Rapid and sensitive allele-specific (AS)-RT-PCR assay for detection of T315I mutation in chronic myeloid leukemia patients treated with tyrosine-kinase inhibitors.
Point mutations in the kinase domain of BCR-ABL were described in 40-90% of patients with chronic myeloid leukemia (CML) resistant to Imatinib. We herein describe the development of a rapid allele-specific (AS)-RT-PCR assay to identify the T315I mutation, which confers full resistance to all available tyrosine-kinase inhibitors (TKI). The mutation status of 65 patients with resistant CML was evaluated, and the T315I was detected in 3/65 (4.6%). Comparisons between sequencing and AS-RT-PCR results, as well as serial dilutions experiments proved that the method is specific and reproducible, with maximum sensitivity of 1 × 10(-3). The developed assay is a convenient and easy tool to be used in research of CML resistance for rapid mutation screening and, together with sequencing, may be included in efficient strategies for early detection of TKI resistance in patients with CML.
Specialized Techniques Laboratory, Molecular Biology Division, Asociación Española Primera de Socorros Mutuos, Bvr. Artigas 1475, CP 11200, Montevideo, Uruguay.
Gonzalo
G
Manrique Arechavaleta
Vanesa
V
Scholl
Verónica
V
Pérez
Roberta
R
Bittencourt
Arthur
A
Moellmann
Rocio
R
Hassan
Héctor N
HN
Seuánez
Jane
J
Dobbin
Lem
L
Martinez
Ilana Zalcberg
IZ
Renault
Rosario
R
Uriarte
D000483:Alleles; D019943:Amino Acid Substitution / Q000235:genetics; D019411:Clinical Laboratory Techniques / Q000379:methods*; D004351:Drug Resistance; D042241:Early Diagnosis; D004791:Enzyme Inhibitors / Q000627:therapeutic use*; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000188:drug therapy*; D020125:Mutation, Missense; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D051562:Proto-Oncogene Proteins c-bcr / Q000037:antagonists & inhibitors*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D004791:Enzyme Inhibitors; D011505:Protein-Tyrosine Kinases; D051562:Proto-Oncogene Proteins c-bcr