Denaturing-HPLC-based assay for detection of ABL mutations in chronic myeloid leukemia patients resistant to Imatinib.
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.
Soverini
Simona
S
Institute of Hematology and Medical Oncology Seràgnoli, University of Bologna, Bologna, Italy.
Martinelli
Giovanni
G
Amabile
Marilina
M
Poerio
Angela
A
Bianchini
Michele
M
Rosti
Gianantonio
G
Pane
Fabrizio
F
Saglio
Giuseppe
G
Baccarani
Michele
M
D000970:Antineoplastic Agents / Q000627:therapeutic use*; D001549:Benzamides; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004252:DNA Mutational Analysis; D019008:Drug Resistance, Neoplasm; D016044:Fusion Proteins, bcr-abl; D016313:Genes, abl; D006801:Humans; D000068877:Imatinib Mesylate; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy* / Q000235:genetics*; D010879:Piperazines / Q000627:therapeutic use*; D017354:Point Mutation; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011743:Pyrimidines / Q000627:therapeutic use*; D012189:Retrospective Studies; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D000970:Antineoplastic Agents; D001549:Benzamides; D010879:Piperazines; D011743:Pyrimidines; D000068877:Imatinib Mesylate; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl