Flow cytometric measurement of phosphorylated STAT5 in AML: lack of specific association with FLT3 internal tandem duplications

STAT5 phosphorylation has been noted in 69-95% of AML cases by Western blotting. We used flow cytometry to measure phosphorylated STAT5 on a semi-quantitative scale. The method was validated on K562 cells, which constitutively express phosphorylated STAT5, but lose this when BCR-abl tyrosine kinase activity is blocked by ST1571. Phosphorylated STAT5 was found to measure 2.22 +/- 0.09 relative fluorescence units (RFU) falling to 0.925 +/- 0.005 RFU in the presence of ST1571. Phosphorylated STAT5 expression was 0.99 to 2.09 RFU in 28 primary AML samples. There was no logical cut-off point between positive and negative fluorescence. FLT3 internal tandem duplications, found in 11/28 samples, were not significantly associated with the level of phosphorylated STAT5 expression. We conclude that STAT5 phosphorylation can be measured sensitively by flow cytometry in AML and that its expression should not be dichotomised as present or absent. (C) 2003 Elsevier Science Ltd. All rights reserved.