Lab Med Online.  2017 Jan;7(1):13-19. 10.3343/lmo.2017.7.1.13.

Fragment Analysis for Detection of the FLT3-Internal Tandem Duplication: Comparison with Conventional PCR and Sanger Sequencing

Affiliations
  • 1Catholic Genetic Laboratory Center, College of Medicine, Seoul St Mary's Hospital, The Catholic University of Korea, Seoul, Korea. microkim@catholic.ac.kr yonggoo@catholic.ac.kr
  • 2Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea.

Abstract

BACKGROUND
We evaluated a sensitive and quantitative method utilizing fragment analysis of the fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD), simultaneously measuring mutant allele burden and length, and verified the analytical performance.
METHODS
The number and allelic burden of FLT3-ITD mutations was determined by fragment analysis. Serial mixtures of mutant and wild-type plasmid DNA were used to calculate the limit of detection of fragment analysis, conventional PCR, and Sanger sequencing. Specificity was evaluated using DNA samples derived from 50 normal donors. Results of fragment analysis were compared to those of conventional PCR, using 481 AML specimens.
RESULTS
Defined mixtures were consistently and accurately identified by fragment analysis at a 5% relative concentration of mutant to wild-type, and at 10% and 20% ratios by conventional PCR and direct sequencing, respectively. No false positivity was identified. Among 481 AML specimens, 40.1% (193/481) had FLT3-ITD mutations. The mutant allele burden (1.7-94.1%; median, 28.2%) and repeated length of the mutation (14-153 bp; median, 49 bp) were variable. The concordance rate between fragment analysis and conventional PCR was 97.7% (470/481). Fragment analysis was more sensitive than conventional PCR and detected 11 additional cases: seven had mutations below 10%, three cases represented conventional PCR failure, and one case showed false negativity because of short ITD length (14 bp).
CONCLUSIONS
The new fragment analysis method proved to be sensitive and reliable for the detection and monitoring of FLT3-ITD in patients with AML. This could be used to simultaneously assess ITD mutant allele burden and length.

Keyword

FLT3-ITD; Quantitative fragment analysis; Mutant allele burden; ITD length; Acute myeloid leukemia; Analytical performance
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