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.

PURPOSE More than 50% of patients with stage IV colorectal cancer (metastatic colorectal cancer [mCRC]) relapse postresection. The efficacy of postoperative systemic treatment is limited in this setting. Thus, these patients would greatly benefit from the use of a reliable prognostic biomarker, such as circulating tumor DNA (ctDNA) to identify minimal or molecular residual disease (MRD). PATIENTS AND METHODS We analyzed a cohort of 112 patients with mCRC who had undergone metastatic resection with curative intent as part of the PREDATOR clinical trial. The study evaluated the prognostic value of ctDNA, correlating MRD status postsurgery with clinical outcomes by using a personalized and tumor-informed ctDNA assay (bespoke multiple PCR, next-generation sequencing assay). Postresection, systemic therapy was given to 39.2% of the patients at the discretion of the treating physician. RESULTS Postsurgical, MRD positivity was observed in 54.4% (61 of 112) of patients, of which 96.7% (59 of 61) progressed at the time of data cutoff (hazard ratio [HR]: 5.8; 95% CI, 3.5 to 9.7; P < .001). MRD-positive status was also associated with an inferior overall survival: HR: 16.0; 95% CI, 3.9 to 68.0; P < .001. At the time of analyses, 96% (49 of 51) of patients were alive in the MRD-negative arm compared with 52.4% (32 of 61) in the MRD-positive arm. Patients who did not receive systemic therapy and were MRD-negative in the combined ctDNA analysis at two time points had an overall survival of 100%. In the multivariate analysis, ctDNA-based MRD status was the most significant prognostic factor associated with disease-free survival (HR: 5.78; 95% CI, 3.34 to 10.0; P < .001). CONCLUSION This study confirms that in mCRC undergoing resection of metastases, postoperative MRD analysis is a strong prognostic biomarker. It holds promises for being implemented in clinical decision making, informing clinical trial design, and further translational research.

102 Background: Numerous studies have shown the clinical utility of ctDNA, a non-invasive biomarker to detect MRD and stratify CRC patients who are more likely to relapse. We present an analysis of MRD detection in CRC patients from a prospective multicentre UK study, who were monitored pre- and post-surgery before adjuvant chemotherapy (ACT). Methods: The study recruited patients diagnosed with stage II-III CRC (n=122), including a subset of rectal patients who underwent tri-modality treatment (TMT). All patients had their primary tumor resected and 56% (68/122) received ACT. Paired plasma samples (n=244) were collected before surgery/neaodjuvant chemoradiotherapy and after surgery; median follow-up for survival was 15.48 months (0.16 - 42.1 months). Individual tumors and matched germline DNA were whole-exome sequenced and somatic single nucleotide variants (SNVs) identified. Multiplex PCR assays were designed to track tumor-specific SNVs (Signatera, bespoke mPCR NGS assay) in plasma samples. The study evaluated ctDNA status and clinical outcomes including radiologic imaging. Cox regression was used to calculate recurrence-free survival (RFS) in patients stratified by post-op ctDNA status. Patients were also stratified into low and high-risk groups based on the clinicopathological features. Multivariate analysis was performed with covariates: ctDNA, age, gender, laterality, stage, number of lymph node resected, MSI & TMB. Results: Pre-treatment ctDNA was detected in 93.4% (100/107) of patients. Post-operative ctDNA status prior to ACT was assessed in 107 patients, of whom, 13% (14/107) were MRD-positive (MRDpos). Of the MRDpos patients 42.9% (6/14) eventually relapsed. In contrast, only 8.6% (8/93) of MRD-negative (MRDneg) cases relapsed (HR: 10; 95% CI: 3.3-30; p<0.001). MRD rates stratified by risk features in each of the stages with respective recurrence rates are shown in Table. In stage III patients (n=64), 45.4% (5/11) of the MRDpos patients relapsed, whilst only 17% (9/53) of the MRDneg cases relapsed (HR: 9; 95% CI:2.6-32; p<0.0001). In the multivariate analysis, ctDNA status was the most significant prognostic factor associated with RFS (HR: 28.8, 95% CI: 3.5-234.1; p<0.001). Conclusions: Postoperative ctDNA analysis with tumor informed assay enables detection of CRC patients at high-risk of recurrence. Early detection of MRD could guide ACT decisions in intervention trials and is currently underway in TRACC. Clinical trial information: NCT04050345. [Table: see text]

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.

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