Expression of Erythropoietin Receptor (EPO-R) and In Vitro Functional Effects of Epoetin in B-Cell Malignancies.

Background

The biological effects of erythropoietin (EPO), the principal regulator of erythropoiesis, may extend to several non-hematopoietic tissues and cells. Recent studies suggest that EPO and erythropoietin receptors (EPO-R) may be expressed by solid tumors including breast, prostate, ovarian, and renal cancers. Studies also suggest that the EPO/EPO-R pathway might regulate survival and growth of cancer cells. Since recombinant human EPO (Epoetin) is frequently used to treat anemia in hematological malignancies, we studied the EPO-R expression as well as the in vitro effects of epoetin on tumor cells from patients with B-cell chronic lymphocytic leukemia (B-CLL), mantle cell lymphoma (MCL) and multiple myeloma (MM).

Methods

The genomic expression of EPO-R was analyzed in highly purified tumor cells by RT-PCR. Flow cytometry was applied to detect EPO-R protein expression on the cell surface (using EPO-dig + anti-dig FITC, F. Hoffmann-La Roche, Basel, Switzerland) and intracellularly (in permeabilized cells, using Santa Cruz polyclonal anti-EPO-R antibody). The functional capability of epoetin alfa, epoetin beta, and darbepoetin alfa to activate tumor cells in vitro (alone, and in combination with CD40L-transfected fibroblasts as co-stimulators) was assessed by proliferation assay (DNA synthesis) and by flow cytometry (CD69).

Results

EPO-R mRNA was detected in peripheral blood mononuclear cells in 32/41 (78%) B-CLL and 5/7 (71%) MCL patients. The positive results were confirmed in highly purified (>95%) tumor cells from 7 B-CLL patients. In MM patients, bone marrow mononuclear cells from 13/14 (93%) patients and highly purified (>98%) plasma cells from 3/3 (100%) patients were positive for EPO-R mRNA. However, surface EPO-R protein could not be detected in any of 9 tested B-CLL patients (5 of which were positive and 4 negative for EPO-R mRNA). In contrast, intracellular EPO-R protein was detected in 8/8 (100%) PCR-positive patients with B-CLL including 5 patients who had a negative surface EPO-R staining. Intracellular EPO-R protein was also found in 4/4 (100%) tested patients with MCL. CD69 expression and [³H]-thymidine incorporation were assessed in 8 patients with B-CLL and 4 patients with MCL after stimulation of tumor cells with epoetin. CD69 expression was not induced and no epoetin-induced proliferation was observed following in vitro stimulation with any of the 3 epoetin preparations.

Conclusion

The results indicate that EPO-R mRNA is expressed by tumor cells in most patients with B-CLL, MCL or MM. However, it is unclear whether the gene expression translates into a fully functional protein. EPO-R protein could be detected intracellularly in most samples but not on the cell surface (as assessed by different flow cytometry techniques). Furthermore, in vitro tumor cell stimulation even with high concentrations of epoetin did not induce proliferation/activation.