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

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