Post-surgery level of circulating DNA in stage III colon cancer patients: Impact on the reliability of minimal residual disease detection.

Minimal residual disease (MRD) assessment using circulating nuclear DNA (cir-nDNA) testing has demonstrated strong prognostic value in patients with operable stage III colon cancer (CC). However, further clinical research is needed to optimize the use of adjuvant therapy in this context. Since the sensitivity of variant allele frequency (VAF)-based MRD detection depends on total cirDNA concentration, we investigated its variation in 67 stage III CC patients over 8 weeks following surgery. A majority of patients showed significantly higher post-surgical cir-nDNA levels compared to their pre-surgical baseline-71.1% during the first month and 51.4% during the second. Cir-nDNA levels tended to considerably vary (up to 18-fold), especially during the first 3 weeks after surgery. We also observed a strong correlation between cir-nDNA levels and neutrophil extracellular traps (NETs) markers throughout the 2-month post-operative period. While previous studies have generally assumed that cir-nDNA levels decrease significantly within 1 month post-surgery in MRD-negative stage III patients, our findings challenge this paradigm. NETs appear to be a substantial source of cir-nDNA and represent a major confounding factor in interpreting total cir-nDNA, given the high inter-individual variability in NETs production after surgery. Our data suggest that defining a single optimal time point for MRD testing may be misleading. Instead, we propose a sampling window between fourth and sixth week post-surgery. Additionally, our results call into question the reliance on VAF as a standalone metric for MRD detection. The most robust strategy would involve integrated monitoring of total cir-nDNA concentration, absolute mutant DNA levels, and NETs-associated inflammation.