Immunocytochemistry reveals RANKL expression of myeloma cells.

Two highly interesting papers related to osteoclastic activation in multiple myeloma were published in the December 15 issue of Blood.1,2 These studies were related to the imbalance of 2 molecules involved in the regulation of bone resorption: receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin. Croucher and colleagues2 found that mouse myeloma cells expressed RANKL mRNA and that RANKL protein could be demonstrated on the cell membrane. Contrary to these findings, Giuliani and colleagues1 could detect neither RANKL mRNA in human myeloma cells nor RANKL protein using immunohistochemistry of formalin-fixed, decalcified, and paraffin-embedded bone marrow specimens in patients with multiple myeloma. Although these discrepancies might let the reader assume that there are crucial differences between the murine model used2 and human myeloma disease, we wish to make the readers aware of our results, which show a strong RANKL expression of plasma cells in patients with multiple myeloma and osteolytic lesions. Pearse et al3 demonstrated that myeloma stimulates osteoclastogenesis by triggering an increase in RANKL and a decrease in osteoprotegerin expression in stromal cells, but RANKL expression could not be demonstrated in myeloma cells.3 On the other hand, human myeloma cell lines have been shown to express RANKL by other investigators.4 The immunohistochemical evaluation of formalinfixed, decalcified, and paraffin-embedded tissue sections may be associated with some limitations, since it is known that decalcification of formalin-fixed tissue samples reduces its antigenicity.5 To avoid the potential problems associated with this technique, we studied the RANKL expression in multiple myeloma patients with osteolytic lesions by immunocytochemistry using bone marrow smears and cytospins. Intracellular staining of bone marrow cells was performed using the same monoclonal mouse antibody against human RANKL as Giuliani and colleagues (clone 70525.11, R&D Systems, Minneapolis, MN). The antibody was applied for 30 minutes in a 1:10 dilution (antibody-diluent with background reducing components, Dako, Carpinteria, CA) for detection of RANKL-expressing cells. Rabbit antimouse (secondary antibody, Dako, Glostrup, Denmark) was applied for 30 minutes in a 1:50 dilution. Visualization was performed by the alkaline phosphatase/anti–alkaline phosphatase double bridge technique (APAAP; Dako). For intensification, the secondary antibody was used again for another 10 minutes of incubation, followed by another APAAP application for 10 minutes (Fuchsin Substrate-Chromogen-System, Dako). Counterstaining was done with hematoxylin. Control stainings were performed the same way but without using the primary antibody or using an irrelevant antibody (pancytokeratin). SaOs-2 cells, a human osteosarcoma cell line, which is known to express RANKL, served as a positive control. We could detect a strong cytoplasmatic expression of RANKL in bone marrow plasma cells in all 6 multiple myeloma patients investigated (Figure 1). Negative controls showed no staining reaction, and SaOs-2 cells were strongly positive for RANKL. Complementary to these results of cytoplasmatic RANKL expression, we performed flow cytometry for the analysis of RANKL expression on the cell surface using the same bone marrow aspirates. Plasma cells were identified as CD38 strongly positive ( ) and CD138 (B-B4) positive ( ) cells as described previously.6 Immunofluorescence staining of RANKL on the cell surface was performed using the same monoclonal mouse antibody against