Fragmentation of cell-free DNA (cfDNA) in locally advanced rectal cancer (LARC).

Abstract

e15665 Background: Liquid biopsy, particularly the analysis of cfDNA or circulating tumor DNA (ctDNA), has become a tool for diagnosis, monitoring, and treatment personalization. However, the extraction of cfDNA in patients with LARC presents challenges, because the cost-effectiveness of ctDNA in LARC is very low compared to metastatic disease. Fragmentomics examines cfDNA lengths, establishing the physiological size at 167 base pairs (bp). This study investigates the association between cfDNA fragmentation and clinical variables in LARC patients undergoing preoperative treatment. Methods: A cross-sectional study included 62 LARC patients treated with preoperative treatment at the Medical Oncology Department diagnosed in 2021-2024. cfDNA was extracted from plasma samples collected prior to neoadjuvant therapy. Fragment length (bp) was analyzed in relation to tumor location, size, T and N stages, EMVI (extramural vascular invasion) score, and circumferential margin involvement. Statistical significance was set at p<0.05 using T-student and ANOVA. Results: Among the 62 patients, 42 males (67.7%) and 20 females (32.3%), with a mean age of 64.5 years (SD 10.73). All were adenocarcinomas: 18 located in the lower rectum (29%), 28 in the middle rectum (45.2%), and 16 in the upper rectum (25.8%). 48 were T3 (77.4%) 8 T4 (12,9%) with nodal involvement in 57 cases (91.9%) and circumferential margin involvement in 22 cases (35.5%). The mean cfDNA fragmentation length was 149.16 bp (SD 13.01). Greater fragmentation was observed in patients with lower rectal tumors, T4 stage, and circumferential margin involvement, although differences were not statistically significant. Multivariate analysis revealed a significant association between tumor size and cfDNA length; for every centimeter increase in tumor size, fragment length decreased 2.6 bp (p=0.007). Conclusions: LARC patients with poor prognostic factors such as tumors in the lower rectum, T4 stage, or margin involvement exhibited greater cfDNA fragmentation. This finding reached statistical significance in patients with larger tumors, suggesting a potential tumor-derived origin of the fragmented cfDNA. Larger studies are required to validate cfDNA fragmentation as a reliable prognostic biomarker in LARC patients. Multiple linear regression for fragment length. Variable B p CI 95% Location 2,503 0,262 (-1,93, 6.41) Size (cm) -2,645 0,007 (-4,520, -0,75) Circumferential margin 4,921 0,158 (-1,994, 11,837) Vascular invasion -6,033 0,259 (-16,668, 4,592) B: Regression coefficient p: statistical significance CI: confidence interval.