Purpose: Minimal residual disease (MRD) assessment guided by circulating DNA (cir-nDNA) testing is strongly prognostic in operable colon cancer patients. However, further clinical research to result in fully changing the clinical management of colon cancer about the use of adjuvant therapy. Experimental Design: In order to improve the analytical signal sensitivity of MRD detection we investigated the sources of cir-nDNA and its concentration variation in 74 stage III colon cancer patients before surgery and up to eight weeks after tumor resection. Results: A majority of stage III colon cancer patients showed significant higher post-surgery cir-nDNA levels as compared to pre-surgery level (82.2% and 64.4% during the first month and second month period). We observed a strong association of two neutrophil extracellular traps (NETs) markers (Myeloperoxidase, MPO and neutrophil elastase, NE) during the two-months post-operative period. Conclusions: Whereas the literature previously assumed that cir-nDNA concentration significantly decreased at most one-month post-surgery in the majority of stage III colon cancer patients, our data challenge this paradigm. NETs appear to constitute a confounding factor in assessing total cir-nDNA concentration since NETs production largely varies post-surgery among patients. Thus, data suggest that it would be misleading to define an optimal post-surgery blood collection time for MRD detection. At best, we estimate that given the high level of cir-nDNA content and large inter-individual variability, the best time range for blood collection could be between the fourth and the sixth week post-surgery. Second, the value of the variant allele frequency (VAF) that is so far a criteria to select mutant cir-nDNA, should be cautiously taken into consideration. We are providing various recommendations in that regards.

The authors have declared no competing interest.

This study was funded by the European ERA-NET grant on Translational Cancer Research (TRANSCAN-2). This study was funded by SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553,by MSD AVENIR [MSD-THRuST grant and by the Societe Francaise des Acides Nucleiques Circulants (SFAC). This study was funded by Spain by Fundacion Cientifica Asociacion Espanola Contra el Cancer (FC -AECC) / Instituto de Salud Carlos III (ISCIII). This study was funded by the Italian Ministry of Health.

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Ethics Committee of Candiolo Cancer Institute FPO-IRCCS gave ethical approval for this work. Ethics Committee at the Hospital Universitari Bellvitge gave ethical approval for this work. Ethics Committee CPP Ouest II of ICM gave ethical approval for this work.Ethics Committee Vall Hebron Ethical Committee gave ethical approval for this work

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