ROS1 fusions are well treatable aberrations in NSCLC. Besides solvent-front mutations (SFM) in resistance to targeted therapy, small-scale ROS1 mutations are largely unknown. We exploratively analyzed the clinicopathological characteristics, survival and therapy of small-scale ROS1 mutations in NSCLC patients without activating ROS1 fusions or SFMs. Next-generation sequencing was performed on tissue samples from NSCLC patients within the Network Genomic Medicine. Patients with ROS1 fusions and SFMs were excluded. We analyzed the characteristics of the patients, their survival and their response to systemic therapy. Of 10,396 patients analyzed, 101 (1.0%) patients harbored small-scale ROS1 mutations. Most patients were male (73.3%) and smokers (96.6%). Nearly half of the patients presented with squamous-cell carcinoma (SqCC, 40.4%). Most mutations were transversions (50.5%), and 66% were in the kinase domain. Besides TP53 mutations (65.3%), KRAS (22.8%), EGFR (5.9%), PIK3CA (9.9%) and FGFR1-4 mutations (8.9%) co-occurred. In 10 (9.9%) patients, ROS1 mutation was the only aberration detected. The median overall survival (mOS) of all stage IV patients was 10.5 months and the survival differed significantly in patients with or without KRAS co-mutations (9.7 vs 21.5 months, p=0.02, HR 2.6, 95% CI: 1.1-5.9). Altough not significant, the most common KRAS mutation in this cohort G12V (n=7/17) worsened survival (4.4 versus 9.7 months). Patients significantly benefited from treatment with immune-checkpoint blockade (21.5 vs 4.4 months, p=0.003, HR 0.14, 95% CI: 0.03-0.6). KRAS co-mutant patients treated with ICB only had an mOS of 8 months. In three patients with co-occurring mutations, qualifying for and having received targeted therapy (2x EGFRmut, 1x MET fusion), mOS was 23.5 months (95% CI: NA). The cohort's clinical characteristics contrasted ROS1-fused cohorts. Co-occurrence of KRAS mutations led to shortened survival and patients benefited from ICB. Our data does not support the idea of ROS1 small-scale mutations as strong oncogenic drivers in NSCLC, but rather as relevant bystanders altering the efficacy of treatment approaches.

The ROS1 proto-oncogene encodes a receptor tyrosine kinase that activates downstream signaling pathways related to cell proliferation and survival. Although ROS1 fusions are well-treatable genetic aberrations, little is known about small-scale ROS1 aberrations. Driving on our previous investigations elucidating their clinical and molecular characteristics, we aimed at analyzing the effect exerted by the detected aberrations with regard to their biological effect on the protein structure. Next-generation sequencing (NGS) was performed on tissue samples from Non-small cell lung cancer patients within the National Network Genomic Medicine. Patients with activating ROS1 fusions and solvent front mutations (SFM) were excluded. We analyzed the molecular aberrations using a mutation mapper, the protein structure prediction software AlphaFold® and the protein database models 3zbf and 4uxl. Of 8072 patients analyzed by NGS between 2018 and 2022, 129 (1.6%) patients harbored ROS1 mutations without having a ROS1 fusion or SFM. In 10.2% of patients ROS1 mutation was the only detected aberration and most ROS1 mutations were transversions (52.1%). 75.4% of all mutations are missense and 7% are truncating mutations. About 95% of all mutations are located in the cytoplasmic domain whereof 57.3% of mutations are located in the protein tyrosine kinase domain. Only 2.4% of mutations are located in the ATP binding site. We did not encounter a specific mutational hotspot. Most importantly, we determined truncating and missense mutations in the DFG-motif which controls the active, type I kinase conformation and the access to the catalytic site. These mutations either truncate the crucial phenylalanine of the motif or substitute its glycine for a valine. Structure predictions revealed considerable deformations in the motif. Furthermore we report several amino acid substitutions in the kinase domain activation loop. This evidence implies a biological impact in silico backing up the previously determined clinical impact. We warrant further research to characterize the biological impact in vitro and in vivo, and the potential to act as a drug target.

Fusions in the ROS1 proto-oncogene are among the best treatable genetic aberrations in Non-small cell lung cancer (NSCLC). Besides the occurrence of solvent-front mutations (SFM) in acquired resistance to targeted therapy, little is known about small-scale ROS1 aberrations. We comprehensively analyzed clinical and molecular characteristics of ROS1 mutations in NSCLC patients without activating ROS1 fusions or SFMs.