Gene expression in signaling subsets of AML blasts induced by G-CSF

While the response to G-CSF is a significant predictor of AML outcome, the nature of cells responding differentially to this and other cytokines remains unclear. Based on gene expression signatures observed in G-CSF Responsive Cells (RC) and non-G-CSF Responsive Cells (NRC) a cohort of AML samples were segregated into two types. In Type1 both cell subsets displayed an undifferentiated phenotype. In the Type2 AML the RC showed a very similar undifferentiated progenitor-like phenotype as in Type1 RC, whereas the NRC exhibited a more differentiated hematopoietic phenotype. The frequency of RC per sample was a determinant of whether a patient belonged to Type1 or Type2. Type2 RC had higher expression of CD321, higher colony formation capacity and greater responsiveness to cytokines secreted by NRC. Further analysis of clinical mRNA databases revealed that CD321 was a prognostic indicator for AML and a potential marker of clonogenic activity associated with this AML cancer lineage. Cryopreserved PBMC from 11 AML patinets were stimulated with G-CSF. Following stimulation cells were fixed, permeabilized and stained for selected surface markers (CD3, CD33, CD45) and and phospho-Stat3/5. After staining subsets positive and negative for phospo-Stat3/5 were sorted out for further microarray analysis. T-cells were sorted from each sample to control for the RNA quality by comparisons to RNA prepared from live T-cells sorted form healthy donors.