Giant cell tumor of bone (GCTB) is a locally aggressive neoplasm composed of mononuclear neoplastic cells with an osteoblastic precursor phenotype, mononuclear macrophage, and osteoclast-like multinucleated giant cells. These typically affect ends of long bones and are generally treated with surgery, either en-bloc resection or curettage with or without local adjuvants. The mononuclear neoplastic cells in GCTB express high levels of receptor activator of nuclear factor NF-K ? ligand (RANKL) that binds to tumor necrosis factor-induced receptor activator of nuclear factor NF-K ? (RANK) receptors expressed on multinucleated osteoclast-like giant cells and its mononuclear precursors.[1] Such interaction of RANK-RANKL leads to recruitment of osteoclast precursors promoting osteoclastogenesis and bone resorption. The RANKL/RANK/OPG system plays an important role in the regulation of bone growth and turnover. Osteoprotegerin (OPG) acts as a decoy receptor for RANKL and inhibits the stimulation of osteoclast differentiation. In normal physiological conditions, there is a balance between RANK, RANKL, and OPG.[2] [3]

Denosumab is a human monoclonal antibody that binds to RANKL with high affinity, preventing its interaction with RANK on the surface of osteoclasts and its precursors.[4] [5] Studies have shown its efficacy in treating unresectable GCTB as well as in neoadjuvant settings. In this study, we have analyzed the histomorphological changes in GCTB following treatment with denosumab. Though literature on histopathology of post-denosumab-treated GCTBs has been published worldwide, very few studies have been reported from India.[4] [6] [7] [8] Moreover, immunohistochemistry (IHC) with antihistone H3.3G34W antibodies, a novel diagnostic marker of GCTB has not been widely performed in this subset of cases. In this article, apart from assessing the histomorphological changes, we also attempted to determine the expression of histone mutation H3.3G34W by using mutant specific antibody.

Materials and Methods

Of the total 109 cases of GCTB encountered during the study period (2018?2020), fourteen (14) cases of Campanacci grade III GCTB who had received neoadjuvant denosumab treatment were analyzed retrospectively to study post-treatment changes. The remaining 95 patients who did not receive denosumab following a histological diagnosis of GCTB were excluded from the study. The demographic data, location of the tumors, imaging findings, and treatment history were retrieved from the medical records. The initial diagnosis of GCTB was made on core needle biopsy (4 patients), curettage specimen (6 patients), and fine needle aspiration smears along with cell block (4 patients). All the cases except one were initially diagnosed outside and referred to our institute for further management. Following confirmation of diagnosis, all the patients received three cycles of 120 mg denosumab subcutaneously on day 0, day 8, and day 15. Patients were followed up for 1 month and repeat radiographs were taken prior to surgery to assess the effectiveness of denosumab therapy. After formation of a bony shell around the tumor on imaging, surgery was done that included resection in eight patients and curettage followed by bone grafting in six patients. The curettage specimens were submitted for histological examination entirely. Representative tumor sections were examined from the excised specimens. The sections were processed for routine paraffin sections after fixation in 10% buffered formalin. Decalcification was performed with 10% nitric acid whenever necessary prior to processing. The histopathological features on routine hematoxylin and eosin-stained sections were analyzed in all the cases. The representative formalin fixed paraffin embedded tissue blocks were chosen for IHC. An attempt was made to exclude the sections subjected to prior decalcification wherever possible and care was taken not to include the hemorrhagic and necrotic areas. In five cases, nondecalcified sections were not available, so IHC was performed on decalcified tissue sections.

IHC was performed using antihistone rabbit monoclonal antibody H3.3G34W (clone RM 263, Medasys Livermore, California, United States; dilution 1:100) on Roche Ventana Benchmark GX fully automated platform. The presence of unequivocal crisp nuclear staining with H3.3G34W in the cells irrespective of intensity and proportion was considered as positive staining. A case of GCTB with classic histomorphology and positive staining on IHC was taken as a positive control.

Postoperative recovery was uneventful in all the cases. None of them received any adjuvant denosumab following surgery. Follow-up details were documented wherever available. Clinical symptoms after denosumab treatment and last follow-up after surgery were noted.

Statistical Analysis

Statistical analysis was limited to descriptive statistics using Microsoft Office Excel 2016, Singapore. No hypothesis testing was performed.

Ethics

The study was approved by Nims Institutional Ethics Committee (EC/NIMS/2338/2019, dated 22.03.2019) after obtaining waiver of informed consent. The procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional) and with the Helsinki Declaration of 1964, as revised in 2013.

|?Fig. 1?(A and B) Pre-treatment radiographs of giant cell tumor of bone involving right proximal tibia. (A) Anteroposterior and (B) lateral radiographs showing a large geographic mildly expansile locally aggressive lytic lesion in proximal tibia with ambiguous lateral margins and fine internal septations. (C and D) Post-denosumab treatment radiographs of the same case. (C) Anteroposterior and (D) lateral radiographs showing development of marginal sclerosis (white arrows) and internal ossification. Note there is a small pathological fracture along the medial tibial cortex (dashed arrow).|

On histopathology, all these cases except three showed prominent newly formed woven bone along with spindle cells arranged in short fascicles and storiform pattern. Areas of fibrosis and hyalinization were noted. The other changes included focal necrosis (5 cases), collection of foamy macrophages (7 cases), inflammatory infiltrate (3 cases), cholesterol clefts (1 case), and foreign body giant cells (1 case). The inflammatory cells chiefly comprised of lymphocytes and plasma cells admixed with few neutrophils. Some of the cases showed prominent ectatic thin walled vessels. ABC-like changes were noted in two patients and benign fibrous histiocytoma (BFH)-like area was seen in one patient. There was marked reduction in osteoclast-like giant cells in five patients, complete absence in six patients, and remaining three patients showed substantial amount of residual osteoclast-like giant cells ([Fig. 2]). The location, imaging findings, and histopathological changes in post-denosumab-treated GCTB are summarized in [Table 1].