Objective: Lung cancer is the most prevalent malignancy among men and women in Türkiye. National guidelines have been established to standardize molecular pathology diagnoses for the detection of driver mutations in non-small cell lung cancer with impact in the management and treatment of the disease.

This study examines ROS1 gene alterations comparing immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) diagnostic techniques in a single-center cohort.

Material and Methods: A total of 200 biopsy specimens, including tissue and cytology samples from Ege University Hospital and external consultations (2019–2022), diagnosed with non-small cell lung cancer (NSCLC) were analyzed. ROS1 SP384 IHC staining patterns (H-Score) and reflex molecular testing for EGFR, ALK, and ROS1 were recorded.

Results: Among 34 ROS1 IHC-positive cases (16.9%), ROS1 FISH positivity was observed in 7 cases (3.5%), including 5 IHC-positive (5/34; 14.7%) and 2 IHC-negative (2/166; 1.2%). The median H-Score of FISH-positive cases was 60.

Conclusion: ROS1 IHC exhibited staining in cases harboring ALK, EGFR, and KRAS mutations, including one case with concurrent EGFR and ROS1 alterations. The ROS1 SP384 clone demonstrated 71.43% sensitivity and 84.97% specificity, with a negative predictive value of 99.3%. ROS1 IHC is a valuable screening modality for molecular alterations. FISH validation is recommended, and discordant cases may require NGS or RT-PCR for rare mutations or unidentified fusion partners.