Clinicopathological characteristics of giant cell tumor of bone and identification of a novel H3F3A p.V36_K37insL mutation.

BACKGROUND: Osteoclast-rich bone lesions present overlapping clinical and radiological features, while their biological behaviors and treatment protocols differ greatly, posing challenges for differential diagnosis. H3.3G34W immunohistochemistry serves as the primary biomarker for giant cell tumor of bone (GCTB), yet this marker is not expressed in all GCTB cases. Denosumab, the first-line targeted drug, induces profound tumour remodelling that raises diagnostic difficulties for post-treatment specimens. METHOD: A total of 45 osteoclast-rich bone lesions collected between 2020 and 2024 at the First Affiliated Hospital of Zhejiang University School of Medicine were retrospectively screened, with 16 cases harbouring H3F3A mutations enrolled for analysis. All hematoxylin and eosin (HE) staining and immunohistochemical (IHC) slides underwent independent review by two senior pathologists, and full imaging datasets were re-assessed by two radiologists. Sanger sequencing was performed on formalin-fixed paraffin-embedded(FFPE) specimens to identify H3F3A gene variants. Clinicopathological and radiological profiles of mutant GCTB were summarised, and histological as well as molecular changes before and after denosumab intervention were compared. RESULT: Among the 45 screened lesions, 16 harboured H3F3A mutations, including 14 canonical p.G34W variants, one p.G34L variant and one previously unreported p.V36_K37insL insertion variant undetectable by routine H3.3G34W immunohistochemistry. Typical osteolytic, eccentric and expansile imaging manifestations were observed in most GCTB cases. Conventional untreated lesions exhibited characteristic evenly distributed mononuclear stromal cells and osteoclast-like giant cells. Marked depletion of giant cells, spindle cell proliferation, woven bone formation and weakened IHC staining were common histological alterations after denosumab therapy, accompanied by reduced mutant allele abundance in partial post-treatment specimens. Two patients developed local tumour recurrence during follow-up. CONCLUSION: Comprehensive integration of clinical, radiological, histological and molecular evidence is essential for accurate differential diagnosis of osteoclast-rich bone lesions after denosumab treatment. The newly identified H3F3A p.V36_K37insL variant may represent a potential rare variant for morphologically‑typical GCTB, and serve as an auxiliary molecular clue for H3.3‑G34W‑immunonegative cases. Further multicenter and functional studies are required to clarify the biological characteristics of this novel variant.