Establishment of a cost-effective method to detect FLT-ITD and D835 mutations in acute myeloid leukemia patients in the Taiwanese population

Hsingjin Eugene Liu^a, Ching-Huai Ko^b, Freda Lam^c, Lawrence Shih-Hsin Wu^d, Kuei-Fang Lee^e

^a Department of Radiation Oncology, Wan Fang Hospital, Taipei, Taiwan
^b Medigen Biotechnology Corp., Taipei, Taiwan
^c Research Development Division, Vita Genomics Inc., Taipei, Taiwan
^d Institute of Medical Sciences, Tzu Chi University, Hualien, Taiwan
^e Laboratory for Cytogenetics, Center for Genetic Counseling, Buddhist Tzu Chi General Hospital, Hualien, Taiwan

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Abstract
Objective

The FMS-related tyrosine kinase 3 (FLT-3) gene is a hematopoietic growth factor receptor gene, an independent negative prognostic factor, which affects the proliferation and differentiation of stem cells or hematopoietic progenitor cells. Patients with FLT-3 gene mutations have a worse prognosis and responsiveness to chemotherapy than those without these mutations. Our study aims to establish a conventional detection method for FLT3-ITD and D835 mutations in patients with acute myeloid leukemia (AML).

Materials and methods

In this study, we recruited 100 patients with AML. Primers were designed to distinguish between wild-type FLT-3, FLT3-ITD, and D835 variants. Methods using a polymerase chain reaction (PCR)-Agilent 2100 Bioanalyzer, PCR-ABI PRISM 3100 Genetic Analyzer, and PCR–agarose gel electrophoresis were compared.

Results

A high-accuracy, easily operated, low-cost technique to detect the FLT-3 variation with 99.9% specificity was established in this study. The PCR platform, the Agilent 2100 Bioanalyzer (plus DNA 1000 LabChip kit) chip analysis platform, and the ABI PRISM 3100 Genetic Analyzer (plus GeneScan-500 size standard) short tandem repeat (STR) fluorescence analysis platform were used in different experimental comparisons. The ABI PRISM 3100 Genetic Analyzer (plus GeneScan-500 size standard) STR fluorescence analysis platform was the most suitable method to detect FLT3 variants. This method has a high degree of sensitivity, accuracy, and a specificity of 99.9%.

Conclusion

AML with the homozygous mutated FLT-3 may have a worse cure rate than AML with heterozygous mutation. This mutation is not related to drug resistance, but is a factor in a high risk of relapse; it is also related to unfavorable overall survival. Our designed detection methods should provide key information to develop personalized medicine for AML patients.

Keywords
Acute myeloid leukemia; FLT3-ITD; FLT3-D835