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Front Pediatr. 2014 Sep 9;2:96. doi: 10.3389/fped.2014.00096. eCollection 2014.

High-resolution melting curve analysis, a rapid and affordable method for mutation analysis in childhood acute myeloid leukemia.

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Department of Hematology/Oncology, Shanghai Children's Medical Center, Shanghai Jiaotong University School of Medicine , Shanghai , China.
Division of Hematology, Oncology and Blood and Marrow Transplantation, Children's Center for Cancer and Blood Disease, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California , Los Angeles, CA , USA.



Molecular genetic alterations with prognostic significance have been described in childhood acute myeloid leukemia (AML). The aim of this study was to establish cost-effective techniques to detect mutations of FMS-like tyrosine kinase 3 (FLT3), nucleophosmin 1 (NPM1), and a partial tandem duplication within the mixed-lineage leukemia (MLL-PTD) genes in childhood AML.


Ninety-nine children with newly diagnosed AML were included in this study. We developed a fluorescent dye SYTO-82 based high-resolution melting (HRM) curve analysis to detect FLT3 internal tandem duplication (FLT3-ITD), FLT3 tyrosine kinase domain (FLT3-TKD), and NPM1 mutations. MLL-PTD was screened by real-time quantitative PCR.


The HRM methodology correlated well with gold standard Sanger sequencing with less cost. Among the 99 patients studied, the FLT3-ITD mutation was associated with significantly worse event-free survival (EFS). Patients with the NPM1 mutation had significantly better EFS and overall survival. However, HRM was not sensitive enough for minimal residual disease monitoring.


High-resolution melting was a rapid and efficient method for screening of FLT3 and NPM1 gene mutations. It was both affordable and accurate, especially in resource underprivileged regions. Our results indicated that HRM could be a useful clinical tool for rapid and cost-effective screening of the FLT3 and NPM1 mutations in AML patients.


FLT3; MLL; NPM1; acute myeloid leukemia; childhood; high-resolution melting curve analysis

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