C-Mpl Is Not Expressed or Functional At Detectable Levels on Primary Chronic Lymphocytic Leukemia (CLL) Tumor Samples

Abstract 2849

The development of thrombocytopenia is associated with progression of CLL to a more advanced stage and is commonly used to classify CLL into the higher disease risk categories. It has been estimated that 5% of CLL patients will present with associated immune thrombocytopenia purpura (ITP) and immune mediated destruction can exacerbate thrombocytopenia related to bone marrow infiltration. Thromopoietin (TPO) mimetics stimulate platelet production though activation of the thrombopoietin receptor (c-Mpl) on megakaryocytes and are currently indicated for chronic ITP. In principle, this approach could be considered for treatment of ITP in CLL patients. In order to rule out the possibility that c-Mpl is functionally expressed in CLL tumor cells; we analyzed expression and TPO-dependent function in primary patient samples.

We have developed assays for the analysis of c-Mpl expression on the surface of B-cells in CLL patient samples and to assess functional response to ex-vivo stimulation of theses primary samples with TPO. Peripheral blood was collected from CLL patients and mononuclear cells isolated by Ficoll separation. This cohort included treatment naïve CLL samples (n=57) and CLL samples collected from subjects undergoing active treatment (n=30). CLL cells were analyzed by flow cytometry and identified based upon viability and CD5+/CD19+ expression. c-Mpl expression was measured using a novel c-Mpl-specific monoclonal antibody that was shown to be specific for c-Mpl by flow cytometry. Residual platelets associated with individual PBMC aliquot served as an internal control for c-Mpl expression. To investigate c-Mpl function, CLL cells were stimulated with rhTPO (10 ng/mL for 10 min) and induction of pSTAT5 was measured to assess a functional response to ex-vivo stimulation of TPO. In addition c-Mpl mRNA expression was surveyed in CLL by using mRNA microarray analyses to correlate c-Mpl mRNA expression with protein and functional expression of c-Mpl.

Robust c-Mpl protein expression was observed in platelets from normal and CLL patients (fold over control -normal: 31.90 ± 6.39, CLL: 26.76 ± 6.57; mean ± 95%CI), no significant expression of c-Mpl was observed on CD5+/CD19+ CLL cells (fold over control −1.06 ± 0.021; mean ± 95%CI). No induction of STAT5 phosphorylation was detected in B-cell CLL cells in any of the samples stimulated with TPO (fold over control - normal: 0.90 ± 0.02, CLL: 1.04 ± 0.034; mean ± 95%CI). Microarray analysis of the CLL samples demonstrated c-Mpl mRNA expression levels equivalent to background across all CLL samples analyzed (intensity score −10.3 ± 3.0; mean ± 95%CI). In conclusion, we demonstrated a lack of cell surface c-Mpl protein expression in CLL cells. The additional data suggesting the lack of evidence for significant expression of c-Mpl mRNA expression supports the hypothesis that CLL cells do not express c-Mpl and unlikely to be stimulated in patients treated with TPO mimetics. This hypothesis will need to be tested in an appropriate clinical trial to assess the potential benefits of TPO mimetics for the treatment of thrombocytopenia in CLL patients.