FLT-3 Activity and Its Response to Drugs Can Be Determined in AML Blast Cells by FLT-3 Phosphorylation Status Using Flow Cytometry.

One of the most common molecular defects identified in acute myeloid leukemia (AML) patients is an activating mutation of FLT3 tyrosine kinase. The identification of activated FLT3 as a contributor to the cause and progression of much leukemia has led to its consideration as a potential target for therapy. Since small molecule FLT3 kinase inhibitors are actually in clinical trials; a robust and standardized method for screening of FLT3 receptor activation is necessary.

We evaluated the expression level of FLT3 receptor (CD135) by Facs analysis. We developed a flow cytometry method to measure FLT3 phosphorylation (P-FLT3) in samples with <10 (e)5 cells. The amount of P-FLT3 in the samples was determined as the mean fluorescence intensity (MFI). The P-FLT3 status of the treated samples was expressed as a percentage of the untreated control (100%). The method was first validated in FLT3 wild-type (HL-60) and mutant (MV4-11/ITD⁺) as well as FLT3 negative (K562) cell lines. The method also provides to be reproducible with samples AML from patients. Analysis was performed after exposure to drugs, in vitro and in vivo. In response to increasing drugs concentration (CEP-701 and SU11657) there was a linear reduction in P-FLT3. The results validate a rapid method to detect P-FLT3 protein at the single cell level by flow cytometry, and enable an accurate assessment of FLT3 kinase activity in blast cells in response to novel tyrosine kinase inhibitors.