Kinetics of postoperative circulating cell-free DNA and impact on minimal residual disease detection rates in patients with resected stage I-III colorectal cancer.

5 Background: A growing body of evidence supports the utility of circulating tumor DNA (ctDNA) as a useful biomarker for detecting molecular residual disease (MRD) in colorectal cancer (CRC). Immediately after surgery or during adjuvant therapy, high levels of cell-free DNA (cfDNA) from normal tissue may limit the detection of tumor-derived ctDNA. The optimal timing of blood collection for reliable MRD detection after surgery or adjuvant therapy remains unclear. Methods: In this retrospective, U.S.-based, multi-institutional study, data from commercial ctDNA testing in 16,347 patients with stage I-III CRC were analyzed. Complete clinical data were available for 417 patients with 2,538 plasma samples collected between 6/2019 and 4/2022. The median follow-up for relapsed and non-relapsed patients was 730 and 615 days, respectively. A personalized, tumor-informed multiplex PCR-based next-generation sequencing assay (Signatera) was used to quantify ctDNA prior to surgery and postoperatively in a longitudinal manner. We analyzed the kinetics of total cfDNA and compared it with the ctDNA MRD positivity rates at various time points after surgery. Results: Among all patients, cfDNA levels were higher immediately after surgery (0-2 weeks) and gradually declined during the subsequent 2-8 weeks (p<0.0001). Despite the higher cfDNA levels, among patients with immediate post-operative draws (0-2 weeks) 30.6% (113/369) of patients were ctDNA positive. Similar ctDNA detection rates were observed with conventional MRD windows after resection, whether the window was defined as 2-6 weeks (20.8%; 957/4605) or 2-8 weeks (20.9%; 1155/5534). In the clinically annotated cohort, ctDNA-positivity during the MRD time window of 2-8 weeks and during surveillance (>6 months post-operatively and subsequent to any adjuvant therapy) was significantly associated with worse recurrence-free survival as compared to ctDNA negative patients (MRD: HR 14.1, 95% CI: 5.8-34; p<0.0001; and surveillance HR 20.6, 95% CI: 10.6-37.6; p<0.0001), respectively. On analyzing cfDNA dynamics during adjuvant therapy, cfDNA levels gradually increased between 8 weeks and 8 months after surgery (p=0.0001), suggesting a potential impact of treatment on cell death and cfDNA shed. Conclusions: This is one of the first, large-scale in-depth studies evaluating treatment-based fluctuations in post-operative cfDNA and its correlation with ctDNA-positivity. Our analyses demonstrated no significant impact of plasma cfDNA levels on ctDNA detection rates across different MRD windows using a tumor-informed approach. This may affect real-world application of personalized ctDNA testing by allowing earlier testing windows, as well as guide clinical trial designs using ctDNA as an integral biomarker.