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Updated: Jan 5, 2026
Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
Detection and Quantification of FLT3 Internal Tandem Duplication Mutations Do Not Vary Significantly Between Whole
Jonathan A Schumacher1, Victoria D Holgard1, Faryal Sial1
1ARUP Laboratories, Salt Lake City, UT.
Objectives:
Many commonly used FLT3 mutational assay protocols require a tedious blast enrichment step. We investigated whether elimination of this step would still give equivalent results and compared the accuracy of variant allele fraction (VAF) between polymerase chain reaction/capillary electrophoresis (PCR/CE) vs next-generation sequencing (NGS) methods.
Methods:
Total leukocyte vs blast-enriched whole-blood aliquots were tested for FLT3 internal tandem duplication (ITD) and tyrosine kinase domain mutations by PCR/CE. VAF of the ITD mutations was also compared with NGS VAF.
Results:
Blast-enriched vs total leukocyte specimens showed 100% concordance in the 25 positive specimens. VAF was consistently lower by NGS, with poorer fidelity to PCR/CE VAF as the ITD size increased.
Conclusions:
Our study supports elimination of the blast enrichment step without compromising results or sensitivity. In addition, since NGS shows a loose correlation with PCR/CE quantitative results, NGS VAF should not be reported for FLT3 ITDs.
