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

• Lee G
• Kim J
• Lee S
• Jang W
• Park J
• Chae H
• Kim M
• Kim Y

• KMID: 2363001
• DOI: http://doi.org/10.3343/lmo.2017.7.1.13

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

MeSH Terms

Alleles
DNA
Humans
Leukemia, Myeloid, Acute
Limit of Detection
Methods
Plasmids
Polymerase Chain Reaction*
Sensitivity and Specificity
Tissue Donors
Vascular Endothelial Growth Factor Receptor-1
DNA
Vascular Endothelial Growth Factor Receptor-1

Figure

• 

  Fig. 1. (A) Linearity of fragment analysis using standard material (66 bp-type), calculating percent ratio for area of peak. (B) Linearity of fragment analysis using standard material (66 bp-type), calculating percent ratio for height of peak. (C) Correlation for fragment analysis using methods (A) and (B).

• 

  Fig. 2. Distribution of standard deviation (SD) values of the mean area ratio by fragment analysis, using patient specimens.

• 

  Fig. 3. Representative examples of limit of detection for each method; the results of fragment analysis [(A) was 3%], conventional PCR [(B) was 10%], Sanger sequencing [(C) was 20%].

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  Fig. 4. fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) analysis in acute myeloid leukemia patients: fragment analysis versus conventional PCR in discrepant case. Fig. 4. fms-like tyrosine kinase 3-internal tandem duplication (FLT3-ITD) analysis in acute myeloid leukemia patients: fragment analysis versus conventional PCR in discrepant case. (A) Fragment analysis for initial sample; the arrows indicating peak size are FLT3-ITDs. (B) Fragment analysis for followup sample of (A); the arrows indicating the smaller peak are the same as the peak size of (A). (C) Conventional PCR for followup of sample of (A), which used the same sample as used in (B); In the photographs, it was difficult to identify the existence of an FLT3-ITD-positive band. (D) Fragment analysis for short-length ITD. (E) For conventional PCR, this is shown as a slightly thick wild-type band when electrophoresis was performed under typical conditions. (F) To confirm the ITD band, we extended the running time of electrophoresis; in doing this, the wild-type band and ITD band could be distinguished.

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