Next-generation sequencing (NGS) has emerged as a molecular technique capable of generating an extensive genomic characterization of non-small cell lung cancer (NSCLC). Because a wide range of biomarker-based therapeutic options now exist, molecular laboratories have become essential in guiding clinical decision-making. In this report, we present the performance of an NGS assay accredited under UNE-EN ISO 15189:2022 in a series of 350 individuals. The most common alterations involved TP53 (51.0%), KRAS (26.6%), and EGFR (12.9%). We also identified patterns of coexistence and mutual exclusivity among variants, together with distinct mutational distributions linked to sex and tobacco exposure. Actionable variants appeared at a significantly higher rate in women (80.5%, p < 0.001) and in patients who never smoked (87.7%, p < 0.001). After NGS became the standard molecular platform, 36.4% of patients accessed at least one targeted therapeutic line. For the subgroup of 200 stage IV cases, use of targeted agents in first-line therapy correlated with extended progression-free survival (PFS) (13.4 months (95% CI, 10.2-16.6) (p = 0.001)). Likewise, overall survival (OS) improved notably among individuals who received any targeted agent (26.2 months (95% CI, 11.8-40.5) (p < 0.001)). Overall, these findings indicate that broader application of NGS within the public system has advanced the incorporation of precision oncology.
