ABSTRACT
RNA processing is an essential post-transcriptional phenomenon that provides the necessary complexity of transcript diversity prior to translation. Aberrations in this process could contribute to tumourigenesis, and we have previously reported increased splicing alterations in giant cell tumor of bone (GCTB), which carries mutations in the histone variant H3.3 encoding glycine 34 substituted for tryptophan (H3.3-G34W). G34W interacts with several splicing factors, most notably the trans-acting splicing factor hnRNPA1L2. To gain a deeper understanding of RNA processing in GCTB and isogenic HeLa cells with H3.3-G34W, we generated RNA-immunoprecipitation sequencing data from hnRNPA1L2 and H3.3-G34W associated RNAs, which showed that 80% overlapped across genic regions and were frequently annotated as E2F transcription factor binding sites. Splicing aberrations in both GCTB and HeLa cells with H3.3-G34W were significantly enriched for known hnRNPA1L2 binding motifs (p value < 0.01). This splicing aberration differed from hnRNPA1L2 knockouts, which showed alterations independent of H3.3-G34W. Of functional significance, hnRNPA1L2 was redistributed to closely match the H3.3 pattern, likely driven by G34W, and to loci not occupied in normal parental cells. Taken together, our data reveal a functional overlap between hnRNPA1L2 and H3.3-G34W with likely significant consequences for RNA processing during GCTB pathogenesis. This provides novel opportunities for therapeutic intervention in future modus operandi.
Subject(s)
Bone Neoplasms , Exons , Giant Cell Tumor of Bone , Histones , Humans , Alternative Splicing , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Giant Cell Tumor of Bone/genetics , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , HeLa Cells , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Heterogeneous Nuclear Ribonucleoprotein A1/genetics , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/metabolism , Heterogeneous-Nuclear Ribonucleoprotein Group A-B/genetics , Histones/metabolism , Histones/genetics , RNA SplicingABSTRACT
BACKGROUND: Giant cell tumor of bone (GCTB) is a locally aggressive bone tumour aggravated by stromal cell proliferation and metastasis. OBJECTIVE: We investigated the mechanism of action of human chorionic gonadotropin (HCG) in mediating GCTB proliferation and invasion. METHODS: The expression of HCG was quantified using quantitative real-time PCR. After the primary stromal cells were isolated and identified, the function of HCG in GCTB was estimated using the cell counting kit-8, flow cytometry, scratch experiment, transwell assay, Western blot, and immunofluorescence. Moreover, the mechanism of HCG was assessed through western blotting. RESULTS: HCG expression was decreased in clinical tissue samples from patients with GCTB. We validated that HCG repressed stromal cell proliferation, migration, invasion, autophagy, and epithelial- mesenchymal transition (EMT) and promoted cell apoptosis in GCTB. We also verified that HCG repressed the autophagy and EMT of stromal cells through the Smad signaling axis in GCTB. HCG inhibited the transduction of the Smad signaling pathway by restraining the binding of the TGF-ß II receptor to ligand Activin A. CONCLUSION: HCG restrained the Smad signaling pathway by antagonizing TGF-ß signaling in GCTB. HCG may serve as a useful patent to treat GCTB.
Subject(s)
Giant Cell Tumor of Bone , Transforming Growth Factor beta , Humans , Giant Cell Tumor of Bone/drug therapy , Giant Cell Tumor of Bone/metabolism , Cell Line, Tumor , Patents as Topic , Signal Transduction , Chorionic GonadotropinABSTRACT
Narlumosbart () is a recombinant, fully human, anti-receptor activator of nuclear factor kappa-Β ligand (RANKL) IgG4 monoclonal antibody being developed by CSPC Pharmaceutical and its wholly owned subsidiary Shanghai Jinmante Biotechnology for the treatment of giant cell tumour of bone (GCTB), bone metastases from solid tumours and osteoporosis. The RANK/RANKL signalling pathway plays a pivotal role in osteoclastogenesis and in the pathogenesis of GCTB. Narlumosbart specifically binds to RANKL and blocks the interaction of RANKL with RANK, thus inhibiting osteoclastogenesis and bone resorption by osteoclasts. In September 2023, narlumosbart received conditional first approval in China for the treatment of adults with GCTB that is unresectable or when surgical resection would result in severe functional disability. Clinical studies of narlumosbart for bone metastases, postmenopausal osteoporosis and glucocorticoid-induced osteoporosis are underway in China. This article summarizes the milestones in the development of narlumosbart leading to this first approval for the treatment of adults with GCTB.
Subject(s)
Bone Density Conservation Agents , Bone Neoplasms , Bone Resorption , Giant Cell Tumor of Bone , Osteoporosis , Adult , Female , Humans , China , Bone Resorption/metabolism , Osteoclasts/metabolism , Osteoclasts/pathology , Bone Neoplasms/drug therapy , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Giant Cell Tumor of Bone/drug therapy , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Bone Density Conservation Agents/therapeutic useABSTRACT
Giant cell tumors of bone (GCTBs) are locally aggressive tumors with the histological features of giant cells and stromal cells. Denosumab is a human monoclonal antibody that binds to the cytokine receptor activator of nuclear factor-kappa B ligand (RANKL). RANKL inhibition blocks tumor-induced osteoclastogenesis, and survival, and is used to treat unresectable GCTBs. Denosumab treatment induces osteogenic differentiation of GCTB cells. In this study, the expression of RANKL, special AT-rich sequence-binding protein 2 (SATB2, a marker of osteoblast differentiation), and sclerostin/SOST (a marker of mature osteocytes) was analyzed before and after treatment with denosumab in six cases of GCTB. Denosumab therapy was administered a mean of five times over a mean 93.5-day period. Before denosumab treatment, RANKL expression was observed in one of six cases. After denosumab therapy, spindle-like cells devoid of giant cell aggregation were RANKL-positive in four of six cases. Bone matrix-embedded osteocyte markers were observed, although RANKL was not expressed. Osteocyte-like cells were confirmed to have mutations, as identified using mutation-specific antibodies. Our study results suggest that treatment of GCTBs with denosumab results in osteoblast-osteocyte differentiation. Denosumab played a role in the suppression of tumor activity via inhibition of the RANK-RANKL pathway, which triggers osteoclast precursors to differentiate into osteoclasts.
Subject(s)
Bone Density Conservation Agents , Bone Neoplasms , Giant Cell Tumor of Bone , Humans , Denosumab/pharmacology , Osteocytes/metabolism , Giant Cell Tumor of Bone/genetics , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Osteogenesis , NF-kappa B , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Bone Density Conservation Agents/pharmacology , RANK Ligand/metabolism , Cell DifferentiationABSTRACT
The main features of a giant cell tumor of bone (GCTB) are frequent recurrence and aggressive osteolysis, which leads to a poor prognosis in patients. Although the treatment methods for a GCTB, such as scraping and resection, effectively inhibit the disease, the tendency toward malignant transformation remains. Therefore, it is important to identify new treatment methods for a GCTB. In this study, we first found high Siglec-15 expression in GCTB tissues, which was significantly associated with Campanacci staging and tumor recurrence. In Spearman's analysis, Siglec-15 expression was significantly correlated with Ki-67 levels in tumor tissues. In vitro, the mRNA and protein levels of Siglec-15 were high in GCTB stromal cells (Hs737. T), and Siglec-15 knockdown inhibited the biological characteristics of GCTB stromal cells. The RNA sequencing results enabled a prediction of the downstream genes by using the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and MCODE analyses, and the findings showed that CXCL8 was significantly regulated by Siglec-15 and might be a promising downstream target gene of Siglec-15. Therefore, Siglec-15 may be a potential immunotherapy target for a GCTB.
Subject(s)
Bone Neoplasms , Giant Cell Tumor of Bone , Humans , Giant Cell Tumor of Bone/genetics , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Bone Neoplasms/genetics , Ki-67 Antigen , RNA, Messenger , Sialic Acid Binding Immunoglobulin-like LectinsABSTRACT
Denosumab is a game-changing drug for giant cell tumor of bone (GCTB); however, its clinical biomarker regarding tumor ossification of GCTB has not been elucidated. In this study, we investigated the relationship between Wnt/ß-catenin signaling and the ossification of GCTB and evaluated whether endogenous nuclear ß-catenin expression predicted denosumab-induced bone formation in GCTB. Genuine patient-derived primary GCTB tumor stromal cells exhibited osteoblastic characteristics. Identified osteoblastic markers and nuclear ß-catenin translocation were significantly upregulated via differentiation induction and were inhibited by treating with Wnt signaling inhibitor, GGTI-286, or selective Rac1-LEF inhibitor, NSC23766. Furthermore, we reviewed the endogenous ossification and nuclear ß-catenin translocation of 86 GCTB clinical samples and elucidated that intra-tumoral ossification was significantly associated with the nuclear translocation. Three-dimensional quantitative analyses (n = 13) of tumoral CT images have revealed that the nuclear ß-catenin translocation of naïve GCTB samples was significantly involved with the denosumab-induced tumor ossification. Our findings suggest a close relationship between the nuclear ß-catenin translocation and the osteoblastic differentiation of GCTB. Investigations of the nuclear ß-catenin in naïve GCTB samples may provide a promising biomarker for predicting the ossification of GCTB following denosumab treatment.
Subject(s)
Bone Neoplasms , Giant Cell Tumor of Bone , Bone Neoplasms/diagnostic imaging , Bone Neoplasms/drug therapy , Bone Neoplasms/metabolism , Cell Differentiation , Denosumab/pharmacology , Denosumab/therapeutic use , Giant Cell Tumor of Bone/diagnostic imaging , Giant Cell Tumor of Bone/drug therapy , Giant Cell Tumor of Bone/metabolism , Humans , Osteoblasts/metabolism , Osteogenesis , beta CateninABSTRACT
Dysregulation of angiogenesis is associated with tumor development and is accompanied by altered expression of pro-angiogenic factors. EGFL7 is a newly identified antigenic factor that plays a role in various cancers such as breast cancer, lung cancer, and acute myeloid leukemia. We have recently found that EGFL7 is expressed in the bone microenvironment, but its role in giant-cell tumor of bone (GCTB) and osteosarcoma (OS) is unknown. The aims of this study are to examine the gene expression profile of EGFL7 in GCTB and OS and compare with that of VEGF-A-D and TNFSF11 using single-cell RNA sequencing data. In-depth differential expression analyses were employed to characterize their expression in the constituent cell types of GCTB and OS. Notably, EGFL7 in GCTB was expressed at highest levels in the endothelial cell (EC) cluster followed by osteoblasts, myeloid cells, and chondrocytes, respectively. In OS, EGFL7 exhibited highest expression in EC cell cluster followed by osteoblastic OS cells, myeloid cells 1, and carcinoma associated fibroblasts (CAFs), respectively. In comparison, VEGF-A is expressed at highest levels in myeloid cells followed by OCs in GCTB, and in myeloid cells, and OCs in OS. VEGF-B is expressed at highest levels in chondrocytes in GCTB and in OCs in OS. VEGF-C is strongly enriched in ECs and VEGF-D is expressed at weak levels in all cell types in both GCTB and OS. TNFSF11 (or RANKL) shows high expression in CAFs and osteoblastic OS cells in OS, and osteoblasts in GCTB. This study investigates pro-angiogenic genes in GCTB and OS and suggests that these genes and their expression patterns are cell-type specific and could provide potential prognostic biomarkers and cell type target treatment for GCTB and OS.
Subject(s)
Bone Neoplasms , Giant Cell Tumor of Bone , Osteosarcoma , Bone Neoplasms/genetics , Bone Neoplasms/pathology , Calcium-Binding Proteins/genetics , EGF Family of Proteins/genetics , EGF Family of Proteins/metabolism , Giant Cell Tumor of Bone/genetics , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Humans , Osteosarcoma/genetics , Sequence Analysis, RNA , Transcription Factors/metabolism , Tumor Microenvironment/genetics , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Surface ultrastructures of giant cells (GCs) may help distinguish an aggressive tumor from an indolent giant cell tumor (GCT). This protocol describes a better way for ultrastructural surface imaging of GC from GCT of bone by scanning electron microscope (SEM). Fresh GCT samples collected in Dulbecco's modified Eagle medium (DMEM) are thoroughly washed to remove the blood and treated with collagenase to isolate the GCs. The collagenase-treated and critical point dried (CPD) samples yield a greater number of isolated GCs with better surface morphology, including membrane folding and micro-vesicular structures on the surface. Collagenase digestion and CPD should be performed for ultrastructural surface imaging of individual giant cells.
Subject(s)
Bone Neoplasms , Giant Cell Tumor of Bone , Bone Neoplasms/diagnostic imaging , Bone Neoplasms/pathology , Bone and Bones/metabolism , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Giant Cell Tumor of Bone/ultrastructure , Giant Cells/metabolism , Humans , Microscopy, Electron, ScanningABSTRACT
To validate the reliability and implementation of an objective diagnostic method for giant cell tumour of bone (GCTB). H3-3A gene mutation testing was performed using two different methods, Sanger sequencing and immunohistochemical (IHC) assays. A total of 214 patients, including 120 with GCTB and 94 with other giant cell-rich bone lesions, participated in the study. Sanger sequencing and IHC with anti-histone H3.3 G34W and G34V antibodies were performed on formalin-fixed, paraffin-embedded tissues, which were previously decalcified in EDTA if needed. The sensitivity and specificity of the molecular method was 100% (95% CI: 96.97-100%) and 100% (95% CI: 96.15-100%), respectively. The sensitivity and specificity of IHC was 94.32% (95% CI: 87.24-98.13%) and 100% (95% CI: 93.94-100.0%), respectively. P.G35 mutations were discovered in 2/9 (22.2%) secondary malignant GCTBs and 9/13 (69.2%) GCTB after denosumab treatment. We confirmed in a large series of patients that evaluation of H3-3A mutational status using direct sequencing is a reliable tool for diagnosing GCTB, and it should be incorporated into the diagnostic algorithm. Additionally, we discovered IHC can be used as a screening tool. Proper tissue processing and decalcification are necessary. The presence of the H3-3A mutation did not exclude malignant GCTB. Denosumab did not eradicate the neoplastic cell population of GCTB.
Subject(s)
Bone Neoplasms/diagnosis , Giant Cell Tumor of Bone/diagnosis , Histones/genetics , Histones/metabolism , Mutation , Adolescent , Adult , Aged , Aged, 80 and over , Bone Neoplasms/drug therapy , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Child , Denosumab/therapeutic use , Diagnosis, Differential , Early Detection of Cancer , Female , Giant Cell Tumor of Bone/drug therapy , Giant Cell Tumor of Bone/genetics , Giant Cell Tumor of Bone/metabolism , Humans , Male , Middle Aged , Paraffin Embedding , Sensitivity and Specificity , Sequence Analysis, DNA , Tissue Fixation , Young AdultABSTRACT
Giant cell tumor of bone (GCTB) is a locally aggressive primary bone neoplasm composed by tumoral stromal cells (SCs) and a reactive component that consists of monocytic/histiocytic cells that give rise by fusion to osteoclast-like multinucleated cells. Recently, specific Histone 3.3 mutations have been demonstrated in SCs of GCTB. Many of the pathways related to bone proliferation and regulation depend on the primary cilium, a microtubule-based organelle that protrudes outside the cell and acts as a sensorial antenna. In the present work, we aimed to study the presence and role of primary cilia in GCTB. Ultrastructural, immunohistochemical, and immunofluorescence studies were performed in order to demonstrate, for the first time, that the primary cilium is located in spindle-shaped SCs of GCTB. Moreover, we showed Hedgehog (Hh) signaling pathway activation in these cells. Hence, primary cilia may play a relevant role in GCTB tumorogenesis through Hh signaling activation in SCs. RESEARCH HIGHLIGHTS: Transmission electron microscopy allows describing and differentiating cellular subpopulations in giant cell tumor of bone (GCTB). The primary cilium is present in some tumoral stromal cells of GCTB. Hedgehog signalling is activated in these cells.
Subject(s)
Bone Neoplasms , Giant Cell Tumor of Bone , Bone Neoplasms/pathology , Cilia/metabolism , Cilia/pathology , Giant Cell Tumor of Bone/genetics , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Hedgehog Proteins/metabolism , Humans , Stromal CellsABSTRACT
OBJECTIVE: To assess P63 expression in giant cell-containing lesions of the bone (GCLB) and to determine its utility in differentiating giant cell tumor of the bone (GCTB) from other GCLBs. MATERIAL AND METHOD: Cases diagnosed as GCLB on histopathology were included in the study. P63 immunohistochemistry was performed in all the cases. The percentage of cells showing nuclear positivity was assessed in the non-giant cell component. Statistical analysis was performed using the Mann-Whitney U test. RESULTS: Of the total 53 cases studied, the majority were GCTBs (23), followed by 12 cases of chondroblastomas (CBL) and 18 other giant cell lesions (GCLs). All giant cell-containing lesions except one case of CBL and brown tumor of hyperparathyroidism (BTH) showed P63 staining in the non-giant cell component. However, the mean P63 labeling of GCT (52.6%) was higher compared to CBL (28.3%), aneurysmal bone cyst (ABC) (15.2%), non-ossifying fibroma (NOF) (24.5%), giant cell lesion of small bones (GCLSB) (11%), BTH (6.8%) and chondromyxoid fibroma (CMF) (12.3%), with a p-value of < 0.001. CONCLUSION: Although p63 was present in majority of the GCLBs, its percentage positivity was significantly higher in GCTB compared to the other GCLBs. The diagnosis of GCTB is likely if cut-off value of > 50% is applied.
Subject(s)
Bone Neoplasms , Chondroblastoma , Giant Cell Tumor of Bone , Bone Neoplasms/pathology , Chondroblastoma/metabolism , Chondroblastoma/pathology , Giant Cell Tumor of Bone/diagnosis , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Giant Cells/metabolism , Giant Cells/pathology , Humans , ImmunohistochemistryABSTRACT
Giant cell tumor of bone (GCTB) is an aggressive osteolytic bone tumor characterized by the within-tumor presence of osteoclast-like multinucleated giant cells (MGCs), which are induced by the neoplastic stromal cells and lead to extensive bone destruction. However, the underlying mechanism of the pathological process of osteoclastogenesis in GCTB is poorly understood. Here we show that the proteoglycan Serglycin (SRGN) secreted by neoplastic stromal cells plays a crucial role in the formation of MGCs and tumorigenesis in GCTB. Upregulated SRGN expression and secretion are observed in GCTB tumor cells and patients. Stromal-derived SRGN promotes osteoclast differentiation from monocytes. SRGN knockdown in stromal cells inhibits tumor growth and bone destruction in a patient-derived orthotopic xenograft model of mice. Mechanistically SRGN interacts with CD44 on the cell surface of monocytes and thus activates focal adhesion kinase (FAK), leading to osteoclast differentiation. Importantly, blocking CD44 with a neutralizing antibody reduces the number of MGCs and suppresses tumorigenesis in vivo. Overall, our data reveal a mechanism of MGC induction in GCTB and support CD44-targeting approaches for GCTB treatment.
Subject(s)
Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Osteogenesis , Proteoglycans/metabolism , Vesicular Transport Proteins/metabolism , Animals , Antibodies, Neutralizing/pharmacology , Carcinogenesis/drug effects , Carcinogenesis/genetics , Carcinogenesis/pathology , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Enzyme Activation/drug effects , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Giant Cell Tumor of Bone/genetics , Giant Cells/drug effects , Giant Cells/metabolism , Giant Cells/pathology , Humans , Hyaluronan Receptors/metabolism , Male , Mice , Mice, Inbred NOD , Mice, SCID , Osteogenesis/drug effects , Osteogenesis/genetics , Osteosarcoma/genetics , Osteosarcoma/pathology , Proteoglycans/genetics , RAW 264.7 Cells , Up-Regulation/drug effects , Up-Regulation/genetics , Vesicular Transport Proteins/geneticsABSTRACT
Cells of the monocyte macrophage lineage form multinucleated giant cells (GCs) by fusion, which may express some cell cycle markers. By using a comprehensive marker set, here we looked for potential replication activities in GCs, and investigated whether these have diagnostic or clinical relevance in giant cell tumor of bone (GCTB). GC rich regions of 10 primary and 10 first recurrence GCTB cases were tested using immunohistochemistry in tissue microarrays. The nuclear positivity rate of the general proliferation marker, replication licensing, G1/S-phase, S/G2/M-phase, mitosis promoter, and cyclin dependent kinase (CDK) inhibitor reactions was analyzed in GCs. Concerning Ki67, moderate SP6 reaction was seen in many GC nuclei, while B56 and Mib1 positivity was rare, but the latter could be linked to more aggressive (p = 0.012) phenotype. Regular MCM6 reaction, as opposed to uncommon MCM2, suggested an initial DNA unwinding. Early replication course in GCs was also supported by widely detecting CDK4 and cyclin E, for the first time, and confirming cyclin D1 upregulation. However, post-G1-phase markers CDK2, cyclin A, geminin, topoisomerase-2a, aurora kinase A, and phospho-histone H3 were rare or missing. These were likely silenced by upregulated CDK inhibitors p15INK4b, p16INK4a, p27KIP1, p53 through its effector p21WAF1 and possibly cyclin G1, consistent with the prevention of DNA replication. In conclusion, the upregulation of known and several novel cell cycle progression markers detected here clearly verify early replication activities in GCs, which are controlled by cell cycle arresting CDK inhibitors at G1 phase, and support the functional maturation of GCs in GCTB.
Subject(s)
Biomarkers, Tumor/metabolism , Bone Neoplasms/pathology , Cell Cycle Proteins/metabolism , Cell Cycle , Cell Proliferation , Giant Cell Tumor of Bone/pathology , Adolescent , Adult , Aged , Bone Neoplasms/metabolism , Female , Follow-Up Studies , Giant Cell Tumor of Bone/metabolism , Humans , Male , Middle Aged , Prognosis , Retrospective Studies , Tumor Cells, Cultured , Young AdultABSTRACT
Giant cell tumor of bone (GCTB) is an intermediate malignant bone tumor that is locally aggressive and rarely metastasizes. Denosumab, which is a receptor activator of nuclear factor kappa B ligand (RANKL) inhibitor, can be used to treat GCTB. We focused on potential immunotherapy for GCTB and investigated the tumor microenvironment of GCTB. Programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) expression and signal-regulatory protein alpha (SIRPα), forkhead box P3 (FOXP3), and cluster of differentiation 8 (CD8) infiltration were assessed by immunohistochemical studies of 137 tumor tissues from 96 patients. Of the naive primary specimens, 28% exhibited PD-L1 expression and 39% exhibited IDO1 expression. There was significantly more SIRPα+, FOXP3+, and CD8+ cell infiltration in PD-L1- and IDO1-positive tumors than in PD-L1- and IDO1-negative tumors. The frequency of PD-L1 expression and SIRPα+ cell infiltration in recurrent lesions treated with denosumab was significantly higher than in primary lesions and recurrent lesions not treated with denosumab. PD-L1 expression and higher SIRPα+ cell infiltration were significantly correlated with shorter recurrence-free survival. PD-L1 and SIRPα immune checkpoint inhibitors may provide clinical benefit in GCTB patients with recurrent lesions after denosumab therapy.
Subject(s)
Antigens, Differentiation/metabolism , B7-H1 Antigen/metabolism , Bone Neoplasms/drug therapy , Denosumab/administration & dosage , Giant Cell Tumor of Bone/drug therapy , Receptors, Immunologic/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Bone Neoplasms/metabolism , Denosumab/pharmacology , Female , Gene Expression Regulation, Neoplastic/drug effects , Giant Cell Tumor of Bone/metabolism , Humans , Indoleamine-Pyrrole 2,3,-Dioxygenase/metabolism , Male , Middle Aged , Retrospective Studies , Survival Analysis , Treatment Outcome , Tumor Microenvironment/drug effects , Young AdultABSTRACT
AIMS: Giant cell tumour of bone (GCTB) is histologically defined as a lesion containing reactive giant cells and a neoplastic mononuclear cell population; in up to 92% of cases, GCTB is characterised by a specific mutation of the histone gene H3F3A. The cellular composition ranges from giant-cell-rich to giant-cell-poor. The diagnosis of GCTB can be challenging, and several other lesions need to be excluded, e.g. aneurysmal bone cysts, non-ossifying fibromas, chondroblastomas, brown tumours, and giant-cell-rich osteosarcomas. Our aim was to analyse the clinical history, imaging, molecular pathology and histology of three H3F3A-mutated bone tumours without detectable giant cells. None of the patients received denosumab therapy. METHODS AND RESULTS: Diagnostic material was obtained by curettage or resection and/or biopsy. Common histomorphological features of all three reported lesions were fibrocytic, oval cells in a background of osteoid and an absence of multinuclear giant cells as confirmed with CD68 immunohistochemistry. We used immunohistochemistry and Sanger sequencing to demonstrate positivity for the H3.3 p.G34W mutation. Differential diagnoses were systematically excluded on the basis of histomorphology, immunohistochemistry, and fluorescence in-situ hybridisation. The imaging (radiography, computed tomography, and magnetic resonance imaging) for all three cases is presented and discussed. CONCLUSIONS: We believe that these GCTBs without giant cells expand one end of the heterogeneous range of GCTB. Because of the lack of giant cells, correct diagnosis of GCTB is challenging or even impossible on histological grounds alone. In these cases, detection of the characteristic H3F3A mutation (G34W-specific antibody RM263 or sequencing) is extremely helpful for diagnosing those lesions without giant cells as giant cell tumours of bone.
Subject(s)
Giant Cell Tumor of Bone , Histones , Adult , Bone Neoplasms/diagnosis , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Bone and Bones/pathology , Chondroblastoma , Diagnosis, Differential , Female , Giant Cell Tumor of Bone/diagnosis , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Giant Cells/pathology , Histones/genetics , Histones/metabolism , Humans , Immunohistochemistry , Male , Mutation , Osteosarcoma , RadiologyABSTRACT
BACKGROUND: Giant cell tumor of bone (GCTB) is a rare osteoclastogenic stromal tumor. GCTB can rarely undergo malignant transformation. This post hoc analysis evaluated and classified malignancies in patients with GCTB who received denosumab. METHODS: This analysis was conducted on patients with pathologically confirmed GCTB and measurable active disease treated with denosumab 120 mg subcutaneously once every 4 weeks, with loading doses on study days 8 and 15, as part of a phase 2, open-label, multicenter study. We identified potential cases of malignancy related to GCTB through an independent multidisciplinary review or medical history, associated imaging or histopathologic reports, and disease course. The findings were summarized and no statistical analysis was performed. RESULTS: Twenty of five hundred twenty-six patients (3.8%) who received at least one dose of denosumab were misdiagnosed with GCTB that was later discovered to be malignancies: five primary malignant GCTB, five secondary malignant GCTB, four sarcomatous transformations, and six patients with other malignancies (giant cell-rich osteosarcoma, undifferentiated pleomorphic sarcoma, spindle cell sarcoma, osteogenic sarcoma, phosphaturic mesenchymal tumor of mixed connective tissue type, and fibrosarcoma/malignant fibrous histiocytoma). Many malignancies were present before denosumab was initiated (8 definitive cases, 7 likely cases), excluding potential involvement of denosumab in these cases. Signs associated with potential misdiagnoses of GCTB included poor mineralization with denosumab treatment, rapid relapse in pain, or a failure of the typical dramatic improvement in pain normally observed with denosumab. CONCLUSIONS: Although rare, GCTB can undergo malignant transformation, and rates in this study were consistent with previous reports. Signs of poor mineralization or lack of response to denosumab treatment may warrant close monitoring. TRIAL REGISTRATION: clinicaltrials.gov , ( NCT00680992 ). Registered May 20, 2008.
Subject(s)
Biomarkers, Tumor/analysis , Bone Density Conservation Agents/therapeutic use , Bone Neoplasms/drug therapy , Denosumab/therapeutic use , Giant Cell Tumor of Bone/drug therapy , Adolescent , Adult , Aged , Aged, 80 and over , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Female , Follow-Up Studies , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/pathology , Humans , Male , Middle Aged , Prognosis , Survival Rate , Young AdultABSTRACT
Giant cell tumors of bone (GCT) are benign tumors that show a locally aggressive nature and affect bones' architecture. Recently, cryoablation and irradiation treatments have shown promising results in GCT patients with faster recovery and less recurrence and metastasis. Therefore, it became a gold standard surgical treatment for patients. Hence, we have compared GCT-untreated, cryoablation, and irradiation-treated samples to identify protein alterations using high-frequency liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Our label-free quantification analysis revealed a total of 107 proteins (p < 0.01) with 26 up-regulated (< 2-folds to 5-fold), and 81 down-regulated (> 0.1 to 0.5 folds) proteins were identified from GCT-untreated and treated groups. Based on pathway analysis, most of the identified up-regulated proteins involved in critical metabolic functions associated with tumor proliferation, angiogenesis, and metastasis. On the other hand, the down-regulated proteins involved in glycolysis, tumor microenvironment, and apoptosis. The observed higher expressions of matrix metalloproteinase 9 (MMP9) and TGF-beta in the GCT-untreated group associated with bones' osteolytic process. Interestingly, both the proteins showed reduced expressions after cryoablation treatment, and contrast expressions identified in the irradiation treated group. Therefore, these expressions were confirmed by immunoblot analysis. In addition to these, several glycolytic enzymes, immune markers, extracellular matrix (ECM), and heat shock proteins showed adverse expressions in the GCT-untreated group were identified with favorable regulations after treatment. Therefore, the identified expression profiles will provide a better picture of treatment efficacy and effect on the molecular environment of GCT.
Subject(s)
Cryosurgery , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/therapy , Proteomics , Tandem Mass Spectrometry , Adolescent , Child , Child, Preschool , Chromatography, Liquid , Down-Regulation , Female , Gene Ontology , Giant Cell Tumor of Bone/radiotherapy , Giant Cell Tumor of Bone/surgery , Humans , Male , Matrix Metalloproteinase 9/metabolism , Middle Aged , Neoplasm Proteins/metabolism , Protein Interaction Maps , Reproducibility of Results , Signal Transduction , Transforming Growth Factor beta/metabolismABSTRACT
The neoplastic stromal cells of giant cell tumor of bone (GCTB) carry a mutation in H3F3A, leading to a mutant histone variant, H3.3-G34W, as a sole recurrent genetic alteration. We show that in patient-derived stromal cells H3.3-G34W is incorporated into the chromatin and associates with massive epigenetic alterations on the DNA methylation, chromatin accessibility and histone modification level, that can be partially recapitulated in an orthogonal cell line system by the introduction of H3.3-G34W. These epigenetic alterations affect mainly heterochromatic and bivalent regions and provide possible explanations for the genomic instability, as well as the osteolytic phenotype of GCTB. The mutation occurs in differentiating mesenchymal stem cells and associates with an impaired osteogenic differentiation. We propose that the observed epigenetic alterations reflect distinct differentiation stages of H3.3 WT and H3.3 MUT stromal cells and add to H3.3-G34W-associated changes.
Subject(s)
Bone Neoplasms/genetics , Giant Cell Tumor of Bone/genetics , Histones/genetics , Osteogenesis , Bone Neoplasms/metabolism , Bone Neoplasms/physiopathology , DNA Methylation , Epigenesis, Genetic , Epigenomics , Giant Cell Tumor of Bone/metabolism , Giant Cell Tumor of Bone/physiopathology , Histones/metabolism , Humans , Mutation, MissenseABSTRACT
OBJECTIVES: This research aimed to investigate the relative level of Runt-related transcription factor 2 (RUNX2) in giant cell tumor of bone (GCTB). Through the histopathological similarities between osteoporosis and GCTB, the biological functions of exogenous RUNXS were demonstrated in GCTB cell lines. This generated awareness of the molecular mechanism of the biogenesis and metastasis of GCTB, as well as showing the pathways and processes involved in this study. This research also expected to provide hints for the clinical treatment of patients with GCTB, to release the tumor burden and reduce the recurrence rate and metastasis of patients with this condition. METHODS: The expression of RUNX2 in the tumors was verified by Western Blot, qRT-PCR and immunohistochemistry, compared with the normal tissues' adjacent tumors. Subsequently, the plasmids expressing RUNX2 were constructed, amplified and transfected into the 0404 cell line through transfection kits (0.4, 0.8, 1.6, 2.4 ng/µl). After that, the proliferation, migration, invasion, cellular viability and apoptosis of 0404 cell lines were examined by EDU assay, wound healing assay, transwell assay, annexin v staining, and CCK8 assay, respectively. RESULTS: The messenger RNA (mRNA) level of RUNX2 in tumors was over 100 folds more than the normal tissues. The protein level of tumors upregulated 8.32(±4.41) folds relatively. After the transfection of RUNX2 overexpressed plasmids into the 0404 cell line, the mRNA level of RUNX2 increased approximately 530.11(±24.87), 1117.96(±77.68), 2835.09(±45.22) and 4781.51(±79.37) folds respectively, and the protein level was upregulated about 4.12(±1.15), 16.73(±1.63), 21.53(±2.41) and 23.39(±0.85) folds respectively. The proliferation of 0404 cells was inhibited by 2.13(±1.02)% of 1.6 ng/µl group and 3.03(±1.76)% of 2.4 ng/µl group. And the migration was inhibited about 45.56(±6.13)%, 50.79(±5.27)%, 63.15(±8.62)% and 93.90(±3.65)% respectively. The invasion was decreased about 14.49(±5.4)%, 37.02(±6.52)%, 42.24(±2.59)% and 48.97(±10.61)% respectively. Meanwhile, FITC Annexin V/PI apoptosis assay demonstrated that RUNX2 plasmids could promote apoptosis rate around 4.15(±0.27)%, 5.07(±0.27)%, 7.61(±0.45)% and 11.32(±1.02)% respectively, and CCK8 proved these plasmids could weaken cellular viability in a concentration-dependent manner with the time passing. CONCLUSIONS: RUNX2 is highly expressed in giant cell tumors of bone. The RUNX2 overexpressed plasmids we constructed could be successfully transfected into 0404 cell line. Far more importantly, the exogenous RUNX2 can seriously block the biological functions of 0404 cell line in a concentration-dependent manner, including proliferation, translocation, invasion, cellular viability, and apoptosis. Meanwhile, the mechanism was hypothesized and discussed in the article.
Subject(s)
Core Binding Factor Alpha 1 Subunit/physiology , Giant Cell Tumor of Bone/metabolism , Adolescent , Adult , Apoptosis , Cell Line, Tumor , Core Binding Factor Alpha 1 Subunit/genetics , Female , Humans , Male , Middle Aged , Plasmids , Transfection , Young AdultABSTRACT
BACKGROUND: Giant cell tumor of bone (GCTB) is considered to be a kind of borderline tumor, which has a tendency to recur and translocate. MicroRNAs are one type of small noncoding RNA, which can inhibit the translation of targeted mRNA through RNA-induced silencing complex. METHODS: Microarray was conducted on three groups of tumor tissues and normal tissues from patients with GCTB, and results showed different expression profiles of miRNAs with Gene Ontology analysis and Kyoto Encyclopedia of Genes and Genomes analysis. The functions of miR-187-5p and miR-1323, which were highly expressed in GCTB, were examined by 5-ethynyl-2'-deoxyuridine (EDU), transwell, and CCK8 assays. RNAhybrid et al. (RNA prediction softwares) predicted that the two microRNAs targeted fibroblast growth factor receptor substrate 2 (FRS2), which was verified by luciferase assay and rescue experiments. RESULTS: miR-187-5p and miR-1323 were highly expressed in tumor tissues. They can jointly regulate the biological functions of GCTB in vitro. Luciferase assay confirmed that the two microRNAs can bind to the 3' untranslated regions (UTR) of mRNA of FRS2. And, rescue experiments verified the relationships between the two microRNAs and FRS2. CONCLUSION: There were some different-expressed microRNAs between GCTB and normal tissues. miR-187-5p and miR-1323 can regulate the biological functions of GCTB through influencing the expression of FRS2.