Receptor activator of NF-kappaB ligand (RANKL) is expressed in chondroblastoma: possible involvement in osteoclastic giant cell recruitment

Abstract

Aims: Chondroblastoma is a rare, locally aggressive bone tumour that causes osteolytic destruction at the epiphyseal end of the affected bone. It is possible that tumour cells may stimulate osteoclastogenesis and osteolytic destruction through the production of receptor activator of NF-kappaB ligand (RANKL), which is a key molecule essential for regulating osteoclast formation and activity. Therefore, the expression of RANKL at both the mRNA and the protein level was investigated in chondroblastoma tumour tissue obtained from patients.

Methods: The expression of RANKL gene transcripts was analysed by the reverse transcription-polymerase chain reaction (RT-PCR), and the cellular localisation of RANKL mRNA and protein was demonstrated by means of in situ hybridisation and immunohistochemistry.

Results: RT-PCR analysis indicated that RANKL mRNA was present in all chondroblastoma specimens and normal cancellous bone samples, but not in normal articular cartilage and chondrosarcoma tissues. In contrast, gene transcripts of osteoprotegerin (OPG), the decoy receptor of RANKL, were detected in all types of tissues. The chondroid origin of neoplastic mononuclear cells in chondroblastoma was confirmed by positive S-100 immunohistochemical staining. Both RANKL mRNA and protein were exclusively expressed in these neoplastic mononuclear cells.

Conclusions: These findings suggest that RANKL may be involved in the tumour cell induced recruitment of osteoclast-like cells and consequent osteolytic bone destruction in chondroblastoma.

Figure 1

Chondroblastoma affecting medial femoral condyle. Computed tomography scan shows a large expansile lesion affecting the medial femoral condyle and extending into the lateral. Cortical thinning expansion and disruption are noted.

Figure 2

Gene expression of receptor activator of NF-κB ligand (RANKL) in chondroblastoma by reverse transcription-polymerase chain reaction. Lane M, 100 bp DNA ladder; lanes 1–3, chondroblastoma; lanes 4–6, chondrosarcoma; lane 7, normal cancellous bone; lane 8, normal cartilage. The sizes of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RANKL, and osteoprotegerin (OPG) PCR products were 206 bp, 486 bp, and 324 bp, respectively.

Figure 3

Cellular localisation of RANKL in chondroblastoma by in situ hybridisation (ISH) and immunohistochemistry (IHC). (A) Histological appearance of chondroblastoma by haematoxylin and eosin staining. The mononuclear cells show distinct cytoplasmic boundaries and focal mineralisation is seen between individual tumour cells. Scattered osteoclast-like giant cells (OC) are also present. (B, C) RANKL gene expression is detected in the neoplastic mononuclear cells by ISH. Hybridisation signals are dark blue in colour. No hybridisation signals were seen in osteoclast-like giant cells (OC). (D) The ISH negative control treated with RNase before hybridisation shows no detectable signals for RANKL gene transcripts. (E) RANKL immunoreactivity is detected in the cytoplasm of neoplastic mononuclear cells. Immunoreactivity is seen as a brown colour. (F) The negative control for RANKL IHC shows the specificity of the primary antibody. (G, H) RANKL positive neoplastic mononuclear cells show a positive reaction for S-100 protein. Immunoreactivity is seen as a brown colour.