectal cancer treated with curative intent at Danish and Spanish hospitals between 2014 and 2019. To quantify ctDNA in plasma samples (n = 1,204), 16 patient-specific somatic single-nucleotide variants were profiled using multiplex-PCR, next-generation sequencing. Results: Detection of ctDNA was a strong recurrence predictor postoperatively [HR = 7.0; 95% confidence interval (CI), 3.7–13.5; P < 0.001] and directly after ACT (HR = 50.76; 95% CI, 15.4–167; P < 0.001). The recurrence rate of postoperative ctDNA-positive patients treated with ACT was 80% (16/20). Only patients who cleared ctDNA permanently during ACT did not relapse. Serial ctDNA assessment after the end of treatment was similarly predictive of recurrence (HR = 50.80; 95% CI, 14.9–172; P < 0.001), and revealed two distinct rates of exponential ctDNA growth, slow (25% ctDNA-increase/month) and fast (143% ctDNA-increase/month; P < 0.001). The ctDNA growth rate was prognostic of survival (HR = 2.7; 95% CI, 1.1–6.7; P = 0.039). Serial ctDNA analysis every 3 months detected recurrence with a median lead-time of 9.8 months compared with standard-of-care computed tomography. Conclusions: Serial postoperative ctDNA analysis has a strong prognostic value and enables tumor growth rate assessment. The novel combination of ctDNA detection and growth rate assessment provides unique opportunities for guiding decision-making. See related commentary by Morris and George, p. 438

3540 Background: Challenges in the postoperative management of stage III colorectal cancer include: 1) selection of high-risk patients for adjuvant chemotherapy (ACT), 2) lack of markers to assess ACT efficacy, 3) assessment of recurrence risk after ACT, and 4) lack of markers to guide treatment decisions for high-risk patients e.g. additional therapy or intensified surveillance. Circulating tumor DNA (ctDNA) is a promising marker with potential to mitigate the challenges. Here we used serial ctDNA measurements to assess the correlation between recurrence and ctDNA detection: postoperative, during and after ACT, and during surveillance; and to assess growth rates of metachronous metastases. Uniquely, we also used concurrent CT scans and ctDNA measurements to compare the sensitivity for detecting recurrence. Methods: Stage III CRC patients treated with curative intent at Danish and Spanish hospitals in 2014-2019 were recruited (n = 166). Blood samples (n = 1227) were collected prior to and immediately after surgery, and every third month for up to 36 months. Per patient 16 personal mutations were used to quantify plasma ctDNA (Signatera, bespoke mPCR NGS assay). Results: Detection of ctDNA was a strong recurrence predictor, both postoperatively (HR 7.2, 95% CI 3.8-13.8, P< 0.001), directly after ACT (HR = 18.2, 95% CI 7.1-46, P < 0.001), and when measured serially after end of treatment (HR = 41, 95% CI 16-100, P < 0.001). The recurrence rate of postoperative ctDNA positive patients treated with ACT was 80% (16/20). Patients who stayed ctDNA positive during ACT all recurred. Serial post-treatment ctDNA measurements revealed exponential growth for all recurrence patients following either a SLOW (26%-increase/month) or a FAST (126%-increase/month) pattern (P < 0.001). From ctDNA detection to radiologic recurrence, ctDNA levels of FAST patients increased by a median 117-fold, and up to 554-fold. The 3-year overall survival was 43% for FAST patients and 100% for SLOW and non-recurrence patients (HR = 41.3, 95% CI 7.5-228, P < 0.001). Coinciding CT scans and ctDNA measurements (n = 113 patients, 235 coinciding events, median 2 per patient) showed a high agreement (92%) and ctDNA either detected residual disease before the CT scan (n = 7 patients) or at the same time (n = 14 patients). The median lead-time was 7.5 months. Conclusions: The study confirmed the prognostic power of serial postoperative ctDNA analysis. Moreover, it provided novel analyses demonstrating that ctDNA is more sensitive for recurrence detection than CT scans and can be used for tumor growth rate assessments. The difference between FAST and SLOW growing tumors suggest that growth rates could guide whom to start on systemic therapy rapidly and whom to send for diagnostic imaging. Altogether, the study highlights many potential utilities of ctDNA in guiding clinical decision-making.

11 Background: Timely detection of recurrence, as well as identification of patients at high risk of recurrence after surgery and after completion of adjuvant therapy, are major challenges in the treatment of colorectal cancer (CRC). Postsurgical circulating tumor DNA (ctDNA) analysis is a promising tool for the identification of patients with minimal residual disease (MRD) and a high risk of recurrence. The objective of this prospective, multicenter study was to determine whether serial postsurgical ctDNA analysis could identify the patients at high risk of recurrence, provide an assessment of adjuvant therapy efficacy and detect relapse earlier than standard-of-care radiological imaging. Methods: The cohort comprises 265 stage I-III CRC patients, the to-date largest cohort assessed for ctDNA. All patients had the tumor resected and a subset of 62.6% (166 /265) was additionally treated with ACT. Plasma samples (n = 1503) were collected at various time points for a median follow-up of 28.4 months (range: 1.2-51.0 months). Individual tumors and matched germline DNA were whole-exome sequenced and somatic single nucleotide variants (SNVs) were identified. Personalized multiplex PCR assays were designed to track tumor-specific SNVs (Signatera, bespoke mPCR NGS assay) in each patient’s plasma sample. Results: Postoperative ctDNA status prior to ACT was assessed in 218 patients, of which 9.17% (20/218) were identified to be MRD-positive and 75% (15/20) eventually relapsed. The remaining 25% (5/20) of MRD-positive patients that did not relapse, received ACT. In contrast, only 13.6% (27/198) of MRD-negative cases relapsed (HR: 11: 95% CI: 5.7-20; p < 0.001). Longitudinal ctDNA-positive status, post-definitive therapy (n = 202) was associated with a HR of 36 (95% CI: 16-81; p < 0.001). For a subset of 155 patients postoperative CEA and ctDNA measurements were compared, wherein, ctDNA-positive status was found to be significantly associated with RFS (HR, 7.1; 95% CI, 3.4-15; P < 0.001) compared to CEA (HR, 1.2; 95% CI, 0.46-3.1; P = 0.73). Serial ctDNA analysis detected MRD up to a median of 8 months (0.56 - 21.6 months) ahead of radiologic relapse. Conclusion: Postoperative ctDNA positive status was associated with markedly reduced RFS compared to CEA. The study also shows that effective therapy can be curative in a portion of MRD-positive patients. In a longitudinal setting, ctDNA analysis predicted the risk of recurrence and is a more reliable biomarker for treatment response monitoring.