FLT3 ITD and TKD mutation screen


FMS-Like Tyrosine kinase 3 (FLT3) is a (transmembrane) receptor tyrosine kinase expressed on a number of hematologic progenitors. Like all such RTKs, ligand binding results in the initiation of JAK, RAS, and PI3K signaling cascades. Relevant to AML, mutations in FLT3 result in constitutive, ligand independent, yet TKI sensitive signaling.

Mutations observed in FLT3 recapitulate those found in other RTKs such as EGFR in lung cancer as well as KIT and PDGFRA in GISTs. Mutations come in two general varieties; internal tandem duplication events (ITD type mutations) and tyrosine kinase domain mutations (TKD mutations). The former is most often an in-frame duplication of between 9 and 45 bases and occurs in approximately 30% of de-novo AML cases. When present at an allelic ratio above 0.5 (VAF: 33%), FLT3-ITD mutations confer an inferior prognosis. TKD mutations occur in approximately 10% of AML cases and while similarly activating the prognostic significance of these remain uncertain.

As of this writing Health Canada has approved midostaurine for use in newly diagnosed, FLT3 mutation positive AML patients and gilteritinib for use in the relapsed/refractory setting (see individual product monographs for approved indications).


Reflex test in patients with a new diagnosis of AML (or query). Diagnoses of APL excepted

Reflex test in patients with documented relapsed or refractory AML

Note: As FLT3 mutations not not felt to be stable biomarkers, FLT3 based MRD testing is not offered.


Any reviewing hematopathologist or treating hematologist


  1. Completed CGL Myeloid Testing requisition form [link]
  2. One of the following specimens (please see our Guidelines/Policies for Samples and Transport)


  • 0.5ml Bone marrow aspirate in ETDA tube (purple top)
    • This specimen may also serve as a gDNA source for myeloid panel based mutation screening


  • 5mL Peripheral Blood in EDTA tubes (purple top) – requires circulating blasts 
    • Peripheral blood derived white blood cells fixed in methanol/acetic acid (residual cytogenetic specimen)
    • Bone marrow specimen fixed in methanol/acetic acid (residual cytogenetic specimen)


Click here for guidelines on transporting specimens.


FLT3-ITD screen

The presence or absence of an FLT3 internal tandem duplication (ITD) was determined by PCR amplification of exons 14 and 15. The presence of an ITD would be evidenced by the appearance of a novel (larger) peak upon capillary gel electrophoresis. Allelic fraction is calculated as the area under the curve (AUC) of the ITD over the AUC of all amplified products. Allelic ratio is calculated as the area under the curve (AUC) of the ITD over the AUC of the wild type peak. The limit of reliable detection of this assay has not been specifically determined though ITDs at a VAF of 5% are routinely detectable.

FLT3-TKD mutation screen

Amplification and subsequent analysis was performed by droplet digital (ddPCR) using Biorad’s QX200 Droplet Digital PCR System. The assay is designed to detect and quantify mutations in codons 835 and 836 of the tyrosine kinase domain (TKD) of the FLT3 gene. The assay can specifically identify the three most common FLT3 TKD mutations being p.D835Y (c.2503G>T); p.D835V (c.2504A>T) and p.D835H (c.2503G>C) while other mutations in codons D835/I836 may be detected but will not be identified. The limit of reliable detection of this assay has been determined to be 1% (with 5 ng of input gDNA).


Identification of an FLT3 mutation allows for the timely introduction of an FLT3 directed TKI into the patient’s therapeutic regimen.


Results are reported within six working days from receipt of specimen and completed requisition form.


NCCN Guidelines – AML v1.2022