Targeted NGS of Preoperative Circulating Cell-Free DNA in Colorectal Cancer.

INTRODUCTION: Circulating tumor DNA is a promising noninvasive tool for tumor genotyping and recurrence monitoring in Colorectal Cancer (CRC), but its role in nonmetastatic disease remains unclear. This cross-sectional study evaluated preoperative plasma circulating cell-free DNA (cfDNA) in patients undergoing complete mesocolic excision (CME) with D3 lymphadenectomy. METHODS: Preoperative plasma samples were collected from 32 patients with predominantly right-sided CRC; 31 with histologically confirmed malignancy were analysed. cfDNA was sequenced using the AVENIO ctDNA Expanded Panel targeting 77 cancer-related genes. Sequencing metrics, Variant Allele Frequency (VAF), variant types, and cfDNA variants were evaluated. RESULTS: High-quality cfDNA was obtained from all patients (mean yield 0.69 ng/μL). Sequencing met performance criteria (>22 million read pairs, >86% mapped reads, depth 6779×/3523×). Variants were detected in 74% of the patients, most frequently in TP53 (n=11), APC (n=8) and KRAS (n=7). The mean VAF was 0.8%, with some variants exceeding 20%. The most frequent hotspot was KRAS c.38G>A (p.Gly13Asp) (n=4). Recurrent variants were observed in IDH2, MET, GNAS, CTNNB1, and PDCD1LG2, indicating molecular heterogeneity. DISCUSSION: Preoperative tumor-naïve cfDNA sequencing enabled detection of established CRC driver alterations and rare variants, supporting its potential for minimally invasive molecular characterization. Compared with tumor-informed approaches, this strategy allows plasma profiling without prior tissue sequencing; however, variant interpretation remains limited without matched tumor and white blood cell DNA. CONCLUSION: Targeted cfDNA sequencing is feasible in preoperative CRC patients undergoing CME and provides a molecular baseline for future perioperative studies. Larger prospective cohorts with longitudinal follow-up are required to define its prognostic and clinical utility.