Test Name FLT3 ITD mutation analysis (qualitative) Assay Type Capillary Electrophoresis This test is performed by using the LeukoStrat® FLT3 Mutation Assay from Invivoscribe. Method Description Primers flanking exons 14 and 15 of the FLT3 gene are used to amplify the patient’s DNA. The forward and reverse PCR primers are fluorescently labelled with different fluorophores that confirm the presence of sample signal. The size of the ITD PCR product is determined by capillary electrophoresis. Wild type FLT3 alleles will amplify and produce a product measured at 327±1 bp as generated by the FLT3 Mutation Assay, while alleles that contain ITD mutations will be greater than or equal to 330 bp. 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 the FLT3 internal tandem duplication 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 | 21 References 1.  Grafone, T. et al. (2012) An overview on the role of FLT3-tyrosine kinase receptor in acute myeloid leukemia: biology and treatment. Oncology Reviews, 6(1), e8. 2.  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. 3.  Gilliland, D.G. et al. (2002) The roles of FLT3 in hematopoiesis and leukemia. Blood 100(5):1532-1542. 4.  Murphy, K.M. et al. (2003) Detection of FLT3 Internal Tandem Duplication and D835 Mutations by a Multiplex Polymerase Change Reaction and Capillary Electrophoresis Assay. Journal of Molecular Diagnostics 5:96-102. FLT3 ITD Clinical Information The fms related tyrosine kinase 3 (FLT3) gene codes for a transmembrane receptor/signaling protein of the tyrosine kinase group and is located on chromosome 13q12, containing 24 exons and spanning about 100 kB1 . Binding of FLT3 ligand to the FLT3 receptor ultimately leads to production of proteins that cause cell growth and inhibit cell death through apoptosis. Mutations in FLT3 have been found in some hematopoietic neoplasms, and are particularly common in adult acute myeloid leukemia (AML), with an overall incidence of approximately 40%1 . The highest mutation rates are seen in adult patients with AML and normal- or intermediate-risk cytogenetics, and patients with acute promyelocytic leukemia2 . The most prevalent and clinically significant type of FLT3 mutation is an internal tandem duplication (ITD), or length mutation (LM) in the juxtamembrane domain of the receptor. Many clinical studies have found FLT3 ITD mutations to be associated with higher numbers of leukemic cells in both blood and bone marrow, increased incidence of relapse, and decreased overall survival. It is thought that FLT3 mutations lead to constitutive FLT3 activation3-4 . In order to determine the best treatment options, it is recommended for patients with AML to be screened for the presence of FLT3 mutations. 20