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Chronic myelogenous leukemia

Sensitivity and reliability of DNA-based mutation analysis by allele-specific digital PCR to follow resistant BCR-ABL1-positive cells

Leukemia volume 35, pages 2419–2423 (2021)Cite this article

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Mutations in the kinase domain (KD) of BCR-ABL1 are known causes of resistance to tyrosine kinase inhibitor (TKI) treatment of chronic myeloid leukemia (CML) and Ph-positive acute lymphoblastic leukemia (Ph+ ALL), and detection is routinely performed at the transcript level. Sequencing remains the preferential technology and enables the identification of mutations at any position in the KD. Next-generation sequencing (NGS) is nearly 1-log more sensitive than standard sequencing, thus significantly facilitating earlier detection of mutations [1,2,3]. Moreover, NGS recognizes the complexity of the mutation landscape and can distinguish between polyclonal and compound mutations, if certain conditions are followed [1, 4]. The most frequently used amplicon-based NGS approaches are based on sequencing of shorter overlapping PCR products covering the entire length of the KD, thus distant mutations that may co-occur within one cell cannot be reliably assessed. There is a possibility of sequencing of long-range PCR product of the whole KD of BCR-ABL1, however there were available limited approaches and capacities of NGS platforms [5]. The promising technology for reliable assessment of compound mutations in the KD of BCR-ABL1 are Oxford Nanopore technology or SMRT PacBio technology.

Determination of the level of the BCR-ABL1 KD mutation is important to follow the kinetics of resistant Ph+ cells and to therapeutically prevent progression [6]. However, an expression-based analysis of mutated BCR-ABL1 may not precisely reflect the quantity of the clones.

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Fig. 1: Comparison of mutated KD of BCR-ABL1 data at the DNA level by AS-ddPCR (BCR-ABL1 DNAmut) and at the mRNA level (BCR-ABL1 mRNAmut) by NGS in 122 samples.
Fig. 2: Clonal evolution of BCR-ABL1 mutated cells in vivo.

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Acknowledgements

This work was funded by the Czech Science Foundation with grant #18-18407S and institutional support #00023736 from Ministry of Health of Czech Republic. We would like to thank to David Kundrat (Institute of Hematology and Blood Transfusion) for the fishplot creation. We would like to acknowledge Pavel Semerak (CLIP laboratories, Faculty Hospital Motol and Second Faculty of Medicine, Charles University, Prague, CZ) for the technical support for single-cell FACS sorting of KCL-22 resistant clones.

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Authors and Affiliations

  1. Institute of Hematology and Blood Transfusion, Prague, Czech Republic

    Vaclava Polivkova, Adela Benesova, Hana Zizkova, Jitka Koblihova, Nikola Curik, Eliska Motlova, Hana Klamova, Cyril Salek & Katerina Machova Polakova

  2. Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic

    Nikola Curik & Katerina Machova Polakova

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  1. Vaclava Polivkova
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Contributions

VP performed majority of analysis and evaluated data; AB contributed to NGS analysis; HZ contributed to ddPCR analysis; JK contributed to panel sequencing and RNA-seq; NC performed in vitro analysis; EM contributed to ddPCR analysis of KCL-22; HK and CS evaluated and provided clinical data of patients; KMP designed the study, interpreted results and wrote the paper; all authors critically reviewed and approved the paper.

Corresponding author

Correspondence to Katerina Machova Polakova.

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KMP and CS—consultancy for Incyte and Angelini.

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This work was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Ethics Committees of the Institute of Hematology and Blood Transfusion, Prague. All patients provided written informed consent for the use of their samples for this research.

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Polivkova, V., Benesova, A., Zizkova, H. et al. Sensitivity and reliability of DNA-based mutation analysis by allele-specific digital PCR to follow resistant BCR-ABL1-positive cells. Leukemia 35, 2419–2423 (2021). https://doi.org/10.1038/s41375-021-01226-0

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