title: Detecting Gene Rearrangements in Patient Populations Through a 2-Step Diagnostic Test Comprised of Rapid IHC Enrichment Followed by Sensitive Next-Generation Sequencing.
abstract: Targeted therapy combined with companion diagnostics has led to the advancement of next-generation sequencing (NGS) for detection of molecular alterations. However, using a diagnostic test to identify patient populations with low prevalence molecular alterations, such as gene rearrangements, poses efficiency, and cost challenges. To address this, we have developed a 2-step diagnostic test to identify NTRK1, NTRK2, NTRK3, ROS1, and ALK rearrangements in formalin-fixed paraffin-embedded clinical specimens. This test is comprised of immunohistochemistry screening using a pan-receptor tyrosine kinase cocktail of antibodies to identify samples expressing TrkA (encoded by NTRK1), TrkB (encoded by NTRK2), TrkC (encoded by NTRK3), ROS1, and ALK followed by an RNA-based anchored multiplex polymerase chain reaction NGS assay. We demonstrate that the NGS assay is accurate and reproducible in identification of gene rearrangements. Furthermore, implementation of an RNA quality control metric to assess the presence of amplifiable nucleic acid input material enables a measure of confidence when an NGS result is negative for gene rearrangements. Finally, we demonstrate that performing a pan-receptor tyrosine kinase immunohistochemistry staining enriches detection of the patient population for gene rearrangements from 4% to 9% and has a 100% negative predictive value. Together, this 2-step assay is an efficient method for detection of gene rearrangements in both clinical testing and studies of archival formalin-fixed paraffin-embedded specimens.
authors.affiliation: *Ignyta Inc., San Diego, CA †ArcherDX Inc., Boulder, CO ‡Samsung Medical Center, Sungkyunkwan University School of Medicine, Kangnamgu, Seoul, Korea.
authors.forename: Danielle A
authors.initials: DA
authors.lastname: Murphy
authors.forename: Heather A
authors.initials: HA
authors.lastname: Ely
authors.forename: Robert
authors.initials: R
authors.lastname: Shoemaker
authors.forename: Aaron
authors.initials: A
authors.lastname: Boomer
authors.forename: Brady P
authors.initials: BP
authors.lastname: Culver
authors.forename: Ian
authors.initials: I
authors.lastname: Hoskins
authors.forename: Josh D
authors.initials: JD
authors.lastname: Haimes
authors.forename: Ryan D
authors.initials: RD
authors.lastname: Walters
authors.forename: Diane
authors.initials: D
authors.lastname: Fernandez
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Stahl
authors.forename: Jeeyun
authors.initials: J
authors.lastname: Lee
authors.forename: Kyoung-Mee
authors.initials: KM
authors.lastname: Kim
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lamoureux
authors.forename: Jason
authors.initials: J
authors.lastname: Christiansen
mesh_terms: D005260:Female; D015321:Gene Rearrangement; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D008297:Male; D016612:Paraffin Embedding; D013194:Staining and Labeling / Q000379:methods*