Detection of heterogeneous flt3 itd mutant variants in
- 1. Dr. Kamal Modi SN Gene lab, Surat
- 2. FLT- 3 Gene fms related tyrosine kinase 3 encodes a class III receptor tyrosine kinase that regulates hematopoiesis.
- 3. Binding of the fms-related tyrosine kinase 3 ligand to the extracellular domain Induces homo-dimer formation in the plasma membrane Auto-phosphorylation of the receptor activated receptor kinase subsequently phosphorylates and activates multiple cytoplasmic effector molecules Apoptosis, Proliferation, and Differentiation of hematopoietic cells in bone marrow
- 4. Mutations that result in the constitutive activation of this receptor result in acute myeloid leukemia and acute lymphoblastic leukemia
- 6. ITD Mutation FLT3/ITD mutations are in-frame duplications that invariably involve the juxtamembrane domain, a domain that exerts a negative regulatory function over the kinase activity.
- 8. Prognostic significance FLT3/ITD mutation is a poor prognostic factor, which occurs frequently in AML, and is associated with higher incidence of early disease relapse and shorter overall survival.
- 9. FLT3 ITD allelic ratio Ratio of mutant and wild type alleles of FLT3 If ratio is <0.5 , outcome will be favorable as compared to others having ratio more then that.
- 10. FLT3 Inhibitors Compete with ATP for binding to the active pocket of the kinases Inability to auto-phosphorylate or phosphorylate substrate proteins Signal transduction initiated by the mutated RTK is interrupted
- 11. Relapsed samples and samples with a high mutant-to-wild type allelic ratio were invariably more responsive to cytotoxicity from FLT3 inhibition compared with the samples obtained at diagnosis or those with a low mutant allelic ratio. Drugs used are : Quizartinib (AC220) Sorafenib , Sunitinib Lestaurtinib and Midostaurin
- 12. Methodology DNA was extracted from EDTA peripheral blood sample Polymerase chain reaction (PCR) was carried out using a set of primers flanking 14 and 15 of the FLT3 gene Capillary electrophoresis of the PCR product carried out in 3500 DX automated genetic analyzer using LIZ600 size standard FLT3-ITD allelic ratio was calculated as the ratio of the peak height of the mutant product to the peak height of the wild-type product
- 13. FLT3 wild type allele was detected at a computed molecular size of 326 bp and with a peak height of 122364 RFU. Along with this, two distinct FLT3 ITD molecular variants, coded V1 and V2 were detected with a molecular signature of 376 and 394 bp respectively and with a peak height of 36037 and 36708 RFU respectively. Result
- 14. Allele type RFU Bp FLT3 WILD TYPE 122364 326 FLT3 ITD (V1) 36037 376 FLT3 ITD (V2) 36708 394 The clonal variants carried a "mutant to wild type" allelic ratio value of 0.295 & 0.299 for V1 and V2 respectively indicating favorable prognosis as compared to have more then 0.5 allelic ratio .
- 15. We hypothesize that successful treatment will eventually eliminate either or both of the mutant variants and conversely a resistant one will enrich any one of them leading to identification of a potential marker linked to therapeutic resistance. Discussion