BackgroundROS1 fusion is an infrequent, but attractive target for therapy in patients with metastatic non- small-cell lung cancer. In studies on mainly late-stage disease, the prevalence of ROS1 fusions is about 1–3%. In early-stage lung cancer ROS1 might also provide a fruitful target for neoadjuvant or adjuvant therapy. In the present study, we investigated the prevalence of ROS1 fusion in a Norwegian cohort of early-stage lung cancer. We also explored whether positive ROS1 immunohistochemical (IHC) stain was associated with certain mutations, clinical characteristics and outcomes.MethodsThe study was performed using biobank material from 921 lung cancer patients including 542 patients with adenocarcinoma surgically resected during 2006–2018. Initially, we screened the samples with two different IHC clones (D4D6 and SP384) targeting ROS1. All samples that showed more than weak or focal staining, as well as a subgroup of negative samples, were analyzed with ROS1 fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) with a comprehensive NGS DNA and RNA panel. Positive ROS1-fusion was defined as those samples positive in at least two of the three methods (IHC, FISH, NGS).ResultsFifty cases were IHC positive. Of these, three samples were both NGS and FISH-positive and considered positive for ROS1 fusion. Two more samples were FISH positive only, and whilst IHC and NGS were negative. These were also negative with Reverse Transcription quantitative real time Polymerase Chain Reaction (RT-qPCR). The prevalence of ROS1 fusion in adenocarcinomas was 0.6%. All cases with ROS1 fusion had TP53 mutations. IHC-positivity was associated with adenocarcinoma. Among SP384-IHC positive cases we also found an association with never smoking status. There was no association between positive IHC and overall survival, time to relapse, age, stage, sex or pack-year of smoking.ConclusionsROS1 seems to be less frequent in early-stage disease than in advanced stages. IHC is a sensitive, but less specific method and the results need to be confirmed with another method like FISH or NGS.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13000-023-01357-1.