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  • Biotechnical Methods Section BTS
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Biotechnical Methods Section (BTS)

Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography

Leukemia volume 18, pages 864–871 (2004)Cite this article

Abstract

Mutations of the ABL kinase domain (KD) are common in patients with chronic myelogenous leukemia (CML) who develop resistance to imatinib. We developed an RT-PCR-based denaturing high-performance liquid chromatography (D-HPLC) assay to detect mutations of the ABL KD. Validation experiments using mixtures of wild type and mutant amplicons showed that the D-HPLC assay could detect mutant transcripts when they represented at least 15% of the total, and was thus twice as sensitive as automated sequencing. When D-HPLC was applied to 30 cDNAs from patients with imatinib resistance that had previously been characterized for KD mutations by direct sequencing of BCR-ABL RT-PCR products, there was concordance in 97% of samples. Resequencing confirmed the original mutations in all cases. In addition, sequencing of individual clones detected a mutation in one sample that had been mutation-positive by D-HPLC but wild type by conventional sequencing. In serial samples from the same individuals, D-HPLC detected mutations as early as 260 days before hematological relapse. D-HPLC is suitable for routine clinical monitoring of CML patients for emergence of KD mutations and may be useful for optimizing therapy. Early detection of emerging mutant clones may aid in guiding decisions regarding alternative treatment options.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (Grants K24 CA82445 and 1R01 CA65823) (BJD), the Leukemia and Lymphoma Society (BJD) and the VA Merit Review System (MCH).

We thank Gerlinde Patzer and Dr Haifa-Kathrin Al-Ali, University of Leipzig, Germany, for help with sequencing and dedicated patient care, respectively.

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Authors and Affiliations

  1. Oregon Health and Science University Cancer Institute, Portland, OR, USA

    M W N Deininger, S Willis, T M Bainbridge, B J Druker & M C Heinrich

  2. Portland VA Medical Center, Oregon Health and Science University Cancer Institute, Portland, OR, USA

    L McGreevey, T M Bainbridge & M C Heinrich

  3. Howard Hughes Medical Institute, Portland, OR, USA

    B J Druker

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  1. M W N Deininger
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  2. L McGreevey
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  3. S Willis
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  4. T M Bainbridge
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  5. B J Druker
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  6. M C Heinrich
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Correspondence to M W N Deininger.

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Deininger, M., McGreevey, L., Willis, S. et al. Detection of ABL kinase domain mutations with denaturing high-performance liquid chromatography. Leukemia 18, 864–871 (2004). https://doi.org/10.1038/sj.leu.2403307

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