A new assay to identify recurrent mutations in acute myeloid leukemia using next-generation sequencing

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Abstract

Introduction: Acute myeloid leukemia (AML) is a heterogeneous disease characterized by a late onset (it is rare in children), aggressive phenotype and dismal prognosis especially in patients in the older group (>65 years). For risk stratification of patients standard cytogenetic is used along with molecular techniques for point mutation identification. Here we describe a new method using next generation sequencing for identification of mutation in 5 AML recurrently mutated genes - RUNX1, FLT3, DNMT3A, IDH1 and IDH2. Materials and methods: Samples from 40 patients with normal karyotype AML referred to Fundeni Clinical Institute were sequenced. Primer design was performed using LaserGene Genomics suit. Next generation sequencing was performed on MiSeq (Illumina) and results were analyzed using LaserGene Genomics suit. Results of next generation sequencing were compared to Sanger sequencing. Results: No additional mutations were identified in samples from nine patients presenting FLT3-ITD and/or NPM1 mutations. In 25 out of 31 patients with normal karyotype and no FLT3-ITD and NPM1 mutations, we identified mutations in one of the 5 aforementioned genes. All these mutations identified by next generation sequencing were confirmed using the classical Sanger sequencing. Conclusions: We validated a very useful method for mutation identification in AML patients using next generation sequencing. There are many advantages exhibited by this method: it is more cost efficient and it has a higher sensitivity of mutation detection than Sanger sequencing, it has been described as being quantitative and in our case it allowed risk stratification for most of the normal karyotype AML samples which were FLT3-ITD and NPM1 negative.

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