Test Name FLT3 ITD Signal Ratio (SR) mutation analysis Assay Type Capillary Electrophoresis This test is performed by using the LeukoStrat® FLT3 Mutation Signal Ratio Assay from Invivoscribe. Method Description Primers flanking exons 14 and 15 of the FLT3 gene are used to amplify DNA extracted from a patient sample. The size of the resulting internal tandem duplication (ITD) PCR product is determined by capillary electrophoresis and the signal ratio (SR) is calculated by dividing the mutant peak area by the wild-type peak area. Indications for Testing • At initial diagnosis of AML • Stratification of high and low risk AML • ITD positive by qualitative FLT3 ITD test •  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 the mutant:wild-type signal ratio of the FLT3 ITD mutation 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 | 23 Clinical Information The FLT3 tyrosine kinase is one of the most commonly mutated genes in acute myeloid leukemia (AML), occurring in approximately 40% of patients at the time of diagnosis1. Although generally associated with normal cytogenetics where patients have standard risk of relapse, FLT3 mutations have also been identified in sub-groups of patients with chromosomal abnormalities that are associated with high risk of disease relapse2-3 . The most prevalent and clinically significant type of FLT3 mutation is an internal tandem duplication (ITD) in the juxtamembrane domain4 . Many clinical studies have found that FLT3 ITD mutations are associated with higher concentrations of leukemic cells in both blood and bone marrow, increased incidence of relapse, and decreased overall survival. For myelodysplastic syndrome, rare FLT3 ITD mutations are also associated with the disease transformation to AML5 . Additionally, the signal ratio of FLT3 ITD mutations compared to wild-type has been demonstrated to provide prognostic value in both adult and pediatric AML patients6-7 . Patients with high FLT3 ITD signal ratios are likely to have less favorable outcomes. In order to determine the best treatment options, it is recommended that patients with AML be screened for the presence of FLT3 mutations. FLT3 ITD Signal Ratio References 1.  Wei, A. et al. (2017) Midostaurin, enasidenib, CPX-351, gemtuzumab ozogamicin, and venetoclax bring new hope to AML. Blood 130(23):2469-2474. 2.  Thiede, C. et al. (2002) “Analysis of FLT3-activating mutations in 979 patients with acute myelogenous leukemia: association with FAB and identification of subgroups with poor prognosis.” Blood 99(12): 4326-35. 3.  Nakao, M. et al. (1996) “Internal tandem duplication of the FLT3 gene found in acute myeloid leukemia.” Leukemia 10(12):1911-18. 4.  Pinheiro, R. et al. (2008) “FLT3 internal tandem duplication during myelodysplastic syndrome follow-up: a marker of transformation to acute myeloid leukemia.” Cancer Genetics & Cytogenetics 183(2) 89-93. 5.  Meshinchi, S. et al. (2006) “Clinical implications of FLT3 mutations in pediatric AML.” Blood 108(12) 3654-61. 6.  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):431-48. 7.  Pratcorona, M. et al. (2013) “Favorable outcome of patients with acute myeloid leukemia harboring a low-allelic burden FLT3-ITD mutation and concomitant NPM1 mutation: relevance to post-remission therapy.” Blood 121(14): 2734-38. 22