title: Monitoring of FLT3 phosphorylation status and its response to drugs by flow cytometry in AML blast cells.
abstract: FLT3 mutation and overexpression in most acute myeloid leukaemia (AML) patients make this tyrosine kinase receptor an attractive therapeutic target. FLT3 kinase inhibitors are actually in clinical trials, thus it is critical to develop a reproducible and standardized method for screening of FLT3 activation and for monitoring its inhibition in response to drug in AML patients. We developed a flow cytometry method to analyse phosphorylated FLT3 (P-FLT3) in samples with <10(5) cells. The method was first validated in FLT3 wild-type (HL60/WT) and mutant (MV4-11/ITD(+)) as well as FLT3 negative (K562) cell lines. The method also proved to be reproducible in AML patient samples. Analysis was performed after exposure to drugs (CEP-701 and SU11657), in vitro and in vivo. In response to increasing drug concentrations, there was a linear reduction in P-FLT3. Intracellular flow cytometry analysis correlated with Western blot and XTT assays; flow cytometry data also correlated with FLT3 mutational status. The results highlight a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry which enables an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.
authors.affiliation: Institute of Hematology 'John Paul II' Centre for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy. tiziana.grafone@rm.unicatt.it
authors.forename: Tiziana
authors.initials: T
authors.lastname: Grafone
authors.forename: Michela
authors.initials: M
authors.lastname: Palmisano
authors.forename: Chiara
authors.initials: C
authors.lastname: Nicci
authors.forename: Alberto Maria
authors.initials: AM
authors.lastname: Martelli
authors.forename: Ottaviani
authors.initials: O
authors.lastname: Emanuela
authors.forename: Sergio
authors.initials: S
authors.lastname: Storti
authors.forename: Michele
authors.initials: M
authors.lastname: Baccarani
authors.forename: Giovanni
authors.initials: G
authors.lastname: Martinelli
mesh_terms: D000328:Adult; D000368:Aged; D045744:Cell Line, Tumor; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D051941:fms-Like Tyrosine Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D047428:Protein Kinase Inhibitors; C497970:FLT3 protein, human; D051941:fms-Like Tyrosine Kinase 3