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).
IDH1 Mutation Screen
IDH1 mutations are present in up to 5% of AML cases an predict response to inhibitor based therapies (Montesinos et al, 2025, Blood Advances).
As of this writing Health Canada has approved ivosidenib in combination with azacitidine for use in newly diagnosed, IDH1 R132 mutation positive AML patients not eligible to receive intensive induction chemotherapy (see product monograph for approved indications).
NPM1 Mutation Screen
NPM1 mutations are present in up to 30% of AML cases and, in the absence of adverse genetic markers, are associated with a favorable risk profile. Targeted therapy with menin inhibitors has shown improved response in patients with NPM1-mutated AML (Issa et al., 2023, Nature). This test was developed to enable rapid detection of NPM1 variants in AML patients at the time of diagnosis. MRD analysis is available for patients with select NPM1 mutations, see here for details.
INDICATION
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
REFERRAL
Any reviewing hematopathologist or treating hematologist
TEST REQUIREMENTS
- Completed CGL Myeloid Testing requisition form
- One of the following specimens (please see our Guidelines/Policies for Samples and Transport)
Preferred:
- 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
Alternative:
- 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)
TRANSPORT
Click here for guidelines on transporting specimens.
METHOD
FLT3-ITD Mutation 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 (or QX600) 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 reportable threshold for this assay has been set to a variant allele fraction (VAF) of 0.5%. Analytically positive results below this threshold are reported as negative.
IDH1 Mutation Screen
Amplification and subsequent analysis was performed by droplet digital (ddPCR) using Biorad’s QX600 Droplet Digital PCR System. The assay is designed to detect and quantify mutations in codon 132 of the IDH1 gene. This assay can specifically identify the five most common IDH1 R132 variants; these being p.R132C (c.394C>T); p.R132H (c.395G>A), p.R132G (c.394C>G), p.R132S (c.394C>A) and p.R132L (c.395G>T). The reportable threshold for this assay has been set to a variant allele fraction (VAF) of 1%. Analytically positive results below this threshold are reported as negative.
NPM1 Mutation Screen
Targeted NPM1 variants with 4bp insertions between positions c.863 and c.864, including major types A, B, and D, which together account for ~ 95% of NPM1 variants, are detected and quantified using RT-ddPCR on Bio-Rad’s QX200 (or QX600) Droplet Digital PCR System. The reportable threshold for this assay has been set to a variant allele fraction (VAF) of 1%. Analytically positive results below this threshold are reported as negative.
CLINICAL UTILITY
Rapid identification of any of the targeted mutations allows for the timely introduction of an appropriately tailored therapy into the patient’s treatment regimen.
TURNAROUND TIME
Results are reported within six calendar days from receipt of specimen and completed requisition form.
SELECTED REFERENCES
NCCN Guidelines – AML 2026