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

Journal of Thoracic Oncology | October, 2018

Kurtis D. Davies, PhD, Anh T. Le, BA, Jamie Sheren, PhD, Hala Nijmeh, PhD, Katherine Gowan, BS, Kenneth L. Jones, PhD, Marileila Varella-Garcia, PhD, Dara L. Aisner, MD, PhD, Robert C. Doebele, MD, PhD

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 test was negative in 4 of 18 tested samples, and the RNA-based NGS test was negative in 3 of 19 tested samples. For all three testing approaches, we identified test 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.

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