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Lee, Kim, Lee, Jang, Park, Chae, Kim, and Kim: Fragment Analysis for Detection of the FLT3-Internal Tandem Duplication: Comparison with Conventional PCR and Sanger Sequencing
Original Article

Lab Med Online 2017;7(1):13-19.

Published online: 17 January 2017

DOI: https://doi.org/10.3343/lmo.2017.7.1.13

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

GunDong Lee, M.T.1, Jeongeun Kim, M.T.1, SangYoon Lee, M.T.1, Woori Jang, M.D.1, 2, Joonhong Park, M.D.1, 2, Hyojin Chae, M.D.1, 2, Myungshin Kim, M.D.1, 2, ∗, , Yonggoo Kim, M.D.1, 2, ∗,

1Catholic Genetic Laboratory Center, College of Medicine, Seoul St Mary's Hospital, The Catholic University of Korea, Seoul, Korea

2Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea

∗ These authors contributed equally to this work.

Corresponding author: Myungshin Kim Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 16591, Korea Tel: +82-2-2258-1645, Fax: +82-2-2258-1719, E-mail: microkim@catholic.ac.kr

Corresponding author: Yonggoo Kim Department of Laboratory Medicine, College of Medicine, The Catholic University of Korea, 222 Banpo-daero, Seocho-gu, Seoul 16591, Korea Tel: +82-2-2258-1642, Fax: +82-2-2258-1719, Email: yonggoo@catholic.ac.kr

Received 8 December 201520 April 2016       Accepted 29 April 2016

Copyright © 2017 The Korean Society for Laboratory Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: FLT3-ITD, Quantitative fragment analysis, Mutant allele burden, ITD length, Acute myeloid leukemia, Analytical performance

REFERENCES

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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).
lmo-7-13f1.tif
Fig. 2.
Distribution of standard deviation (SD) values of the mean area ratio by fragment analysis, using patient specimens.
lmo-7-13f2.tif
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%].
lmo-7-13f3.tif
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.
lmo-7-13f4.tif
Table 1.
Distribution and length of FLT3-ITD mutations found in 193 samples by fragment analysis
FLT3-ITD Length (bases) Number of positive samples Number of positive patients
1–19 13 10
20–29 35∗† 29
30–39 27∗ 21
40–49 25 21
50–59 22 19
60–69 34† 22
70–79 14 11
80–89 9 7
90–99 8 6
100–109 3 3
≥110 5 5

∗ One sample contained two ITD products with lengths of 25 and 37 bases;

† One sample with ITD mutations yielded two peaks (20 and 63 bases).

Table 2.
Comparison of fragment analysis with conventional PCR for FLT3-ITD testing
Variable FLT3-ITD test
Wild type Mutant type
Normal donor sample (N=50) Fragment analysis 50 0
Patient sample (N=481)
Fragment analysis 288 (59.9%) 193 (40.1%)
Conventional PCR 299 (62.2%) 182 (37.8%)
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