Comparison of Molecular Testing Modalities for Detection of ROS1 Rearrangements in a Cohort of Positive Patient Samples

J Thorac Oncol. 2018 Oct;13(10):1474-1482. doi: 10.1016/j.jtho.2018.05.041. Epub 2018 Jun 20.


Introduction: ROS1 gene fusions are a well-characterized class of oncogenic driver found in approximately 1% to 2% of NSCLC patients. ROS1-directed therapy in these patients is more efficacious and is associated with fewer side effects compared to chemotherapy and is thus now considered standard-of-care for patients with advanced disease. Consequently, accurate detection of ROS1 rearrangements/fusions in clinical tumor samples is vital. In this study, we compared the performance of three common molecular testing approaches on a cohort of ROS1 rearrangement/fusion-positive patient samples.

Methods: Twenty-three ROS1 rearrangement/fusion-positive clinical samples were assessed by at least two of the following molecular testing methodologies: break-apart fluorescence in situ hybridization, DNA-based hybrid capture library preparation followed by next-generation sequencing (NGS), and RNA-based anchored multiplex polymerase chain reaction library preparation followed by NGS.

Results: None of the testing methodologies demonstrated 100% sensitivity in detection of ROS1 rearrangements/fusions. Fluorescence in situ hybridization results were negative in 2 of 20 tested samples, the DNA-based NGS assay was negative in 4 of 18 tested samples, and the RNA-based NGS assay was negative in 3 of 19 tested samples. For all three testing approaches, we identified assay characteristics that likely contributed to false-negative results. Additionally, we report that genomic breakpoints are an unreliable predictor of breakpoints at the transcript level, likely due to alternative splicing.

Conclusions: ROS1 rearrangement/fusion detection in the clinical setting is complex and all methodologies have inherent limitations of which users must be aware to correctly interpret results.

Keywords: FISH; ROS1; lung cancer; molecular testing; next-generation sequencing.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cohort Studies
  • Female
  • Gene Fusion / genetics*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • In Situ Hybridization, Fluorescence / methods*
  • Male
  • Protein-Tyrosine Kinases / genetics*
  • Proto-Oncogene Proteins / genetics*


  • Proto-Oncogene Proteins
  • Protein-Tyrosine Kinases
  • ROS1 protein, human