Searching for mutations in the kinase domain (KD) of BCR-ABL causing drug resistance in patients with chronic myeloid leukemia by ultra-deep sequencing (UDS)


Adéla Broučková (1), Hana Klamová (1), Caterina De Benedittis (2), Michele Baccarani (2), Giovanni Martinelli (2), Simona Soverini (2), Kateřina Machová Poláková (1)


  1. Institute of Hematology and Blood Transfusion, Prague, Czech Republic
  2. Department of Hematology/Oncology L.e A. Seragnoli, University of Bologna, Bologna, Italy

Mutations in the KD of the fusion protein BCR-ABL are the most common mechanism of resistance to the treatment of chronic myeloid leukemia by tyrosinkinase inhibitors. An early knowledge of the type of mutation may have a crucial impact on the therapy modification. Until now it has been published more than 100 types of mutations in the BCR-ABL KD in relation to the disease treatment.
Currently, the Sanger sequencing for mutation detection in the cDNA of BCR-ABL KD is used as the gold standard. The sensitivity of the Sanger sequencing is approximately 20% of mutation present in the sample with a minimum of 100 copies of BCR-ABL transcripts. UDS is based on the clonal amplification of each molecule and its subsequent sequencing on the principle of pyrosequencing. Thus, a huge amount of reads of every single molecule is achieved giving a higher sensitivity than Sanger sequencing.
Retrospectively, we analyzed altogether 40 samples from 4 patients who developed KD mutations in BCR-ABL during the first line treatment with imatinib. We followed the kinetics of mutations from diagnosis to the development of resistance to imatinib. In patients who were subsequently switched to the treatment with second-generation inhibitors, we monitored the rate of eradication of the mutant clone and the presence of other, hidden mutations. Interestingly, at the time of diagnosis, we detected a series of minor variants, similarly as we found in the kinase domain of non-fused ABL gene of healthy donors. Furthermore, we found that using UDS we are able to detect mutations causing resistance to the treatment much earlier (by more than 1 year) than with Sanger sequencing.
From our preliminary data it seems that UDS is a very capable tool for the detection of BCR-ABL KD mutations at ultra-low levels and to detect mutations in the samples with less than 100 copies of BCR-ABL transcript. UDS may provide very early information about a possible drug resistance development with an impact in the therapy individualization. To determine the impact of UDS of KD mutations in BCR-ABL into the clinical practice, as well as for understanding of mutations development in the leukemic clones we plan to retrospectively and prospectively analyze a broader set of patient samples.
Supported by MZČR IGA NT11555

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