Fragment Analysis as a Standard Method for FLT3 Mutation Detection Endorsed by International Guidelines

FLT 3 and mutations affecting prognosis of acute myeloid leukaemia

FMS-like Tyrosine kinase 3 (FLT3) belongs to the class III tyrosine kinase family and is expressed on hematopoietic stem progenitor cells for cell survival and development. In acute myeloid leukaemia (AML) patients, FLT3 is overexpressed in most cases. Common mutations, such as internal tandem duplication (ITD) at exon 12 to 13 and D835 mis-sense mutation, are associated with 20 ~30% and 7% AML cases, respectively. These mutations result in constitutive tyrosine kinase activity of FLT3 and are associated with poor prognosis. The test of these mutations is critical for newly diagnosed AML and for target therapy with FLT3 inhibitors such as type I inhibitors, midostaurin and gilteritinib. These inhibitors have recently been approved by the U.S. FDA for treatment of adult AML.

The FLT-3 standard test: fragment analysis

 FLT3 testing using fragment analysis is currently the standard method developed in 2003. ITD is in-frame duplication in the juxtamembrane region which generates 6 to 180 bases. To test for this mutation, PCR covering the duplication region generates a 330 bp fragment for the wild-type but a fragment >330 bp for ITD. The D835 mutation can be identified by EcoRV digestion of a PCR fragment covering the mutation. The wild-type will be detected as an 80 bp fragment while the D835 mutants as a 129 bp fragment. Both mutation areas can be covered in one fragment analysis.

Although the PCR fragments can be detected by regular agarose gel without using fluorescent labelling, the sensitivity is much lower than fragment analysis.  In some cases, the limitation of the sensitivity can miss these important mutations and may lead to a wrong target therapy strategy.

NGS as a tool for the identification of ITD and D835 mutations

In the past 15 years, NGS quickly gets adapted by clinical labs for gene mutation testing that generally uses Sanger sequencing or fragment analysis. However, the NGS technique requires higher set-up cost and complex bioinformatics analysis. Without large volume of testing samples and bioinformatics expertise in house, NGS is not always easy to replace the conventional tests.

Recently, NGS has been used to detect ITD. Although the author’s lab successfully identified the mutation with a 100% concordance with fragment analysis, it remains to be seen whether every single lab could have this capability to apply NGS for FLT3 mutation detection in the near future.


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