Invivoscribe 2019 | 85 Gel & Capillary CE-IVD Assays 84 Gel & Capillary | CDx IVD Assay - Japan TM JM TK1 TK2 Wild Type = 327 bases Internal Tandem Duplication 330 bases D835/I836 EcoRV digest Undigested = 145 bases or 147 bases Wild Type digested = 79 bases Mutant digested, point mutation = 127 bases Mutant digested, deletion = 124 bases GATATC _ > Available in Japan CDx FLT3 Mutation Assay Intended Use The LeukoStrat® CDx FLT3 Mutation Assay is a PCR-based in vitro diagnostic test designed to detect internal tandem duplication (ITD) mutations and tyrosine kinase domain (TKD) 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 with AML for whom Gilteritinib Fumarate treatment is being considered. Summary and Explanation of the Test AML in general has a poor prognosis. Assessment of the mutation status of the FLT3 (fms related tyrosine kinase 3) receptor gene in karyotype normal AML is the most important prognostic indicator of disease outcome, which is often substantial, as many studies in AML have shown that the presence of FLT3 activating mutations portends a poor prognosis1,2 . The LeukoStrat CDx FLT3 Mutation Assay targets regions of the FLT3 gene to identify ITD mutations and TKD mutations, such as the D835 and I836 mutations, and has been validated in an international clinical trial. The LeukoStrat CDx FLT3 Mutation Assay includes reagents, equipment, software and procedures for isolating mononuclear cells and extracting DNA from patient specimens to determine if FLT3 mutations are present. DNA is amplified via PCR and the amplicons are detected via capillary electrophoresis. FLT3 mutation status is determined by the LeukoStrat CDx FLT3 Software. A FLT3 ITD and/ or TKD mutation is reported as Positive if the mutant:wild-type signal ratio meets or exceeds the clinical cutoff of 0.05. Method Description 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 330±1 bp (Figure 1). 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). Reference 1. Murphy KM et al., A Clinical PCR/Capillary Electrophoresis Assay for the Detection of Internal Tandem Duplication and Point Mutation of the FLT3 Gene. J. Mol. Diag. 5:96-102 (2003). 2. Yamamoto, Y., et al., Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood, 97(8):2434-9 (2001). Ordering Information Catalog # Products Quantity K-412-0331 LeukoStrat® CDx FLT3 Mutation Assay (Japan) 33 reactions K-412-0341 LeukoStrat® CDx FLT3 Mutation Assay Software (Japan) 1 CD complimentary with purchase These are in vitro diagnostic products, and are available for sale or use within Japan. 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. Reagents Reagent Name Units in Assay FLT3 Extraction Control 1 x 1800 μL tube FLT3 ITD Master Mix 1 x 1500 μL tube FLT3 TKD Master Mix 1 x 1500 μL tube FLT3 ITD Positive Control 1 x 100 μL tube FLT3 TKD Positive Control 1 x 100 μL tube FLT3 No Template Control 1 x 200 μL tube Taq DNA Polymerase Enzyme 1 x 200 μL tube EcoRV Enzyme 1 x 200 μL tube All reagents should be stored at -15 to -30 degrees C. The only internationally standardized assay for FLT3 Signal Ratio mutation analysis for selection of acute myeloid leukemia (AML) patients eligible for treatment with Gilteritinib Fumarate. Figure 1 リューコストラット CDX FLT3 変異検査 Gel & Capillary IVD Assays