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Oncogenes, Fusion Genes and Tumor Suppressor Genes

Flow cytometric immunobead assay for the detection of BCR–ABL fusion proteins in leukemia patients

Leukemia volume 23, pages 1106–1117 (2009)Cite this article

Abstract

BCR–ABL fusion proteins show increased signaling through their ABL tyrosine kinase domain, which can be blocked by specific inhibitors, thereby providing effective treatment. This makes detection of BCR–ABL aberrations of utmost importance for diagnosis, classification and treatment of leukemia patients. BCR–ABL aberrations are currently detected by karyotyping, fluorescence in situ hybridization (FISH) or PCR techniques, which are time consuming and require specialized facilities. We developed a simple flow cytometric immunobead assay for detection of BCR–ABL fusion proteins in cell lysates, using a bead-bound anti-BCR catching antibody and a fluorochrome-conjugated anti-ABL detection antibody. We noticed protein stability problems in lysates caused by proteases from mature myeloid cells. This problem could largely be solved by adding protease inhibitors in several steps of the immunobead assay. Testing of 145 patient samples showed fully concordant results between the BCR–ABL immunobead assay and reverse transcriptase PCR of fusion gene transcripts. Dilution experiments with BCR–ABL positive cell lines revealed sensitivities of at least 1%. We conclude that the BCR–ABL immunobead assay detects all types of BCR–ABL proteins in leukemic cells with high specificity and sensitivity. The assay does not need specialized laboratory facilities other than a flow cytometer, provides results within ∼4 h, and can be run in parallel to routine immunophenotyping.

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Acknowledgements

We thank Dr Jean Luc Sanne of the European Commission for his support in initiating and monitoring the EuroFlow project. We are grateful to Dr Frans Nauwelaers, Dr Alice Ho, Dr Charlene Bush-Donovan, Dr Rudi Varro, Dr Mark Herberger, Dr JoAnn Boland and Dr Vera Imper (BD Biosciences) for their continuous support and collaboration in the development of the BCR–ABL immunobead assay. We thank Thorsten Raff (Klinik Schleswig-Holstein, Kiel, Germany) for his support in the PCR studies of the German samples. We thank Mrs Bibi van Bodegom and Caroline Linker for secretarial support.

This study was supported by the European Commission through the STREP grant EU-FP6, LSHB-CT-2006-018708, entitled EuroFlow, ‘Flow cytometry for fast and sensitive diagnosis and follow-up of haematological malignancies’. Work carried out at Salamanca was supported in part by a grant (EST-08-90881) from the Fondo de Investigaciones Sanitarias (FIS) of the Instituto de Salud Carlos III (Ministerio de Ciencia e Innovación, Madrid, Spain).

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Authors and Affiliations

  1. Department of Immunology, Erasmus MC, Rotterdam, The Netherlands

    F Weerkamp, E Dekking, Y Y Ng, V H J van der Velden, P G Hoogeveen, K A J Brouwer-De Cock, A van der Linden, A L Noordijk, W M Comans-Bitter, F J T Staal & J J M van Dongen

  2. Dynomics, Rotterdam, The Netherlands

    F Weerkamp, E Dekking, Y Y Ng, K A J Brouwer-De Cock, A van der Linden & A L Noordijk

  3. BD Biosciences, San Jose, CA, USA

    H Wai & R Chen

  4. 2nd Department of Medicine, University Klinik Schleswig-Holstein, Kiel, Germany

    S Böttcher & M Brüggemann

  5. Dutch Childhood Oncology Group, The Hague, The Netherlands

    A J van der Sluijs & A Koning

  6. Department of Laboratory Medicine, Hematology, University Hospitals Leuven, Leuven, Belgium

    N Boeckx & N Van Poecke

  7. Department of Hematology, Instituto Portugues de Oncologia, Lisbon, Portugal

    P Lucio & A Mendonça

  8. Department of Pediatric Hematology and Oncology, Medical University of Silesia, Zabrze, Poland

    L Sedek & T Szczepański

  9. Department of Pediatric Hematology and Oncology, Charles University, Prague, Czechoslovakia

    T Kalina & M Kovac

  10. Department of Medicine, Cancer Research Centre (IBMCC-CSIC-USAL) and Cytometry Service, University of Salamanca, Salamanca, Spain

    J Flores-Montero & A Orfao

  11. Department of Hematology, Hôpital Necker, Paris, France

    E Macintyre & L Lhermitte

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  1. F Weerkamp
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On behalf of the EuroFlow Consortium (EU-FP6, LSHB-CT-2006-018708)

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Correspondence to J J M van Dongen.

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Weerkamp, F., Dekking, E., Ng, Y. et al. Flow cytometric immunobead assay for the detection of BCR–ABL fusion proteins in leukemia patients. Leukemia 23, 1106–1117 (2009). https://doi.org/10.1038/leu.2009.93

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