Phospho-flow cytometry allows simultaneous quantitation of signalling pathway checkpoints in individual cells within a heterogeneous mixture.

Previously we demonstrated that IL6-induced pSTAT3 is associated with a better prognosis in multiple myeloma (MM). We now compare expression of pSTAT3/5 in malignant and non-malignant cells in MM, acute myeloid leukemia (AML) and chronic lymphoid leukemia (CLL) to investigate the hypothesis that elevated pSTAT3/5 expression is a hallmark of haematological malignancy.

Bone marrow cells (BM) from patients with MM, AML and blood from CLL were analysed for expression of pSTAT3/5 and CD130 (IL6 receptor) in malignant and autologous normal T and B cells.  Constitutive pSTAT3/5 were increased on 0%/0% of CLL, 27%/65% MM, 9%/64% AML samples. IL6-induced pSTAT3 were in 87% of MM patient BM, 100% AML and 27% of CLL samples.

In MM, constitutive and IL6-induced pSTAT3 expression were similar in the malignant clones and bystander T-cells, MM supernatants induced T-cell pSTAT3, and intact T-cell signalling was associated with better prognosis.

Higher IL6 receptor was only on MM cells. In AML and B-CLL, IL6R levels tended to be lower on malignant cells than autologous T-cells. CD8 cells had a lower response to IL6 correlating with a lower level of IL6R. There was a significantly worse prognosis for the patients whose cells were resistant to IL6 stimulation (Log-rank8.3; p<0.05), this included MM patients with T-cells sensitive to IL6 (Log-rank 6.5; p<0.01). In conclusion, we have compared pSTAT3 and pSTAT5 expression in malignant and non-malignant cells and demonstrated that constitutive expression and IL6 sensitivity are not unique to the malignant clone but are present in bystander immune cells.