Molecular Diagnostics | FLT3 (ITD and TKD) Mutation Detection

UWHC Lab Test Details
Test NameFLT3 (ITD and TKD) Mutation Detection
Test Code(s)FLT3MUT
CPT Code(s)81245 - FLT3 ITD 81246 - FLT3 TKD variants (D835, I836)
MethodologyReal-Time PCR followed by Direct Sequencing
Clinical Information

FLT3 in Acute Myeloid Leukemia:

The most common activating FLT3 alterations include internal tandem duplication (FLT3-ITD) within the juxtamembrane domain that occur in ~20-30% of adult acute myeloid leukemias (AMLs) and point mutations in the tyrosine kinase domain (FLT3-TKD; ~7% of adult AMLs), most commonly at codon D835. While both FLT3-ITD and FLT3-TKD mutations are common in AML with a normal karyotype, these mutations are also identified in AML with various karyotypic abnormalities. In general, AML patients with intermediate-risk cytogenetics and with a FLT3-ITD mutation have a significantly poorer prognosis with an increased relapse risk and decreased overall survival. However, the overall prognostic significance of FLT3-ITD mutations may be affected by the presence or absence of additional mutations, such as commonly associated NPM1 mutations, and potentially also by the FLT3-ITD allelic ratio. The prognostic significance of FLT3-TKD mutations remains controversial. See below for therapeutic implications.



FLT3 Targeted Therapy and Allelic Ratio:

The FLT3 receptor tyrosine kinase is a target of several different tyrosine kinase inhibitors, including FDA-approved midostaurin. The presence of FLT3-TKD mutations may have implications for targeted therapy, as not all FLT3 tyrosine kinase inhibitors have demonstrated activity against various FLT3-TKD mutations. A phase III RATIFY clinical trial (CALGB 10603) has shown that the addition of the FLT3 inhibitor midostaurin to standard chemotherapy significantly prolonged overall and event-free survival among AML patients with a FLT3 mutation, including FLT3-ITD as well as the FLT3-TKD. In this trial, a ratio of mutant to wild-type alleles of 0.05 or greater was used as the positive cut-off value for FLT3 analysis. A ratio of mutant to wild-type alleles is calculated by dividing the mutant allele signal by the wild-type allele signal and is not equivalent to mutant allele frequency or fraction (the percentage or fraction of mutant alleles within all of the alleles present in a given sample).

Days PerformedMon-Fri, dayshift.
In-Lab Turnaround Time2 - 3 days.
Collection Requirements
SpecimenBlood or bone marrow
Collection Container
Collection ContainerLavender top
Collection Instructions

Blood and bone marrow must be aseptically collected.


Collection Volume2 mL
Pediatric Collection Volume1.2 mL
Specimen Processing Requirements
Stability Ambient

72 hours

Stability Refrigerated

7 days

Stability Frozen

Not acceptable

Sample AnalyzedWhole Blood or Bone Marrow
Testing Volume1 mL
Pediatric Testing Volume0.3 mL
Specimen Processing

Refrigerate specimen. Do not freeze or centrifuge.

Outreach Specimen Processing

Refrigerate specimen. Do not freeze or centrifuge.

Specimen Transport

Transport specimen to laboratory immediately. Refrigerate specimen if not delivered immediately. Do not freeze sample.

Unacceptable Criteria

Frozen or centrifuged specimens

Interpretive Guidelines

A written interpretive report is provided by the laboratory.

Test Limitations

This test is designed and validated to detect FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations at codons 835 and 836 in genomic DNA extracted from peripheral whole blood or bone marrow aspirates. A negative test result does not exclude the presence of FLT3-ITD or TKD mutations below the detection limits of the assay or the presence of less common FLT3 mutations not detectable by this test. Specifically, mutations within the tyrosine kinase domain of FLT3 outside of codons D835 and I836 will not be reliably detected by this assay.


Genomic DNA is isolated from either peripheral whole blood or bone marrow by automated nucleic acid extraction. Separate polymerase chain (PCR) reactions targeting regions flanking exons 14 and 15 encoding the juxtamembrane region (ITD) and exon 20 for the kinase domain (TKD) of FLT3 are conducted. The amplification products are differentially labeled through the use of fluorescent primers. Following amplification, the FLT3-TKD PCR product is digested with EcoRV restriction endonuclease. Fluorescent products for the juxtamembrane (ITD) and the digested TKD products are detected and sized using capillary electrophoresis relative to a 600 base standard. The ITD allelic ratio is determined as the sum of fluorescent signal(s) for all FLT3-ITD products detected > 326 bases divided by the fluorescent signal of the normal sized wildtype product (324-326 bases). The sensitivity of FLT3-ITD detection is approximately 0.01 ITD allelic ratio. To detect alterations at codons 835 and 836 in the tyrosine kinase domain of FLT3, the lengths of the restriction digested TKD fluorescent amplification products are analyzed. Nucleotide changes at codons 835 and/or 836 inactivate EcoRV restriction within the tyrosine kinase domain for the FLT3 gene. The sensitivity of detecting TKD mutations at codons 835 and 836 is estimated to be at approximately a 0.02 mutant allele ratio. A negative test result does not exclude the presence of FLT3-ITD or TKD mutations below the detection limits of the assay or the presence of less common FLT3 mutations not detectable by this test.

Additional Information

The performance characteristics of this test were validated by UWHC Clinical Laboratories. The U.S. Food and Drug Administration (FDA) has not approved or cleared this test; however, FDA approval or clearance is currently not required for clinical use of this test. The results are not intended to be used as the sole means for clinical diagnosis or patient management decisions. The UWHC Clinical Laboratories is authorized under Clinical Laboratory Improvement Amendments (CLIA) to perform high-complexity testing.


A professional fee is associated with this test.(CPT Code G0452).