In vivo and ex vivo bioassay of FLT3 inhibition by lestaurtinib in 2 representative patients. (A) Direct in vivo bioassay. FLT3 was immunoprecipitated from BM MNCs, and sequential Western blot was performed using 4G10 antiphosphotyrosine antibodies and anti-FLT3 antibodies. (B) “Ex vivo” FLT3 PIA assay. TF/ITD cells were exposed to patient plasma obtained 12 hours after lestaurtinib administration on trial days 14, 28, and 56, then lysed and analyzed by sequential immunoblotting with 4G10 and anti-FLT3 antibodies. Densitometric analysis of phosphorylated FLT3, normalized for the amount of total FLT3, is displayed graphically. Patient 13, a FLT3 WT patient, made no clinical response to lestaurtinib, with direct assay revealing no evidence of in vivo FLT3 inhibition, and ex vivo assay confirming incomplete inhibition of TF/ITD FLT3 by patient plasma at all timepoints. In contrast, Patient 19, a clinically responsive patient with a FLT3 point mutation, showed significant in vivo reduction in FLT3 phosphorylation by day 14. Reductions in blast numbers limited the validity of the direct assay on days 28 and 56, by which time the FLT3 signal had become almost undetectable. Ex vivo assay, however, confirmed the presence of sufficient continuing FLT3 PIA on days 14 and 28 to almost fully inhibit TF1/ITD FLT3 phosphorylation.