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Dear Editor, Next-generation sequencing (NGS) is rapidly being adapted in clinical practice, and numerous clinical laboratories are using this technology to assess patients with acute myeloid leukemia (AML). Among recurrent somatic mutations in AML, the FLT3 internal tandem duplication (ITD) has major clinical implications and is associated with adverse outcomes [1-3]. Identification of the mutation is critical for risk stratification and the decision of early hematopoietic stem cell transplantation [2, 3]. Furthermore, as activated kinase targets such as FLT3 and JAK are under active clinical investigation [4, 5], it has become increasingly important to identify activating FLT3 mutations. During NGS, however, accurate mapping and consequent variant calling is not always possible because the duplication size could be small or large, and the inserted segment is essentially, but not necessarily, a part of the normal reference sequence. We performed NGS for patients with AML. Bone marrow aspirates of 229 patients diagnosed as having AML between March 2017 and February 2018 in Severance Hospital, Seoul, Korea, were prospectively collected after obtaining informed consent. The study was approved by our Institutional Review Board (42016-0869). Thirty patients harbored the FLT3 ITD mutation, identified by PCR and fragment analysis. To determine whether FLT3 ITD mutations could be accurately identified from targeted multigene NGS data, we analyzed the NGS data with different algorithms and compared the results with those of PCR and fragment analysis, a current standard test. NGS was performed using the NextSeq 550 instrument (Illumina, San Diego, CA, USA), with custom probes (Celemics, Seoul, Korea) for AML. The Burrows-Wheeler alignment tool (0.7.12) was used for sequence alignment [6]. GATK MuTect2 (3.8-0), and Pindel (0.2.0) algorithms were used to detect FLT3 ITD mutations [7, 8], which were implemented in the DxSeq algorithm (Dxome, Seoul, Korea). PCR amplification and fragment detection analyses were performed using a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and GeneMapper 3.2 software (Applied Biosystems), with a modified protocol based on that previously described [9]. The median depth of the FLT3 gene was 4,412×, ranging from 2,626× to 7,595×. GATK MuTect2 detected FLT3 ITD mutations in 15 of 30 cases, most of which had a relatively short duplicated segment. The Pindel algorithm detected FLT3 ITD mu-

annals of laboratory medicine

<i>FLT3 </i>Internal Tandem Duplication in Patients With Acute Myeloid Leukemia Is Readily Detectable in a Single Next-Generation Sequencing Assay Using the Pindel Algorithm

FLT3 Internal Tandem Duplication in Patients With Acute Myeloid Leukemia Is Readily Detectable in a Single Next-Generation Sequencing Assay Using the Pindel Algorithm