Denaturing-HPLC-Based Assay for Detection of ABL Mutations in Chronic Myeloid Leukemia Patients Resistant to Imatinib

Clinical Chemistry - Tập 50 Số 7 - Trang 1205-1213 - 2004
Simona Soverini1,2, Giovanni Martinelli2, Marilina Amabile2, Angela Poerio2, Michele Bianchini2, Gianantonio Rosti2, Fabrizio Pane3, Giuseppe Saglio4, Michele Baccarani2
1Center for Applied Biomedical Research (CRBA), St. Orsola-Malpighi Hospital, Bologna, Italy
2Institute of Hematology and Medical Oncology “Seràgnoli”, University of Bologna, Bologna, Italy
3CEINGE Advanced Biotechnologies and Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Naples, Italy
4Division of Hematology and Internal Medicine, Department of Clinical and Biological Sciences, University of Turin, Turin, Italy

Tóm tắt

Abstract Background: Despite the efficacy of the BCR-ABL tyrosine kinase inhibitor Imatinib mesylate for the treatment of chronic myeloid leukemia (CML), resistance has been observed in a proportion of cases, especially those with advanced stages of the disease. Point mutations within the ABL kinase domain are emerging as the most frequent mechanism for reactivation of kinase activity within the leukemic clone. Methods: We developed a denaturing-HPLC (D-HPLC)-based assay for screening for ABL point mutations. For each sample, two partially overlapping fragments of 393 and 482 bp corresponding to the kinase domain were amplified by nested reverse transcription-PCR and analyzed under selected temperature and acetonitrile gradient conditions. Fifty-one bone marrow and/or peripheral blood specimens from 27 CML patients who showed cytogenetic resistance to Imatinib were screened in parallel by D-HPLC and by direct sequencing. Results: In 12 of 27 (44%) patients, D-HPLC showed an abnormal elution profile suggesting the presence of a nucleotide change. Direct sequencing confirmed the presence of a point mutation in all cases. Conversely, all samples scored as wild type by D-HPLC showed no evidence of mutations by direct sequencing. In two cases, novel amino acid substitutions at codons already known for being hot-spots of mutation were identified (F311I and E355D). Conclusions: The proposed D-HPLC-based assay is highly specific and at least as sensitive as sequencing; with respect to the latter, it provides a much faster and less expensive semiautomated system for mutational screening. It may therefore potentially be a valuable tool for regular, large-scale testing of patients undergoing Imatinib treatment.

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Thông tin xuất bản

Clinical Chemistry

Tập 50 Số 7

1205-1213

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