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

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.

An NGS based MRD evaluation from acute myeloid leukemia patients

“…36 With the development of the new bioinformatics analysis, the hybridization capture-based NGS achieved 99.6% coincidence rate with fragment analysis for FLT3-ITD follow up. 37 In this study we demonstrated that NRAS was the most com- This panel was able to detect more positive patients with less cost.…”

“…However, patient‐specific primers have to be designed from the tumor samples 36 . With the development of the new bioinformatics analysis, the hybridization capture‐based NGS achieved 99.6% coincidence rate with fragment analysis for FLT3‐ ITD follow up 37 …”

Using targeted second-generation sequencing technique to guide clinical diagnosis and the effect of medication on the therapeutic effect and prognosis of respiratory tract infection in children: An observational study

“…Due to the limitations of existing microbiological detection methods (low culture sensitivity, long time required; limited number of microorganisms detected by serology and polymerase chain reaction), its diagnosis is still challenging. [ 4 , 5 ] In addition, the lungs are not aseptic and there are microbial colonies in both healthy and diseased conditions. How to distinguish pathogens from colonized bacteria is the main challenge in pathogen diagnosis of LRTI.…”

Revealing molecular architecture of FLT3 internal tandem duplication: Development and clinical validation of a web‐based application to generate accurate nomenclature

“…Novel bioinformatic analysis is required to bridge this clinical gap, improve the sensitivity and accuracy of ITD characterization, [23][24][25][26] as well as accurately calculate the allele ratio. 21,22,27,28 In the published studies, 13,21,22 multiple steps and tools are usually needed to create a final report of FLT3-ITD. An NGS assay using the Archer ® VariantPlex ® Myeloid panel, coupled with the proprietary Archer ® Analysis pipeline (ArcherDX, Inc., Boulder, CO), is a highly sensitive assay for detecting FLT3-ITD, especially for intermediate to long ITDs, compared to traditional PCR-based fragment length analysis (Ding and Zhang, unpublished clinical validation data).…”

“…However, it is a well‐known challenge that insertion mutations over 25 bp length are difficult to analyze and many times being missed by most bioinformatic pipelines. Novel bioinformatic analysis is required to bridge this clinical gap, improve the sensitivity and accuracy of ITD characterization, 23–26 as well as accurately calculate the allele ratio 21,22,27,28 . In the published studies, 13,21,22 multiple steps and tools are usually needed to create a final report of FLT3 ‐ITD.…”