title: Clonal distribution of BCR-ABL1 mutations and splice isoforms by single-molecule long-read RNA sequencing.
abstract: BACKGROUND
  The evolution of mutations in the BCR-ABL1 fusion gene transcript renders CML patients resistant to tyrosine kinase inhibitor (TKI) based therapy. Thus screening for BCR-ABL1 mutations is recommended particularly in patients experiencing poor response to treatment. Herein we describe a novel approach for the detection and surveillance of BCR-ABL1 mutations in CML patients.
  
  
  METHODS
  To detect mutations in the BCR-ABL1 transcript we developed an assay based on the Pacific Biosciences (PacBio) sequencing technology, which allows for single-molecule long-read sequencing of BCR-ABL1 fusion transcript molecules. Samples from six patients with poor response to therapy were analyzed both at diagnosis and follow-up. cDNA was generated from total RNA and a 1,6 kb fragment encompassing the BCR-ABL1 transcript was amplified using long range PCR. To estimate the sensitivity of the assay, a serial dilution experiment was performed.
  
  
  RESULTS
  Over 10,000 full-length BCR-ABL1 sequences were obtained for all samples studied. Through the serial dilution analysis, mutations in CML patient samples could be detected down to a level of at least 1%. Notably, the assay was determined to be sufficiently sensitive even in patients harboring a low abundance of BCR-ABL1 levels. The PacBio sequencing successfully identified all mutations seen by standard methods. Importantly, we identified several mutations that escaped detection by the clinical routine analysis. Resistance mutations were found in all but one of the patients. Due to the long reads afforded by PacBio sequencing, compound mutations present in the same molecule were readily distinguished from independent alterations arising in different molecules. Moreover, several transcript isoforms of the BCR-ABL1 transcript were identified in two of the CML patients. Finally, our assay allowed for a quick turn around time allowing samples to be reported upon within 2 days.
  
  
  CONCLUSIONS
  In summary the PacBio sequencing assay can be applied to detect BCR-ABL1 resistance mutations in both diagnostic and follow-up CML patient samples using a simple protocol applicable to routine diagnosis. The method besides its sensitivity, gives a complete view of the clonal distribution of mutations, which is of importance when making therapy decisions.
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. lucia.cavelier@igp.uu.se.
authors.forename: Lucia
authors.initials: L
authors.lastname: Cavelier
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. adam.ameur@igp.uu.se.
authors.forename: Adam
authors.initials: A
authors.lastname: Ameur
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. susana.haggqvist@igp.uu.se.
authors.forename: Susana
authors.initials: S
authors.lastname: Häggqvist
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. Ida.hoijer@igp.uu.se.
authors.forename: Ida
authors.initials: I
authors.lastname: Höijer
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. nicola.cahill@igp.uu.se.
authors.forename: Nicola
authors.initials: N
authors.lastname: Cahill
authors.affiliation: Department of Medical Sciences, Haematology, Uppsala University, Uppsala, Sweden. ulla.olsson-stromberg@medsci.uu.se.
authors.forename: Ulla
authors.initials: U
authors.lastname: Olsson-Strömberg
authors.affiliation: Department of Immunology, Genetics and Pathology, Science for Life Laboratory, Uppsala University, Uppsala, Sweden. monica.hermanson@igp.uu.se.
authors.forename: Monica
authors.initials: M
authors.lastname: Hermanson
mesh_terms: D000328:Adult; D000368:Aged; D000483:Alleles; D017398:Alternative Splicing; D060965:Clonal Evolution / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005260:Female; D016044:Fusion Proteins, bcr-abl / Q000235:genetics*; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D059368:RNA Isoforms; D012680:Sensitivity and Specificity; D017423:Sequence Analysis, RNA
chemical_list: D047428:Protein Kinase Inhibitors; D059368:RNA Isoforms; D016044:Fusion Proteins, bcr-abl