H3F3A (Histone 3.3) G34W Immunohistochemistry: A Reliable Marker Defining Benign and Malignant Giant Cell Tumor of Bone

Abstract

Giant cell tumor of bone (GCTB) is a locally aggressive subarticular tumor. Having recently reported that H3.3 G34W mutations are characteristic of this tumor type, we have now investigated the sensitivity and specificity of the anti-histone H3.3 G34W rabbit monoclonal antibody in a wide variety of tumors including histologic mimics of GCTB to assess its value as a diagnostic marker. We also determined the incidence of H3.3 G34 mutations in primary malignant bone tumors as assessed by genotype and H3.3 G34W immunostaining. A total of 3163 tumors were tested. Totally, 213/235 GCTB (90.6%) showed nuclear H3.3 p.G34W immunoreactivity. This was not the case for the rare variants, p.G34L, M, and V, which occurred most commonly in the small bones of the hands, patella, and the axial skeleton. If these sites were excluded from the analysis, H3.3 G34W expression was found in 97.8% of GCTB. Malignant bone tumors initially classified as osteosarcomas were the only other lesions (n=11) that showed G34W expression. Notably an additional 2 previously reported osteosarcomas with a p.G34R mutation were not immunoreactive for the antibody. A total of 11/13 of these malignant H3.3-mutant tumors exhibited an osteoclast-rich component: when imaging was available all but one presented at a subarticular site. We propose that subarticular primary malignant bone sarcoma with H3.3 mutations represent true malignant GCTB, even in the absence of a benign GCTB component.

FIGURE 1

Photomicrographs (A and B) and radiographs (C) of a GCTB with extensive secondary aneurysmal bone cyst change, in areas indistinguishable from a primary aneurysmal bone cyst. H3.3 G34W is diffusely expressed by the stromal cells. D and E represent photomicrographs, of a different case (biopsy sample) showing a small isolated fragment of an osteoclast-rich lesion within blood clot. Strong H3.3 G34W (E) nuclei expression confirms the suspicion of a GCTB. The osteoclasts are negative for H3.3 G34W expression.

FIGURE 2

Photomicrographs of a malignant GCTB (M-07: initially classified as osteoclast-rich osteosarcoma) of the distal tibia showing morphologic variation including areas composed of epithelioid cells arranged in cords (A–C), osteoid deposition (B and C), tumor necrosis (C), and spindle cells with moderate nuclear atypia and numerous interspersed osteoclasts (D–F). Diffuse expression of H3.3 G34W is seen in the stromal cells (F).

FIGURE 3

A and B, Resection specimen and corresponding coronal T1 MRI scan showing a lytic hemorrhagic lesion in the subarticular area of the distal femur with a surrounding low signal (sclerotic) component at the proximal aspect of the lesion. Photomicrographs of the subarticular component shows a conventional GCTB with diffuse H3.3 G34W expression (inset) (C). The proximal component comprises a bone-forming tumor with a spindle cell stroma showing a permeative growth pattern (D and E) (H3.3 G34W expression shown on inset D) case M10.