Abstract
BackgroundCurrently, FLT3 internal tandem duplication (ITD) is tested by fragment analysis. With next-generation sequencing (NGS), however, not only FLT3 ITD but also other mutations can be detected, which can provide more genetic information on disease.MethodsWe retrospectively reviewed the results of two tests—fragment analysis and a custom-designed, hybridization capture-based, targeted NGS panel—performed simultaneously. We used the Pindel algorithm to detect FLT3 ITD mutations.ResultsAmong 277 bone marrow aspirate samples tested by NGS and fragment analysis, the results revealed 99.6% concordance in FLT3 ITD detection. Overall, the allele frequency (AF) attained by NGS positively correlated with the standard allelic ratio (AR) attained by fragment analysis, with a Spearman correlation coefficient (r) of 0.757 (95% confidence interval: 0.627–0.846; p < 0.001). It was concluded that an AF of 0.11 attained by NGS is the most appropriate cutoff value (with 85.3% sensitivity and 86.7% specificity) for high mutation burden criterion presented by guidelines.ConclusionSensitive FLT3 ITD detection with comprehensive information of other mutation offered by NGS could be a useful tool in clinical laboratories. Future studies will be needed to evaluate and standardize NGS AF cutoff to predict actual clinical outcomes.
Highlights
Internal tandem duplication (ITD) in the Fms-related receptor tyrosine kinase 3 (FLT3) gene is found in approximately 20 to 30% of cases of normal karyotype acute myeloid leukemia (AML) [1]
We retrospectively analyzed the clinical data of patients who underwent FLT3 internal tandem duplication (ITD) tests using fragment analysis and next-generation sequencing (NGS) applied at the primary source to assess the correlation between these two methods of mutation detection and burden measurement
Capillary electrophoretic fragment analysis Polymerase chain reaction (PCR) was performed on genomic DNA using an AccuPower HotStart PCR Premix (Bioneer, Daejeon, Korea) and specific primers [11] to amplify FLT3 exon 14 and 15 regions with following conditions: 94 °C for 5 min followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 60 s, and final extension at 72 °C for 7 min
Summary
Internal tandem duplication (ITD) in the Fms-related receptor tyrosine kinase 3 (FLT3) gene is found in approximately 20 to 30% of cases of normal karyotype acute myeloid leukemia (AML) [1]. Kim et al Diagnostic Pathology (2022) 17:14 current standard method for the detection and quantification of FLT3 ITD [6,7,8]. Clinical guidelines, such as the European LeukemiaNet (ELN) and National Comprehensive Cancer Network consensus (NCCN) guidelines, define a FLT3 ITD allelic ratio cutoff of 0.5 as measured by fragment analysis as the optimal riskstratification criterion in AML [6, 9]. We retrospectively analyzed the clinical data of patients who underwent FLT3 ITD tests using fragment analysis and NGS applied at the primary source to assess the correlation between these two methods of mutation detection and burden measurement. With next-generation sequencing (NGS), FLT3 ITD and other mutations can be detected, which can provide more genetic information on disease
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