LeukoStrat® CDx FLT3 Mutation Assay

The only internationally standardized CE-IVD assay for FLT3 Signal Ratio mutation analysis.


For selection of acute myeloid leukemia (AML) patients eligible for treatment with midostaurin.

Intended Use

The LeukoStrat® CDx FLT3 Mutation Assay is a PCR-based in vitro diagnostic test designed to detect internal tandem duplications (ITD) FLT3 mutations and tyrosine kinase domain (TKD) FLT3 mutations in genomic DNA extracted from mononuclear cells obtained from peripheral blood or bone marrow aspirates of patients diagnosed with acute myelogenous leukemia.

The LeukoStrat® CDx FLT3 Mutation Assay is used as an aid in the assessment of patients for whom midostaurin treatment is being considered.

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Background

Primers flanking exons 14, 15 and the activation loop region of exon 20 of the FLT3 gene are used to amplify DNA extracted from a patient sample. The forward and reverse PCR primers are fluorescently labeled with different fluorophores that serve to confirm the presence of sample signal.

The size of the ITD PCR product is determined by capillary electrophoresis and the signal ratio (SR) compares the signal intensity of the mutant to the wild-type. FLT3 TKD PCR product is digested with EcoRV and the presence of the mutation is further assessed using capillary electrophoresis and the signal ratio (SR) compares the signal intensity of the mutant to the wild-type.

The FLT3 Mutation Assay includes reagents, software, and procedures for isolating mononuclear cells and extracting DNA from patient specimens to determine if FLT3 mutations are present.

Specimen Requirement

At least 1 mL of peripheral blood or 0.25 mL of bone marrow anticoagulated with sodium heparin.

Method

Internal Tandem Duplication (ITD) Mutations of FLT3

The LeukoStrat® CDx FLT3 Mutation Assay uses fluorescently labeled primers that are in the JM region. Wild-type FLT3 alleles will amplify and produce a product at 327±1 bp as measured by this assay, while alleles that contain ITD mutations will produce a product that exceeds 327±1 bp (Figure 1).

Tyrosine Kinase Domain (TKD) Mutations of FLT3

The LeukoStrat® CDx FLT3 Mutation Assay uses primers that lie on either side of the TKD region. The FLT3 target region is amplified using PCR and then an EcoRV restriction digest is performed. Wild-type alleles of the FLT3 gene yield digestion products of 79±1 bp whereas mutant alleles yield products of 125±1 bp or 127±1 bp from the original undigested amplicon product of 145±1 bp or 147±1 bp, as measured by this assay (Figure 1).

Principle of FLT3 Mutation Analysis



Figure 1: Depicted is a representation of the FLT3 juxtamembrane (JM) region (TM = transmembrane) and the activating loop of the tyrosine kinase (TK) domain. Black arrows represent the relative positions of primers that target the JM region for ITD or the activating loop of the kinase domain for TKD. Colored dots represent fluorophores on labeled primers. The yellow box has vertical black lines that represent the position of the EcoRV restriction digest sites.


References

  1. Murphy KM, et al. Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Change Reaction and Capillary Electrophoresis Assay. Journal of Molecular Diagnostics, 2003, 5:96-102.
  2. Yamamoto, Y, et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 2001, 97(8):2434-9.
Cat. No.
DescriptionAmount
K-412-0291
LeukoStrat® CDx FLT3 Mutation Assay
33 reactions
K-412-0281
LeukoStrat® CDx FLT3 Mutation Assay Software
1 CD complimentary with purchase

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Source:
Product flyer: LeukoStrat® CDx FLT3 Mutation Assay

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Brands: Invivoscribe
​Product groups: Assays
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