title: Next-generation deep sequencing improves detection of BCR-ABL1 kinase domain mutations emerging under tyrosine kinase inhibitor treatment of chronic myeloid leukemia patients in chronic phase.
abstract: OBJECTIVE
  Here, we studied whether amplicon next-generation deep sequencing (NGS) could improve the detection of emerging BCR-ABL1 kinase domain mutations in chronic phase chronic myeloid leukemia (CML) patients under tyrosine kinase inhibitor (TKI) treatment and discussed the clinical relevance of such sensitive mutational detection.
  
  
  METHODS
  For NGS data evaluation including extraction of biologically relevant low-level variants from background error noise, we established and applied a robust and versatile bioinformatics approach.
  
  
  RESULTS
  Results from a retrospective longitudinal analysis of 135 samples of 15 CML patients showed that NGS could have revealed emerging resistant mutants 2-11 months earlier than conventional sequencing. Interestingly, in cases who later failed first-line imatinib treatment, NGS revealed that TKI-resistant mutations were already detectable at the time of major or deeper molecular response. Identification of emerging mutations by NGS was mirrored by BCR-ABL1 transcript level expressed either fluctuations around 0.1 %(IS) or by slight transcript level increase. NGS also allowed tracing mutations that emerged during second-line TKI therapy back to the time of switchover. Compound mutants could be detected in three cases, but were not found to outcompete single mutants.
  
  
  CONCLUSIONS
  This work points out, that next-generation deep sequencing, coupled with a robust bioinformatics approach for mutation calling, may be just in place to ensure reliable detection of emerging BCR-ABL1 mutations, allowing early therapy switch and selection of the most appropriate therapy. Further, prospective assessment of how to best integrate NGS in the molecular monitoring and clinical decision algorithms is warranted.
authors.affiliation: Institute of Hematology and Blood Transfusion, Prague, Czech Republic, katerina.machova@uhkt.cz.
authors.forename: Katerina
authors.initials: K
authors.lastname: Machova Polakova
authors.forename: Vojtech
authors.initials: V
authors.lastname: Kulvait
authors.forename: Adela
authors.initials: A
authors.lastname: Benesova
authors.forename: Jana
authors.initials: J
authors.lastname: Linhartova
authors.forename: Hana
authors.initials: H
authors.lastname: Klamova
authors.forename: Monika
authors.initials: M
authors.lastname: Jaruskova
authors.forename: Caterina
authors.initials: C
authors.lastname: de Benedittis
authors.forename: Torsten
authors.initials: T
authors.lastname: Haferlach
authors.forename: Michele
authors.initials: M
authors.lastname: Baccarani
authors.forename: Giovanni
authors.initials: G
authors.lastname: Martinelli
authors.forename: Tomas
authors.initials: T
authors.lastname: Stopka
authors.forename: Thomas
authors.initials: T
authors.lastname: Ernst
authors.forename: Andreas
authors.initials: A
authors.lastname: Hochhaus
authors.forename: Alexander
authors.initials: A
authors.lastname: Kohlmann
authors.forename: Simona
authors.initials: S
authors.lastname: Soverini
mesh_terms: D000328:Adult; D000368:Aged; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001549:Benzamides / Q000627:therapeutic use*; D019295:Computational Biology; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000068877:Imatinib Mesylate; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000235:genetics*; D008137:Longitudinal Studies; D008297:Male; D008875:Middle Aged; D009154:Mutation / Q000187:drug effects*; D010879:Piperazines / Q000627:therapeutic use*; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011743:Pyrimidines / Q000627:therapeutic use*; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl