Comprehensive Molecular Diagnostic Tests in Non-Small Cell Lung Cancer: Frequency of ALK, ROS1, RET, and Other Gene Fusions/Rearrangements in a Romanian Cohort.

Background/Objectives: Lung cancer remains among the most frequently diagnosed malignancies in Romania, with a high mortality rate. Beyond EGFR mutations, clinically relevant genetic alterations in non-small cell lung cancer (NSCLC) include fusions involving ALK, ROS1, RET, and NTRK1/2/3. This study aimed to determine the prevalence of these mutations in a Romanian cohort and evaluate their associations with clinicopathological features. Methods: DNA and RNA were simultaneously extracted from formalin-fixed, paraffin-embedded (FFPE) tissue sections using the Genexus Purification System (ThermoFisher Scientific). Concentrations were quantified fluorometrically, and gene fusions were analyzed with Ion Torrent NGS (Ion GeneStudio S5) with the Oncomine Focus Assay (ThermoFisher Scientific). Library preparation was automated with the Ion Chef System, and data interpretation was conducted using Ion Reporter. Results: Among 721 newly diagnosed NSCLC patients, 28 (3.88%) harbored gene fusions. Adenocarcinoma prevailed among fusion-positive cases (85.7%). The subgroup included 15 males and 13 females, with a mean age of 63.25 years (range 43-83). ALK fusions were most frequent (1.66% of the cohort; 42.86% of positives), predominantly EML4::ALK. ROS1 fusions were detected in five patients (0.7%), most frequently CD74::ROS1. RET fusions occurred in 1.11%. Rare fusions included one ETV6::NTRK3, one PTPRZ1::MET, and one FGFR3::TACC3 co-occurring with EGFR L858R. Conclusions: Gene fusions were present in a minority of NSCLC cases, with ALK, ROS1, and RET being the most clinically relevant. These alterations were mutually exclusive with common drivers such as EGFR or KRAS. Detection of rare fusions highlights the therapeutic potential of comprehensive NGS profiling in Romanian NSCLC patients.