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Highly sensitive and quantitative detection of BCR-ABL kinase domain mutations by ligation PCR

Leukemia volume 22, pages 2288–2291 (2008)Cite this article

Acquired resistance to imatinib in patients with chronic myeloid leukaemia (CML) or Philadelphia-positive (Ph+) acute lymphoblastic leukaemia frequently arises due to point mutations of the BCR-ABL kinase domain (KD)1, 2 so that, as new inhibitors with differential activity against KD mutant BCR-ABL become available, early detection and precise quantification of key mutations are likely to make an increasingly important contribution to patient-specific treatment decisions. A number of approaches have been developed in recent years to detect these mutations based on the techniques of DNA sequencing, denaturing high-performance liquid chromatography (DHPLC), mutation-specific PCR and mass array genotyping. However, although some of these assays can detect mutants in wild-type allele frequencies as low as 0.1% and others are quantitative, the combination of sensitivity and quantification has remained elusive. Reliable quantification has been provided to date by pyrosequencing, which has a detection limit of around 10% mutants in wild-type allele, and by some PCR-based approaches.3 The potential importance of the quantitative detection of low-level mutations has become clear from the use of PCR-based approaches using mutation-specific primers, which have identified low-level KD mutations in the majority of Ph+ acute lymphoblastic leukaemia patients before therapy and revealed that almost all of those harbouring a low-level T315I or p-loop mutation at diagnosis relapsed with the same mutation, even after achieving a molecular remission on imatinib and chemotherapy.2 Similar techniques have identified low-level, pretherapeutic KD mutations in CML, although the relevance to resistance and progression remains unclear.4 Accurate quantification of low-level mutations is therefore likely to make an important contribution to our understanding and monitoring of disease progression.

In an attempt to combine high sensitivity and reliable quantification, we have now adapted the principle of allele-specific PCR by adding a ligation step to increase the specificity of the assay for the detection of mutant versus wild-type BCR-ABL. This allows the subsequent amplification procedure to be optimized for sensitivity without affecting specificity. On the basis of this development, we report here a new, highly sensitive and quantitative technique to detect BCR-ABL KD mutations and apply the technique for the detection of the key mutations, T315I (codon change ACT to ATT) and E255K (codon change GAG to AGG).

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Acknowledgements

We thank Dr K Krohn (Core unit ‘DNA Technologies’, IZKF Leipzig, Germany) for the sequence analysis. This work was supported by Novartis Pharma GmbH (Nürnberg, Germany).

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Authors and Affiliations

  1. Department of Hematology/Oncology, University of Leipzig, Leipzig, Germany

    O Pelz-Ackermann, M Cross, S-Y Wang, H K Al-Ali, D Niederwieser & T Lange

  2. Department of Hematology/Oncology, Center for Internal Medicine, Johann Wolfgang Goethe University, Frankfurt/Main, Germany

    H Pfeifer

  3. Oregon Health & Science University Cancer Institute, Portland, OR, USA. E-mail: langet@medizin.uni-leipzig.de,

    M Deininger

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  1. O Pelz-Ackermann
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Pelz-Ackermann, O., Cross, M., Pfeifer, H. et al. Highly sensitive and quantitative detection of BCR-ABL kinase domain mutations by ligation PCR. Leukemia 22, 2288–2291 (2008). https://doi.org/10.1038/leu.2008.180

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