Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter to the Editor
  • Published:

Quantitative monitoring of cell clones carrying point mutations in the BCR–ABL tyrosine kinase domain by ligation-dependent polymerase chain reaction (LD–PCR)

Leukemia volume 22, pages 1956–1961 (2008)Cite this article

Tyrosine kinase (TK) inhibitors directed against the BCR–ABL TK domain have become the standard of treatment in patients with chronic myeloid leukemia (CML). In a proportion of patients, however, resistance to TK inhibitors may occur, which, in the case of imatinib, has been associated with point mutations within the ABL TK domain in 40–90% of instances, depending on the CML phase, the methodology of detection and the definition of resistance.1 Mutations conferring resistance frequently occur at the ATP-binding site (P-loop) or at other sites directly affecting drug binding or conformation of the kinase (activation loop, catalytic domain). The level of resistance to individual TK inhibitors may vary from slightly reduced sensitivity to total insensitivity. In patients receiving treatment with imatinib, mutations conferring only moderately impaired sensitivity may be approached by increasing the dose, while the presence of mutations associated with a high level of resistance provides an indication for switching treatment to second generation compounds, such as dasatinib or nilotinib.2 Point mutations reported to confer high levels of resistance to imatinib include for example, Y253F/H, E255K/V, T315I, or H396P/R.2 Dasatinib and nilotinib were shown to be instrumental against most of these mutations,3 but the AA position 315 ( → T315I) remains problematic because none of the currently approved TK inhibitors are effective in this instance.4 Moreover, recent data indicate that a range of specific mutations other than the T315I may occur before and during treatment with dasatinib (V299L, T315A, F317I/S/V, F317L) or nilotinib (Y253H, E255V, F359C).5 These observations underscore the importance of monitoring patients on treatment with TK inhibitors for the presence of mutations. A number of studies in CML patients report on the occurrence of mutations in the entire BCR–ABL TK domain. Recent studies in patients treated with imatinib indicate that E250K, Y253F, E255K, T315I, M351T and F359V account for 60–70% of all mutations. Mutations located within the P-loop appear to be most frequent and are suggested to correlate with poor outcome.6 A number of methods have been employed to detect mutant clones including, for example, PCR amplification coupled with direct sequencing of the BCR–ABL TK domain, high-performance liquid chromatography, the SEQUENOM MassARRAY system, or allele-specific oligonucleotide-PCR amplification. These techniques display different detection limits for the identification of mutant clones ranging from 0.01–30%. Detection of a mutation within the ABL TK domain does not necessarily imply impending onset of clinically resistant disease, particularly if the size of a mutant clone is small.7 Mutant clones have been reported to disappear spontaneously which may, at least in part, be attributable to the occurrence of mutations in cells with restricted proliferative capacity.8, 9 Qualitative methods for mutational analysis may therefore have limited potential to reliably assess the risk of clinically resistant disease, especially if mutant clones present at very low levels are detected.8 It is currently a matter of discussion whether the detection of small mutant BCR–ABL clones, for example, below the level of 1%, is clinically useful. The clinical benefit of sensitive techniques for mutational analysis could be increased if the size of mutant clones could be monitored, to facilitate timely detection of clonally expanding mutant cells during treatment.8 Current approaches to assessing the size of mutant clones include for example, pyrosequencing,9 SEQUENOM MassARRAY analysis,10 or the polymerase colony assay.11 These techniques, however, have limitations with regard to broad application in clinical diagnosis, such as the requirement of very expensive equipment,10 rather complex and laborious design,11 or the apparent inability to quantify mutant clones in the range below 20%.9 We have therefore established a relatively simple method facilitating quantitative analysis of single nucleotide polymorphisms. The technique is based on ligation-dependent competitive PCR (LD–PCR) and represents a modification of the MLPA technology.12 The LD–PCR technique presented displays a detection limit for BCR–ABL TK mutations in a range of apparent clinical relevance, and permits accurate quantitative analysis of mutant clones. In a pilot study, we demonstrate that the technique can be readily applied to the detection and monitoring of clones exhibiting specific mutations within the BCR–ABL TK domain in patients with CML.

The panel of quantification assays presented covers 18 different mutations including all important base substitutions located in the P-loop of the tyrosine kinase domain, and other mutations known to confer a high level of resistance to imatinib, dasatinib and nilotinib (Table 1). The current panel of LD–PCR assays permits detection and quantitative monitoring of the following mutations: M244V, L248V, V299L-C, V299L-T, G250E, Q252H-C, Q252H-T, Y253F, Y253H, E255K, T315A, T315I, F317C, F317I, F317L-A, F317L-G, F317V and M351T (Table 1).

Table 1 LD–PCR detection systems for 18 mutations in the BCR–ABL TK domain
Full size table

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3

References

  1. Quintas-Cardama A, Kantarjian H, Cortes J . Flying under the radar: the new wave of BCR-ABL inhibitors. Nat Rev Drug Discov 2007; 6: 834–848.

    Article  CAS  PubMed  Google Scholar 

  2. Hehlmann R, Hochhaus A, Baccarani M . Chronic myeloid leukaemia. Lancet 2007; 370: 342–350.

    Article  CAS  PubMed  Google Scholar 

  3. Hochhaus A, Kantarjian HM, Baccarani M, Lipton JH, Apperley JF, Druker BJ et al. Dasatinib induces notable hematologic and cytogenetic responses in chronic-phase chronic myeloid leukemia after failure of imatinib therapy. Blood 2007; 109: 2303–2309.

    Article  CAS  PubMed  Google Scholar 

  4. Shah NP, Tran C, Lee FY, Chen P, Norris D, Sawyers CL . Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 2004; 305: 399–401.

    Article  CAS  PubMed  Google Scholar 

  5. Soverini S, Gnani A, Colarossi S, Castagnetti F, Palandri F, Giannoulia P et al. Philadelphia chromosome-positive leukemia patients who harbour imatinib-resistant mutations have a higher likelihood of developing additional mutations associated with resistance to novel tyrosine kinase inhibitors. Blood 2007; 110 abstract [322].

  6. Soverini S, Martinelli G, Rosti G, Bassi S, Amabile M, Poerio A et al. ABL mutations in late chronic phase chronic myeloid leukemia patients with up-front cytogenetic resistance to imatinib are associated with a greater likelihood of progression to blast crisis and shorter survival: a study by the GIMEMA Working Party on Chronic Myeloid Leukemia. J Clin Oncol 2005; 23: 4100–4109.

    Article  CAS  PubMed  Google Scholar 

  7. Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations and for expressing results. Blood 2006; 108: 28–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Willis SG, Lange T, Demehri S, Otto S, Crossman L, Niederwieser D et al. High-sensitivity detection of BCR-ABL kinase domain mutations in imatinib-naive patients: correlation with clonal cytogenetic evolution but not response to therapy. Blood 2005; 106: 2128–2137.

    Article  CAS  PubMed  Google Scholar 

  9. Khorashad JS, Anand M, Marin D, Saunders S, Al-Jabary T, Iqbal A et al. The presence of a BCR-ABL mutant allele in CML does not always explain clinical resistance to imatinib. Leukemia 2006; 20: 658–663.

    Article  CAS  PubMed  Google Scholar 

  10. Vivante A, Amariglio N, Koren-Michowitz M, Ashur-Fabian O, Nagler A, Rechavi G et al. High-throughput, sensitive and quantitative assay for the detection of BCR-ABL kinase domain mutations. Leukemia 2007; 21: 1318–1321.

    Article  CAS  PubMed  Google Scholar 

  11. Nardi V, Raz T, Cao X, Wu CJ, Stone RM, Cortes J et al. Quantitative monitoring by polymerase colony assay of known mutations resistant to ABL kinase inhibitors. Oncogene 2008; 27: 775–782, .

    Article  CAS  PubMed  Google Scholar 

  12. Schouten JP, McElgunn CJ, Waaijer R, Zwijnenburg D, Diepvens F, Pals G . Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification. Nucleic Acids Res 2002; 30: e57.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Lundberg E, De Mare J . Interval estimates in the spectroscopy calibration problem. Scand J Statistics 1980; 7: 40–42.

    Google Scholar 

  14. Branford S, Hughes T . Detection of BCR-ABL mutations and resistance to imatinib mesylate. Methods Mol Med 2006; 125: 93–106.

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Molecular Microbiology and Development of Genetic Diagnostics, Children's Cancer Research Institute (CCRI), Vienna, Austria

    S Preuner, D Denk, M Nesslboeck & T Lion

  2. Department of Statistics and Decision Support, University of Vienna, Austria

    F Frommlet

Authors
  1. S Preuner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. D Denk
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. F Frommlet
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Nesslboeck
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. T Lion
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to T Lion.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Preuner, S., Denk, D., Frommlet, F. et al. Quantitative monitoring of cell clones carrying point mutations in the BCR–ABL tyrosine kinase domain by ligation-dependent polymerase chain reaction (LD–PCR). Leukemia 22, 1956–1961 (2008). https://doi.org/10.1038/leu.2008.97

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2008.97

This article is cited by

Search

Advanced search

Quick links