title: Comparative quantitative analysis of BCR-ABL transcripts with the T315I mutant clone by polymerase chain reaction (PCR)-Invader method.
abstract: Drug resistance is a serious complication in the treatment of chronic myeloid leukemia (CML). The most common and best-characterized mechanism of secondary imatinib resistance in CML is the development of kinase domain mutations in the BCR-ABL gene. Second-generation tyrosine kinase inhibitors, such as dasatinib or nilotinib, overcome most of these mutations, but they are not effective against the T315I mutant. To determine whether these mutations contribute to clinical resistance, it is necessary to monitor the ratio of the mutant and wild-type forms. Here, we developed a polymerase chain reaction (PCR)-Invader assay for comparative quantitative analysis (qPI assay) of BCR-ABL transcripts with the T315I mutant clone. T315I ratios were calculated for the wild-type and mutant fold-over-zero (FOZ) values. In examination with 2 kinds of plasmids containing wild-type or T315I mutant PCR amplicons, mutant FOZ values were detected down to 1% of the total. The results of 12 serial samples from 2 patients (case A: Philadelphia-positive acute lymphoblastic leukemia and case B: CML) with the T315I mutant clone were compared with those of direct sequencing or 2 kinds of allele-specific oligonucleotide (ASO)-PCR. All samples showed the T315I mutation by qPI assay and ASO-PCR, and 10 samples showed it by direct sequencing. Significant correlation (correlation coefficient; r2 = 0.951) was noted between the qPI assay and quantitative ASO-PCR to analyze T315I mutant ratios. Thus, the qPI assay is a useful method for evaluating the T315I mutant clone in BCR-ABL transcripts.
authors.affiliation: Development of Clinical Genomics, BML, Inc., Saitama, Japan. tado-k@bml.co.jp
authors.forename: Kenichi
authors.initials: K
authors.lastname: Tadokoro
authors.forename: Maho
authors.initials: M
authors.lastname: Ishikawa
authors.forename: Makoto
authors.initials: M
authors.lastname: Suzuki
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Saito
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Yamaguchi
authors.forename: Fumiharu
authors.initials: F
authors.lastname: Yagasaki
mesh_terms: D000368:Aged; D000970:Antineoplastic Agents / Q000627:therapeutic use; D001483:Base Sequence; D001549:Benzamides; D004252:DNA Mutational Analysis / Q000379:methods* / Q000706:statistics & numerical data; D004273:DNA, Neoplasm / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D005260:Female; D016313:Genes, abl; D006801:Humans; D000068877:Imatinib Mesylate; D015466:Leukemia, Myeloid, Chronic-Phase / Q000188:drug therapy / Q000235:genetics; D008297:Male; D008875:Middle Aged; D015345:Oligonucleotide Probes / Q000235:genetics; D010879:Piperazines / Q000627:therapeutic use; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000188:drug therapy / Q000235:genetics; D011743:Pyrimidines / Q000627:therapeutic use; D012334:RNA, Neoplasm / Q000235:genetics; D057170:Translational Research, Biomedical
chemical_list: D000970:Antineoplastic Agents; D001549:Benzamides; D004273:DNA, Neoplasm; D015345:Oligonucleotide Probes; D010879:Piperazines; D011743:Pyrimidines; D012334:RNA, Neoplasm; D000068877:Imatinib Mesylate