Rapid detection of FLT3-ITD (exon14-15) gene mutations analysis in acute myelogenous leukemia patients by High Resolution Melting analysis

Abstract

Background: Molecular genetic characterization allows prognostic stratification and can potentially alter treatment choices in acute myeloid leukemia (AML). Activating mutations of the Fsm-like tyrosine kinase3 (FLT3) gene, especially FLT3-ITD mutations, have been associated with an adverse prognosis in AML. Therefore, FLT3 mutation detection becomes essential for AML patients. High Resolution Melting (HRM) analysis is an alternative method for the rapid and affordable detection of gene mutations. Objectives:To establish and evaluate a rapid and affordable method for detecting mutations of FLT3-ITD (exon14-15) gene in acute myeloid leukemia by High Resolution Melting analysis. Materials and Methods: Thirty-five patients with newly diagnosed AML from Maharaj Nakorn Chiang Mai Hospital were included in this study. FLT3-ITD mutation screening was performed by HRM analysis, and the results were compared with the data obtained using conventional PCR with gel electrophoresis and direct sequencing. Results: Among the 35 AML patients studied, 6 patients were scored positively for FLT3-ITD mutation in the conventional PCR, whereas HRM analysis identified 7 out of 35 patients who were positive for FLT3-ITD mutation, which was concordant with direct sequencing results. Interestingly, one sample that was positive by HRM analysis was scored by conventional PCR as negative. Therefore, HRM analysis is more sensitive than conventional PCR. Conclusion: HRM analysis is a rapid and promising screening method for FLT3-ITD mutation, enabling the real-time evaluation of AML progression, which is significant for decision-making regarding treatment. Our results showed that HRM analysis could be a useful clinical tool for the rapid and affordable screening of FLT3-ITD mutation in AML patients.