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. 2022 Jan 26;17(1):14.
doi: 10.1186/s13000-022-01202-x.

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

Jin Ju Kim  1 , Kwang Seob Lee  1 , Taek Gyu Lee  2 , Seungjae Lee  1 , Saeam Shin  3 , Seung-Tae Lee  1
Affiliations

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

Jin Ju Kim et al. Diagn Pathol. .

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords: Acute myeloid leukemia; Allele ratio; FLT3 internal tandem duplication; Fragment analysis.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Correlation of the FLT3 ITD mutant burden as assessed by the two study methods; Allelic ratio (AR) values from fragment analysis and allele frequency (AF) values from NGS are indicated, respectively. Abbreviations: AF, allelic frequency; NGS, next generation sequencing; AR, allelic ratio; FA, fragment analysis
Fig. 2
Fig. 2
ROC curves were calculated for revealing a high FLT3 ITD burden using AF as determined by NGS. A high FLT3 ITD burden was indicated by an AR value of greater than 0.5 by fragment analysis. By applying the cutoff value of 0.11, the AUC was 0.926 (95% CI: 0.833–0.977). ROC curve (solid line) with 95% confidence interval (dashed lines). The dotted line is a diagonal line
Fig. 3
Fig. 3
Change in AF values of somatic mutations in an AML case with discordant results from NGS and fragment analysis, respectively (Patient 7). The FLT3 ITD AR was calculated by fragment analysis was converted into an AF value according to the following equation; AF(%) = AR/(1 − AR)*100. The box represents the blast percentage observed in a bone marrow aspirate smear sample
See this image and copyright information in PMC

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