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J Mol Diagn. 2014 Jul;16(4):405-17. doi: 10.1016/j.jmoldx.2014.03.006. Epub 2014 May 9.

Detection of gene rearrangements in targeted clinical next-generation sequencing.

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Department of Genetics, Washington University, St. Louis, Missouri.
Department of Pathology and Immunology, Washington University, St. Louis, Missouri.
Department of Laboratory Medicine, University of Washington, Seattle, Washington.
Department of Pathology, University of Utah and ARUP Laboratories, Salt Lake City, Utah.
ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
Department of Pathology and Immunology, Washington University, St. Louis, Missouri. Electronic address:


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 ALK and KMT2A (MLL) rearrangement detection by next-generation sequencing in the clinical laboratory. We analyzed a series of seven ALK rearranged cancers, six KMT2A rearranged leukemias, and 77 ALK/KMT2A rearrangement-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 ALK rearrangements in seven of seven FISH-positive cases and KMT2A rearrangements in six of six FISH-positive cases. Among the 77 ALK/KMT2A FISH-negative cases, no false-positive identifications were made by Breakdancer or ClusterFAST. Further, we identified one ALK rearranged 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.

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