Nocturnal rise in circulating tumor DNA in a subset of metastatic colorectal cancer patients

Background: Practical use of ctDNA tests is complicated because many tumors shed into the bloodstream vanishingly low amounts of DNA. Significant efforts have been invested in the identification of factors that influence the performance of ctDNA assays. A recent study demonstrated a nocturnal peak in the amount of circulating tumor cells (CTC) during the deep sleep phase (~4 a.m.). This study questioned whether the same peak is characteristic of circulating tumor DNA (ctDNA). Methods: Plasma samples were collected from 19 RAS/RAF -mutated colorectal cancer (CRC) patients at 12 p.m. (day 1), 4 a.m. (night), and 12 p.m. (day 2). 14 subjects provided a single triplet for the study, and five patients underwent the above procedure twice or thrice. KRAS, NRAS , and BRAF mutations in plasma were quantified via droplet digital PCR (ddPCR). Results: RAS/RAF mutations (>10 copies/mL) were detected in at least one plasma sample in 20 of 25 (80%) triplets. Among these, 15 showed detectable ctDNA at all time points, with intra-individual variation ranging from 1.7% to 74.8%. In five triplets, ctDNA was undetectable during the daytime but present at night. A nocturnal ctDNA peak occurred in 7 of 9 triplets collected during disease control, while all 10 triplets obtained during tumor progression showed other temporal patterns (p = 0.005, Fisher's exact test). Patients sampled multiple times displayed differing ctDNA fluctuation patterns depending on disease status. Conclusions: ctDNA levels exhibit circadian variation, with a 4 a.m. peak observed in patients with disease control. The results align with prior reports on nocturnal surges in CTC in breast cancer patients and suggest that liquid biopsy results are modulated by circadian or sleep-related physiological changes. This work has been supported by the Russian Science Foundation (grant number 25-45-01051)