H3F3A mutation in giant cell tumour of the bone is detected by immunohistochemistry using a monoclonal antibody against the G34W mutated site of the histone H3.3 variant

Abstract

We conclude that positive H3.3 G34W staining is a specific and sensitive method for detection of H3F3A-mutated GCTB. Denosumab treatment leads to a pathomorphosis of the lesion characterized by matrix and osteoid producing H3.3 G34W-negative stromal cells.

Giant cell tumours of bone treated with denosumab: histological, immunohistochemical and H3F3A mutation analyses

“…The primary antibodies used were anti‐NFATc1, an antibody against a master regulator for terminal osteoclastic differentiation (mouse monoclonal, clone 7A6, sc‐7294, dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti‐RUNX2, an antibody against an osteoblastic marker (mouse monoclonal, clone CL0232, AMAb90591, dilution 1:400; Sigma‐Aldrich, St Louis, MO, USA); anti‐histone H3.3 G34W (rabbit monoclonal, clone RM263, 31‐1145‐00, dilution 1:400; RevMAb Biosciences, San Francisco, CA, USA); and anti‐CD68 (mouse monoclonal, clone PG‐M1, GA61361‐2, dilution 1:100; Agilent, Santa Clara, CA, USA). Antigen retrieval was performed in citrate buffer (pH 6) with a microwave oven for anti‐NFATc1, anti‐RUNX2, and anti‐CD68; EDTA buffer (pH 8) and a water bath were used for anti‐G34W . The Real EnVision Detection System, Peroxidase/DAB+, Rabbit/Mouse (Agilent) was used for secondary antibody treatment and protein visualisation.…”

“…Recently, an immunohistochemical antibody against histone H3.3 G34W (G34W) was shown to recognise the p.Gly34Trp (p.G34W) mutation of H3F3A . This antibody is expected to be a useful tool for the diagnosis of GCTB . At present, very limited information is available regarding the genetic and immunohistochemical features of GCTB after denosumab therapy.…”

Epigenetic Alterations in Bone and Soft Tissue Tumors

“…77 The presence of specific recurrent mutations has led to the development of novel diagnostic tests either in the form of molecular analysis of the DNA sequence, 78,79 or immunohistochemistry using mutation-specific antibodies (Figure 2 C&D). 80,81 These novel diagnostic tests are likely to be particularly useful on needle core biopsy specimens, or in cases with atypical clinical presentations. Immunohistochemistry in particular, which may be employed in most histologic labs, is highly promising (Figure 2C and 2D).…”

“…Mutant-specific antibodies—for H3K36M mutant protein in chondroblastoma and H3G34W mutation in giant cell tumor–have shown both excellent sensitivity and specificity. 80,81 …”

“…The vast majority harbor G34W substitutions (>95% of total mutations identified), but other substitutions, including G34V, G34R, G34L and S28N have been found in rare cases. 77,79,80 Nearly all of the alterations affect the G34 position. While this residue itself is not involved in histone post-translational modifications, it is adjacent to H3K36, which can be post-translationally modified by methylation or acetylation as discussed above.…”