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Dynamics of the emergence of dasatinib and nilotinib resistance in imatinib-resistant CML patients

Leukemia volume 26, pages 172–177 (2012)Cite this article

In an important minority of patients with chronic myeloid leukaemia (CML) on imatinib therapy, resistance has been observed, in most cases due to selection of cell clone variants that express BCR–ABL mutants with weakened imatinib-binding interactions. As part of efforts to combat resistance, second-generation tyrosine kinase inhibitors (TKIs) have been developed that are optimised for binding to mutated, imatinib-resistant BCR–ABL variants (dasatinib and nilotinib).1, 2 However, these drugs also apply selection pressure on BCR–ABL clones and can generate resistance, whereby the spectrum of resistance mutations is distinct from, but can overlap with, that of imatinib.3, 4 Thus, upon shifting treatment to a new drug, secondary ‘escape-mutant’ clones of BCR–ABL-positive cells may emerge during treatment to become predominant in the total leukaemic population. In this scenario, heterogeneous cancer cell populations co-exist and undergo both selective and deselective processes upon drug exposure, but may not be eradicated completely even if a single clone achieves dominance. In this study, we evaluate the dynamics of variant BCR–ABL clones upon exposure to second-line drugs. The analysis of imatinib-resistant subjects at transition from first- to second-line TKI appeared particularly attractive for study, as the situation at the initiation of the salvage therapy comprises multiple BCR–ABL clones with diverse degrees of resistance that could be traced upon drug exposure.

The study was conducted as a retrospective, molecular analysis of 200 blood samples from 40 randomly selected CML patients that had been treated with 400–800 mg of imatinib and had acquired imatinib resistance as diagnosed by European LeukemiaNet (ELN) recommendations.5 The patient data, derived from a comparative analysis of four genotyping approaches published recently,6 comprise analyses of blood RNA samples taken before and 3, 6, 9 and 12 months after onset of second TKI. Complementary DNA synthesis was performed using random hexamer and MMLV reverse transcriptase.7 BCR–ABL and beta-glucuronidase (GUS) transcripts were quantified as described previously.8 Importantly, for the current study, mutations were considered to be significant only if detected by two independent methods (D-HPLC/DS, ARMS or L-PCR). Findings from only one method were excluded from further analysis. The significant mutations were analysed by quantitative real-time ARMS-PCR as described previously9, 10 to determine the copy number of mutated transcripts. According to procedures established for assessment of leukaemic burden in CML (ratio BCR–ABL/GUS), mutated transcript burden was expressed as the ratio BCR–ABLmutated/GUS in percent. As all PCR reactions used in analysis were nearly 100% efficient,9, 10 the calculated ratios were directly comparable, allowing a direct overlay of the resulting curves. Ratios derived from mutational analysis describe the absolute amount of transcription per cells harbouring a resistance mutation, rather than expressing resistance in relation to BCR–ABL-positive cells. The latter represents the most common way of analysing resistance in CML. Thus, following this novel approach we were able to describe absolute changes of the resistance burden conferred by a specific mutation (analysed by the BCR–ABLmutant/GUS ratio) in direct relation to changes in leukaemic burden (analysed by the BCR–ABLtotal/GUS ratio).

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Author notes

  1. F X Gruber and T Ernst: These authors contributed equally to this study.

Authors and Affiliations

  1. Department of Pharmacy, Section of Pharmacology, University of Tromsø, Tromsø, Norway

    F X Gruber & I Mikkola

  2. Abteilung Hämatologie/Onkologie, Klinik für Innere Medizin II, Universitätsklinikum Jena, Jena, Germany

    T Ernst & A Hochhaus

  3. University Hospital of Helsinki, Helsinki, Finland

    K Porkka

  4. Department of Chemistry, The Norwegian Structural Biology Centre, University of Tromsø, Tromsø, Norway

    F X Gruber & R A Engh

  5. Abteilung für Hämatologie, Onkologie und Hämostaseologie, Universitätsklinikum Leipzig, Leipzig, Germany

    J Maier & T Lange

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Correspondence to F X Gruber.

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Gruber, F., Ernst, T., Porkka, K. et al. Dynamics of the emergence of dasatinib and nilotinib resistance in imatinib-resistant CML patients. Leukemia 26, 172–177 (2012). https://doi.org/10.1038/leu.2011.187

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