Detection of Gene Rearrangements in Targeted Clinical Next-Generation Sequencing

The identification of recurrent gene rearrangements in the clinical laboratory is the cornerstone for risk stratification and treatment decisions in many malignant tumors. Studies have reported that targeted next-generation sequencing assays have the potential to identify such rearrangements; however, their utility in the clinical laboratory is unknown. We examine the sensitivity and specificity of ALKand KMT2A (MLL)rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALKrearranged cancers, six KMT2Arearranged leukemias, and 77 ALK/KMT2Arearrangement–negative cancers, previously tested by fluorescence in situ hybridization (FISH). Rearrangement detection was tested using publicly available software tools, including Breakdancer, ClusterFAST, CREST, and Hydra. Using Breakdancer and ClusterFAST, we detected ALKrearrangements in seven of seven FISH-positive cases and KMT2Arearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2AFISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALKrearranged case with a noncanonical intron 16 breakpoint, which is likely to affect its response to targeted inhibitors. We report that clinically relevant chromosomal rearrangements can be detected from targeted gene panel–based next-generation sequencing with sensitivity and specificity equivalent to that of FISH while providing finer-scale information and increased efficiency for molecular oncology testing.