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2.
Sci Rep ; 12(1): 13057, 2022 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-35906470

RESUMO

The screening of the BCR::ABL1 kinase domain (KD) mutation has become a routine analysis in case of warning/failure for chronic myeloid leukemia (CML) and B-cell precursor acute lymphoblastic leukemia (ALL) Philadelphia (Ph)-positive patients. In this study, we present a novel DNA-based next-generation sequencing (NGS) methodology for KD ABL1 mutation detection and monitoring with a 1.0E-4 sensitivity. This approach was validated with a well-stablished RNA-based nested NGS method. The correlation of both techniques for the quantification of ABL1 mutations was high (Pearson r = 0.858, p < 0.001), offering DNA-DeepNGS a sensitivity of 92% and specificity of 82%. The clinical impact was studied in a cohort of 129 patients (n = 67 for CML and n = 62 for B-ALL patients). A total of 162 samples (n = 86 CML and n = 76 B-ALL) were studied. Of them, 27 out of 86 harbored mutations (6 in warning and 21 in failure) for CML, and 13 out of 76 (2 diagnostic and 11 relapse samples) did in B-ALL patients. In addition, in four cases were detected mutation despite BCR::ABL1 < 1%. In conclusion, we were able to detect KD ABL1 mutations with a 1.0E-4 sensitivity by NGS using DNA as starting material even in patients with low levels of disease.


Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva , Leucemia-Linfoma Linfoblástico de Células Precursoras , DNA , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/genética , Genômica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inibidores de Proteínas Quinases/farmacologia
3.
Biomed Chromatogr ; 27(4): 502-8, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23034891

RESUMO

We have developed a method of liquid chromatography in tandem with mass spectrometry to monitor therapeutic levels of imatinib in plasma, a selective inhibitor of protein tyrosine kinase. After solid-phase extraction of plasma samples, imatinib and its internal standard, imatinib-D8, were eluted with Zorbax SB-C18 at 60 °C, under isocratic conditions through a mobile phase consisting of 4 mm ammonium formate, pH: 3.2 (solution A) and acetonitrile solution B. The flow rate was 0.8 mL/min with 55% solution A + 45% solution B. Imatinib was detected and quantified by mass spectrometry with electrospray ionization operating in selected-reaction monitoring mode. The calibration curve was linear in the range 10-5000 ng/mL, the lower limit of quantitation being 10 ng/mL. The method was validated according to the recommendations of the Food and Drug Administration, including tests of matrix effect (bias < 10%) and recovery efficiency (>80 and <120%). The method is precise (coefficient of variance intra-day <2% and inter-day <7%), accurate (95-108%), sensitive and specific. It is a simple method with very fast recording time (1.2 min) that is applicable to clinical practice. This will permit improvement of the pharmacological treatment of patients.


Assuntos
Antineoplásicos/sangue , Benzamidas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Piperazinas/sangue , Inibidores de Proteínas Quinases/sangue , Pirimidinas/sangue , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Monitoramento de Medicamentos/métodos , Humanos , Mesilato de Imatinib , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Limite de Detecção
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