Test Name FLT3 TKD mutation analysis (qualitative) Assay Type Capillary Electrophoresis This test is performed by using the LeukoStrat® FLT3 Mutation Assay from Invivoscribe. Method Description Fluorescently labeled PCR primers are used to amplify the activation loop region of exon 20. The activation loop targeted PCR includes a mutagenic primer that creates an additional EcoRV restriction site. The amplicon is digested with EcoRV restriction enzyme, which digests the PCR product at two sites from normal individuals , and at a single site from individuals with the TKD mutation. This assay tests for TKD region mutations that disrupt the EcoRV enzyme’s restriction site sequence, and therefore it is not limited to the D835 or I836 mutation detection. Indications for Testing • At initial diagnosis of AML • Stratification of high and low risk AML •  Recurrence of leukemia after induction therapy on patients not initially screened for FLT3 mutations Interpretation Turn-around Time Specimen Requirements Shipping Conditions Storage Conditions An interpretive report will be issued indicating whether a FLT3 TKD mutation was detected 1-3 business days •  3 mL peripheral blood in EDTA, ACD or Heparin •  1 mL bone marrow in EDTA, ACD or Heparin •  250 ng of previously isolated DNA  Ambient or Cool;  Do not freeze • Room Temp up to 72 hours •  2-8 °C up to 7 days Molecular Diagnostic Tests LabPMM Services Catalog 2019 | 25 Clinical Information Acute myeloid leukemia (AML) generally has a poor prognosis. Several studies have described mutation of the FLT3 receptor as an important prognostic factor in AML, as FLT3 mutants have a worse outcome and response to standard chemotherapeutic interventions1-2 . The FLT3 gene encodes a tyrosine kinase receptor that plays a key role in controlling survival, proliferation and differentiation of hematopoietic cells. Mutations in this gene are critical in causing a deregulation of the delicate balance between cell proliferation and differentiation. The second most common mutation type in the FLT3 gene is a tyrosine kinase domain (TKD) point mutation in the codon for an aspartate (D835) or an isoleucine (I836) residue that is located in the activation loop of the FLT3 protein. FLT3 TKD mutations are caused by nucleic acid substitutions and/or deletions that result in a change in the amino acid sequence in this highly conserved catalytic center. TKD mutations, such as D835 substitutions and deletions, result in constitutive autophosphorylation and activation of FLT33-4 . In order to determine the best treatment options, it is recommended that patients with AML be screened for the presence of FLT3 mutations. FLT3 TKD References 1.  Mrozek, K. et al. (2007) Clinical relevance of mutations and gene-expression changes in adult acute myeloid leukemia with normal cytogenetics: are we ready for a prognostically prioritized molecular classification. Blood 109(2):431-448. 2.  Yanada M, et al. (2005) Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations for acute myeloid leukemia: a meta-analysis. Leukemia 19 (8): 1345-1349. 3.  Yamamoto, Y. et al. (2001) Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 97(8):2434-9. 4.  Gilliland, D.G. et al. (2002) The roles of FLT3 in hematopoiesis and leukemia. Blood 100(5):1532-154 . 24