A comparative study of next-generation sequencing and fragment analysis for the detection and allelic ratio determination of FLT3 internal tandem duplication.

Currently, 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. We 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. Among 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. Sensitive 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. © 2022. The Author(s).

Citation

Jin Ju Kim, Kwang Seob Lee, Taek Gyu Lee, Seungjae Lee, Saeam Shin, Seung-Tae Lee. A comparative study of next-generation sequencing and fragment analysis for the detection and allelic ratio determination of FLT3 internal tandem duplication. Diagnostic pathology. 2022 Jan 26;17(1):14

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PMID: 35081962

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