This study aimed to assess the reliability of an objective diagnostic approach for giant cell tumour of bone (GCTB) by evaluating H3-3A gene mutations through Sanger sequencing and immunohistochemistry (IHC). The cohort comprised 214 patients: 120 diagnosed with GCTB and 94 with other giant cell-rich lesions of bone. Analyses were conducted on formalin-fixed, paraffin-embedded samples, with EDTA-based decalcification applied where required.
Sanger sequencing demonstrated 100% sensitivity (95 percent CI: 96.97–100 percent) and 100 percent specificity (95% CI: 96.15–100%). IHC using antibodies targeting histone H3.3 G34W and G34V variants showed 94.32% sensitivity (95 percent CI: 87.24–98.13 percent) and 100 percent specificity (95% CI: 93.94–100%). P.G35 mutations were identified in 2 of 9 (22.2 percent) secondary malignant GCTBs and 9 of 13 (69.2%) post-denosumab GCTB cases. In this extensive patient series, direct sequencing of H3-3A mutational status proved to be a highly dependable diagnostic tool for GCTB and warrants integration into standard diagnostic protocols. IHC serves effectively as a screening method, though accurate tissue handling and decalcification are essential. The detection of H3-3A mutations does not preclude malignancy in GCTB, and denosumab therapy does not eliminate the neoplastic cell population.
