Detection of FLT3 (FMS-like Tyrosine Kinase) Internal Tandem Duplication (ITD) Mutation using Next Generation Sequencing Technology and Nested PCR

Abstract

Introduction: Sensitive detection of internal tandem duplication (ITD) mutation of FLT3 is very important in acute myeloid leukemia. Conventional PCR methods are not satisfactory in detecting relevant mutations in patients harboring the mutations. To increase detection sensitivity of FLT3-ITD, I developed new detection algorithm using next generation sequencing (NGS) data. I validated results using nested polymerase chain reaction (PCR) methods. I compared results of NGS data, nested PCR and conventional PCR methods. Methods: First, using whole exome sequencing data of 81 AML patients, I applied calling algorithm for FLT3-ITD. Briefly, to detect ITDs with NGS data, the reads are aligned to a reference sequence (UCSC hg19), with BWA which is a read aligner allowing soft-clipping. Some reads can be an indication of the occurrence of ITD and BWA aligns those reads as soft-clipped. Second, I designed two types of primer for Nested PCR. The first primer was targeted wildly for between exon14 and exon15 of FLT3 gene. Nested PCR primer was designed to target previously reported regions in which ITD mutations were frequently found. PCR reactions of two steps were performed using the PCR primers sequentially. Results: In these 81 patients, FLT3-ITD was positive only in 7 patients when tested by conventional PCR methods. When NGS detection method was applied, FLT3-ITD was positive in 11 patients (11/81, 13%). When validation was performed using nested PCR, FLT3-ITD was confirmed in all 11 patients. Nested PCR detected additional 4 patients positive for FLT3-ITD in this population. For 65 patients, FLT3-ITD was negative by both NGS and nested PCR method. One patient with FLT3-ITD by conventional PCR was not defined clearly by nested PCR and NGS method because of smear band and low depth coverage (coverage depth of exon 14 was 42). Overall, NGS method improved sensitivity of FLT3-ITD detection by 57% in this population compared with currently used conventional PCR method. The concordance rate of NGS method and nested PCR was 95% (77/81). Conclusions: FLT3-ITD is a very important genetic factor, guiding a therapeutic direction for AML patients. Here, I have developed more sensitive alternative detection methods for FLT3-ITD based on NGS. All of detection results were validated by nested PCR. NGS method is not only more sensitive than conventional PCR but also capable of determining FLT3-ITD size and amount in AML patients.