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Use of Denaturing HPLC for Detection of Mutations in the BCR-ABL Kinase Domain in Patients Resistant to Imatinib

¹ Leukaemia Research Fund Molecular Pharmacology Laboratory and² Department of Haematology, School of Clinical and Laboratory Sciences, The Medical School, University of Newcastle, Newcastle, UK;

^aaddress correspondence to this author at: Northern Institute for Cancer Research, Cookson Building, Medical School, Newcastle NE2 4HH, UK; fax 44-191-222-7556, e-mail j.a.e.irving@ncl.ac.uk

The first 300 words of the full text of this article appear below.

The ABL tyrosine kinase inhibitor imatinib (STI571, Glivec, Gleevec) has produced dramatic clinical responses in most patients with chronic myeloid leukemia (1)(2)(3)(4). However, drug resistance has arisen as a result of increased expression of the BCR-ABL oncogene or the emergence of clones of cells harboring mutations in the ABL portion of the gene that reduce drug binding while retaining aberrant kinase activity (5). Original observations suggested that one specific substitution, T315I, was responsible (6). However, subsequent reports have demonstrated that this is not the case (7)(8)(9)(10)(11)(12)(13).

To monitor the emergence of drug resistance in patients treated with imatinib, there is a need to develop a reliable method for the screening of mutations in the BCR-ABL oncogene (5)(7). We report here the use of denaturing HPLC (DHPLC) as a method to screen for mutations in exons 4 and 6 of the ABL gene. Although originally described more than 6 years ago, the widespread application of this technique has only recently become possible with the introduction of commercially available specialized HPLC instrumentation dedicated to the performance of mutation analysis (14). Previous reports have demonstrated that the sensitivity and specificity of the technique are consistently high (15)(16).

Peripheral blood samples were obtained, after receipt of informed consent, from 22 patients with accelerated phase or blast crisis chronic myeloid leukemia. Genomic DNA from control samples was obtained from the Westlakes Research Institute at Cumbria after appropriate ethical consent. Genomic DNA was extracted by a standard phenol-chloroform method. PCR was performed with 50–100 ng of DNA, 2.5 mM MgCl₂, 100 µM deoxynucleotide triphosphates, 0.2 µM each of forward . . . [Full Text of this Article]

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