Diagnostic utility of histone H3.3 G34W, G34R, and G34V mutant-specific antibodies for giant cell tumors of bone

Giant cell tumors of bone (GCTBs) are characterized by mononuclear stromal cells and osteoclast-like giant cells; up to 95% have H3F3A gene mutation. The RANKL inhibitor denosumab, when used for the treatment of GCTB, leads to histological changes such as new bone formation and giant cell depletion. Here we assessed the diagnostic utility of immunohistochemical staining with the antibodies against histone H3.3 G34W, G34R and G34V mutant proteins for GCTB and other histologically similar bone and joint lesions. H3.3 G34W, G34R and G34V expressions were detected in mononuclear stromal cells in 47/51 (92%), 1/51 (2%) and 3/51 (6%) cases of primary GCTBs, respectively, in a mutually exclusive manner. All recurrent/metastatic GCTBs (n=14), post-denosumab GCTBs (n=8) and secondary malignant GCTBs (n=2) were positive for H3.3 G34W. The immunohistochemical results were essentially correlated with the H3F3A genotype determined by mutation analysis. In post-denosumab GCTBs, H3.3 G34W expression was seen in immature bone-forming cells. H3.3 G34W, G34R and G34V were negative in 121/122 cases of non-GCTB, including chondroblastoma, osteosarcoma, primary aneurysmal bone cyst and other giant cell–rich lesions. The exception was a single case of undifferentiated high-grade pleomorphic sarcoma that was positive for H3.3 G34W, suggesting the possibility of sarcomatous overgrowth of primary malignant GCTB. Therefore, H3.3 G34W/R/V mutant-specific antibodies are useful surrogate markers for the H3F3A genotype and helpful for the diagnosis of GCTB and its variants. The expression of H3.3 G34W mutant protein in post-denosumab GCTB suggests that neoplastic stromal cells may play a role in new bone formation. •Giant cell tumors of bone (GCTBs) frequently have mutations of the H3F3A gene.•Nuclear expression of H3.3 G34W mutant protein was observed in the majority of GCTB.•Minor subsets of GCTBs were immunohistochemically positive for H3.3 G34R or H3.3 G34V.•The immunohistochemical results were validated by H3F3A gene mutation analyses.•Diagnostic utility of H3.3 G34W/R/V immunohistochemistry for GCTB was demonstrated.