A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing

J Mol Diagn. 2016 Mar;18(2):165-75. doi: 10.1016/j.jmoldx.2015.10.002. Epub 2015 Dec 30.


Chromosomal rearrangements that result in oncogenic gene fusions are clinically important drivers of many cancer types. Rapid and sensitive methods are therefore needed to detect a broad range of gene fusions in clinical specimens that are often of limited quantity and quality. We describe a next-generation sequencing approach that uses a multiplex PCR-based amplicon panel to interrogate fusion transcripts that involve 19 driver genes and 94 partners implicated in solid tumors. The panel also includes control assays that evaluate the 3'/5' expression ratios of 12 oncogenic kinases, which might be used to infer gene fusion events when the partner is unknown or not included on the panel. There was good concordance between the solid tumor fusion gene panel and other methods, including fluorescence in situ hybridization, real-time PCR, Sanger sequencing, and other next-generation sequencing panels, because 40 specimens known to harbor gene fusions were correctly identified. No specific fusion reads were observed in 59 fusion-negative specimens. The 3'/5' expression ratio was informative for fusions that involved ALK, RET, and NTRK1 but not for BRAF or ROS1 fusions. However, among 37 ALK or RET fusion-negative specimens, four exhibited elevated 3'/5' expression ratios, indicating that fusions predicted solely by 3'/5' read ratios require confirmatory testing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brain / physiology
  • Colon / physiology
  • Gene Expression Profiling / methods
  • Gene Fusion*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Limit of Detection
  • Lung / physiology
  • Multiplex Polymerase Chain Reaction / methods
  • Neoplasms / genetics*
  • Paraffin Embedding
  • Real-Time Polymerase Chain Reaction / methods
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Workflow