Investigation of thrombopoietin (TPO) function in primary chronic lymphocytic leukemia cells.

Abstract

6613 Background: Immune thrombocytopenia purpura (ITP) is associated with poor prognosis of chronic lymphoid leukemia (CLL). TPO mimetics have been approved for the treatment of chronic ITP and have been suggested for the use in CLL-related ITP. Before testing this hypothesis in the clinic, the hypothesized role of TPO function in CLL cells was investigated. TPO mimetics stimulate platelet production via activation of the TPO receptor, c-Mpl, thus we analyzed c-Mpl cell surface expression and functional response to TPO in primary CLL cells in samples collected from early and late stage CLL patients. Methods: Peripheral blood was collected from CLL patients and mononuclear cells isolated by Ficoll separation. CLL cells were analyzed by flow cytometry and gated based upon viability and CD5+/CD19+ expression. c-Mpl expression was measured using a tested and validated novel c-Mpl-specific monoclonal antibody by flow cytometry. CLL samples were stimulated with TPO (10 ng/mL for 10 min) and induction of pSTAT5 was measured by intracellular flow cytometry. Platelets (CD41a+) provided internal controls for c-Mpl expression and response to TPO and phorbol myristate acetate was used as a control stimulation to demonstrate that CLL cells were responsive. Results: Analysis of expression and function of c-Mpl was performed on CLL samples (n=89) including 14 patients with thrombocytopenia. While robust c-Mpl expression was observed consistently in platelets from normal and CLL patients (normal: 31.90 ± 2.83, CLL: 26.76 ± 3.31; mean ± SEM), no significant expression of c-Mpl was observed on CD5+/CD19+ CLL cells (1.06 ± 0.01; mean ± SEM). No activation of downstream signaling (pSTAT5) was detected in CLL cells in any of the samples stimulated with TPO (normal: 0.90 ± 0.007, CLL: 1.04 ± 0.02; mean ± SEM), while robust pSTAT5 was detected in platelets (normal: 10.22 ± 0.29, CLL: 6.21 ± 0.23; mean ± SEM). Conclusions: A robust/sensitive platform was developed to profile biologically relevant c-Mpl expression and function in platelets. Using this platform we demonstrate a lack of c-Mpl expression and functional response to TPO challenge in CLL cells suggesting that CLL cells are unlikely to be activated in patients treated with TPO mimetics.