Age-Related Multigene Analysis of Colorectal Cancer Using Next-Generation Sequencing

Background: Colorectal cancer (CRC) remains a major global health problem, with rising incidence among younger individuals. The implementation of next-generation sequencing (NGS) has enabled comprehensive multigene analysis to identify cancer-predisposing variants and molecular alterations in tumors. However, data on age-related genetic differences in CRC from Central and Eastern European populations, including Poland, remain limited. Methods: This study aimed to explore molecular differences in CRC between patients aged ≤50 and >50 years in a Polish cohort. Tumor DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tissue samples obtained from 54 treatment-naive patients. Targeted sequencing of hot spot regions of 50 genes with known association to cancer was performed using an AmpliSeq for Illumina Cancer Hotspot Panel v2. Results: Variant frequencies in younger vs. older patients were: TP53 (71.4% vs. 57.6%), APC (57.1% vs. 45.5%), KRAS (28.1% vs. 72.7%), NRAS (28.6% vs. 0%), SMAD4 (9.5% vs. 12.1%), PIK3CA (14.3% vs. 24.2%), and FBXW7 (4.8% vs. 14.7%). Co-occurrence of APC/KRAS/TP53 variants was observed in 20% of cases. KRAS mutations were significantly more frequent in older patients (p-value = 0.001), while NRAS mutations occurred exclusively in younger patients (29% vs. 0%, p = 0.021). Overall, 46% of patients exhibited multiple gene alterations (≥3 mutations). Notably, IDH1 and CTNNB1 variants were found only in patients with better prognosis, whereas TP53 variants were nearly five times more frequent in patients with worse outcomes. Conclusions: Multigene panel sequencing revealed distinct age-related molecular patterns in CRC. Younger patients were more likely to harbor NRAS variants, whereas KRAS alterations predominated in older individuals. These findings underscore the relevance of NGS-based multigene profiling for risk stratification and personalized therapy in colorectal cancer.