The prognostic value of cell‐free DNA dynamics during chemoradiotherapy in squamous cell carcinomas of the anus

Reliable biomarkers for assessing prognosis and monitoring response to chemoradiotherapy (CRT) are lacking in squamous cell carcinomas of the anus (SCCA). Liquid biopsies measuring cell‐free DNA (cfDNA) by direct fluorescent assay (DFA) provide a simple, non‐invasive approach. This study aims to investigate the prognostic value of cfDNA dynamics during CRT. Blood samples and clinical data were prospectively collected from SCCA patients undergoing CRT. Serum cfDNA levels (ng/μL) were quantified using DFA at baseline, mid‐therapy, and end of treatment (EOT). Baseline levels were analyzed in relation to patient characteristics using Mann–Whitney U test and over time with Wilcoxon signed‐rank test. Survival was evaluated using Kaplan–Meier curves and log‐rank test. In total, 126 patients were included, with cfDNA measurements at baseline ( n = 126), mid‐therapy ( n = 103) and EOT ( n = 108). Median cfDNA levels were 0.78 ng/μL 95% CI (0.72–0.85), 0.62 ng/μL 95% CI (0.56–0.72), and 0.66 ng/μL 95% CI (0.58–0.74), respectively. Baseline cfDNA levels were higher in patients with high‐risk disease (T3‐T4, N+ or M+) and performance status 1–2 ( p < .05), but this did not appear to affect outcomes. cfDNA levels declined from baseline to mid‐therapy and EOT ( p < .001). A lower percentage decline was observed in non‐responders (−9% 95% CI (−24; 33) and −37% 95% CI (−44; −29), p = .002) and treatment failures (−18% 95% CI (−36; 9) and −36% 95% CI (−44; −28), p = .02). Failure to eliminate below the baseline 75th percentile was associated with inferior disease‐free survival (HR = 4.23 95% CI (1.50–11.93), p = .003). In conclusion, cfDNA quantification by DFA is feasible in SCCA. Low cfDNA elimination during CRT was associated with poorer outcomes, highlighting the clinical potential of cfDNA dynamics as a prognostic biomarker.