Laboratory-Developed NGS Panel for Characterization of Advanced Colorectal Cancer, Real-World Data from a Tertiary Referral Centre

Background/Objectives: Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies and a leading cause of cancer-related mortality worldwide. Its development and progression are driven by a complex interplay of genetic and epigenetic alterations that contribute to marked biological heterogeneity and variable clinical outcomes. Comprehensive molecular profiling is therefore essential for accurately characterizing CRC and guiding personalized therapeutic strategies. In this context, next-generation sequencing (NGS) has emerged as a powerful tool, enabling high-throughput, simultaneous detection of multiple clinically relevant genomic alterations in a single assay. Methods: We report our experience with a laboratory-designed NGS panel for the routine assessment of advanced-stage CRC. A total of 611 colorectal cancer specimens were analyzed using a laboratory-developed multi-gene NGS panel targeting 229 amplicons across 25 oncogenes and tumor suppressor genes. Results: The panel demonstrated analytical feasibility, with unsuccessful results observed in only 1.3% of the analyzed samples, due to low-quality/quantity DNA. RAS genes (KRAS and NRAS, altered in 51.2% and 2.7% of cases, respectively), together with BRAF (10.6%), PIK3CA (16.1%), SMAD4 (11.8%), and TP53 (64.3%), represented the most frequently altered molecular markers in the studied cohort. The frequency of major actionable genetic alterations in CRC was comparable between primary (n = 482) and metastatic (n = 121) lesions, suggesting similar mutation frequencies across the groups. Conclusions: This study presents a laboratory-developed multi-gene NGS panel specifically designed for molecular profiling of CRC in routine clinical practice.