122 Gel & Capillary | RUO Assays Invivoscribe 2019 | 123 Gel & Capillary RUO Assays FLT3 Mutation Assays Assay Use FLT3 Mutation Assays are useful for the study of: • Identifying FLT3 mutations in patients with AML • Discriminating between high and low risk patients. FLT3 mutations portend a worse prognosis for patients with AML. Therefore patients testing positive for FLT3 mutations may benefit from a more aggressive treatment regimen Summary and Explanation of the Test Acute myeloid leukemia (AML) in general has a poor prognosis. Recent studies have described mutation of the FLT3 (fms-related tyrosine kinase 3) receptor to be the most important prognostic factor in AML, with FLT3 mutants having a worse outcome and response to standard chemotherapeutic interventions. Accordingly, identification of an FLT3 mutation in AML may indicate a need to reassess and modify standard treatment options. All types of AML can have activating mutations in the FLT3 gene. Mutation of the FLT3 receptor, either by internal tandem duplication (ITD) of the juxtamembrane domain or by point mutation of the aspartic acid residue (D835) or isoleucine (I836) in the activation loop of the kinase domain, causes constitutive activation of the FLT3 receptor. This test kit includes 3 master mixes. The ITD and D835 master mixes target the juxtamembrane and kinase domain regions (respectively). The third master mix, the Specimen Control Size Ladder, targets multiple genes and generates a series of amplicons of 100, 200, 300, 400, and 600 base pairs to ensure that the quality and quantity of input DNA is adequate to yield a valid result. PCR products can be analyzed by capillary electrophoresis with use of ABI instruments or standard agarose TBE gel electrophoresis with ethidium bromide staining. Background Mutations in the fms related tyrosine kinase 3 (FLT3) gene are the most common mutations found in acute myeloid leukemia (AML), occurring in approximately 30% of patients at the time of diagnosis, and are characterized by an aggressive phenotype with a high prevalence of relapse1,2,3 . 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. The second most common mutation type in the FLT3 gene is a tyrosine kinase domain (TKD) point mutation in aspartate (D835) or isoleucine (I836). TKD mutations result in constitutive autophosphorylation and activation of FLT35,6 . TKD mutations have been linked to poor overall survival, but to a lesser extent as compared to ITD mutations. Specimen Requirements 1. 5 cc of peripheral blood, bone marrow biopsy, or bone marrow aspirate anti-coagulated with heparin or EDTA; or, 2. Minimum 5 mm cube of tissue; or, 3. 2 μg of genomic DNA; or, 4. Formalin-fixed, paraffin-embedded tissue or slides. Reference 1. Acute Myeloid Leukemia, Clinical Practice Guidelines in Oncology, National Comprehensive Cancer Network (v.2.2014). 2. Lowenberg, B. et al. “Acute myeloid leukemia.” N Engl J Med 341(14):1051-62 (1999). 3. Thiede, C. et al. “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 (2002). 4. Nakao, M. et al. “Internal tandem duplication of the FLT3 gene found in acute myeloid leukemia.” Leukemia 10(12):1911-18 (1996). 5. Yamamoto, Y et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 97(8):2434-9 (2001). 6. Gilliland, DG et al. The roles of FLT3 in hematopoiesis and leukemia. Blood 100(5):1532-154 (2002). Reagents Controls Concentration Units in 33 Reaction Assay Units in 330 Reaction MegaKit IVS-0017 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-P001 Clonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes IVS-0000 Polyclonal Control DNA 200 μg/mL 1 x 100 μL tube 5 x 100 μL tubes Master Mixes Target Units in 33 Reaction Assay Units in 330 Reaction MegaKit FLT3 ITD Master Mix FLT3 ITD 1 x 1500 μL tube 10 x 1500 μL tubes FLT3 D835 Master Mix FLT3 TKD 1 x 1500 μL tube 10 x 1500 μL tubes Specimen Control Size Ladder Multiple Genes 1 x 1500 μL tube 10 x 1500 μL tubes Gel Electrophoresis Detection Data was generated using the FLT3 ITD Master Mix and amplified products were run on a 2% agarose TBE gel. Lane 1 is a FLT3 ITD control*; lane 2 is a 10% dilution of a FLT3 ITD control; and lane 3 is IVS-0000, which is representative of a WT product. A standard 100 bp DNA size ladder was run in the lanes flanking the test samples. *IVS-0050 performs comparable to IVS-0017 clonal control DNA, which is included in the kit as the positive control. Capillary Electrophoresis Detection (ABI) Data was generated using the FLT3 ITD Master Mix and amplified products were run on an ABI 3100 instrument. Panel 1 is the recommended FLT3 ITD positive control; panel 2 is data generated testing a 10% dilution of the positive control; and Panel 3 is IVS-0000, which is representative of a WT product. Ordering Information Catalog # Products Quantity 1-412-0010 FLT3 Mutation Assay - Gel Detection 33 reactions 1-412-0020 FLT3 Mutation Assay MegaKit - Gel Detection 330 reactions 1-412-0031 FLT3 Mutation Assay - ABI Fluorescence Detection 33 reactions 1-412-0041 FLT3 Mutation Assay MegaKit - ABI Fluorescence Detection 330 reactions These products are intended for Research Use Only. Not for use in diagnostic procedures. Use of this product may require nucleic acid amplification methods such as Polymerase Chain Reaction (PCR). Any necessary license to practice amplification methods or to use amplification enzymes or equipment covered by third party patents is the responsibility of the user and no such license is granted by Invivoscribe, Inc., expressly or by implication. These products are not currently available for sale or use in the United States.