title: Detection of BCR-ABL1 mutations that confer tyrosine kinase inhibitor resistance using massively parallel, next generation sequencing.
abstract: Detection of BCR-ABL1 mutations that confer resistance to tyrosine kinase inhibitors is important for management of patients with t(9;22);BCR-ABL1-positive (Ph+) leukemias. Testing is often performed using Sanger sequencing (SS) which has relatively poor sensitivity. Given the widespread adoption of next generation sequencing (NGS), we sought to reevaluate the testing in the context of NGS methods. We developed an NGS-based BCR-ABL1 mutation test on the Ion Torrent Personal Genome Machine (PGM) to test for resistance mutations, primarily in the kinase domain in BCR-ABL1. We analyzed 508 clinical samples from patients with Ph+ leukemias. In a subset of these samples (n = 97), we conducted a comparison of the NGS results to a classical SS-based test. NGS facilitated detection of low-level mutations (<20 % allele frequency) that were not detectable by SS. In a subset of cases with multiple mutations, NGS was also able to determine if two mutations were on the same molecule (compound) or on separate molecules (polyclonal) but this was limited by the distance between mutated positions and by the effects of apparent distance-dependent PCR recombination. We found 22 compound mutations that centered on one or two key residues including two novel compound mutants: Q252H/Y253H and F311Y/F359I. The advantages of NGS make it a superior method for inventorying BCR-ABL1 resistance mutations. However, data analysis may be complicated by short read lengths and the effects of PCR recombination.
authors.affiliation: ARUP Laboratories, Salt Lake City, UT, USA.
authors.forename: Philippe
authors.initials: P
authors.lastname: Szankasi
authors.affiliation: ARUP Laboratories, Salt Lake City, UT, USA.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Schumacher
authors.affiliation: Department of Pathology, University of Utah, Salt Lake City, UT, 84112, USA. todd.kelley@path.utah.edu.
authors.forename: Todd W
authors.identifier: http://orcid.org/0000-0002-3620-7017
authors.initials: TW
authors.lastname: Kelley
mesh_terms: D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D005787:Gene Frequency / Q000235:genetics; D006801:Humans; D009154:Mutation / Q000235:genetics*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C581634:BCR-ABL1 fusion protein, human; D047428:Protein Kinase Inhibitors; D016044:Fusion Proteins, bcr-abl
keywords: BCR-ABL1; Chronic myelogenous leukemia; Lymphoblastic leukemia; Molecular testing; Next generation sequencing; Philadelphia chromosome; Tyrosine kinase inhibitors