Scanning Electron Microscopy of Giant Cells from Giant Cell Tumor of Bone.

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.

Spectrum of histological features of Denosumab treated Giant Cell Tumor of Bone; potential pitfalls and diagnostic challenges for pathologists.

OBJECTIVES: Denosumab is Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL) inhibitor which is being used in the treatment of locally advanced, recurrent and metastatic Giant Cell Tumor of Bone (GCTB). It causes reduction in monocyte recruitment and Osteoclast-Like Giant Cell (OLGC) formation which limits bone destruction. After Denosumab treatment, GCTB exhibit diverse morphological features which can pose diagnostic challenge. Our aim was to study the spectrum of histologic features seen in Denosumab treated GCTB which could be helpful in establishing correct diagnosis. METHODS: We retrieved and reviewed H&E stained microscopic glass slides of 38 GCTB cases who received Denosumab as neoadjuvant treatment. These cases were treated at different institutes and diagnosed at our institute between January 2017 and October 2019. Morphologic features such as presence of residual OLGC, appearances of mononuclear stromal and bony components were assessed along with other non-specific features. RESULTS: Patients' median age was 29 years. Male to female ratio was 1.53:1. Femur was the most commonly involved bone. Microscopically, peripheral shell of reactive bone was observed in all cases. In 20 (52.6%) cases, there was complete elimination of OLGC. Mononuclear stromal cells were predominantly bland spindle shaped and arranged in fascicular and storiform patterns. Focal atypia was noted in 3 cases. Bony component manifested as trabeculae of woven bone with osteoblastic rimming and immature trabeculae of unmineralized osteoid with haphazardly present osteoblasts. Spectrum of stromal changes included cystic spaces, foamy macrophages, inflammatory infiltrate, hemangiopericytoma-like (HPC-like) vessels, hyalinization, edematous areas and hemosiderin pigment. The tumors showed areas which resembled other bony and soft tissue lesions such non-ossifying fibroma, fibrous dysplasia, osteoblastoma, sclerosing epithelioid fibrosarcoma and osteosarcoma. CONCLUSION: Denosumab treatment induces a variety of changes in GCTB. Clinical history and knowledge of these features are necessary for excluding differential diagnoses and avoiding misdiagnosis.

Myofibroblastic differentiation of stromal cells in giant cell tumor of bone: an immunohistochemical and ultrastructural study.

The nature of the mononuclear stromal cells (MSCs) in giant cell tumor of bone (GCTB) has not been thoroughly investigated. The purpose of this study was to evaluate the degree and significance of myofibroblastic differentiation in 18 cases of GCTB by immunohistochemistry (IH) and/or electron microscopy (EM). All immunostained cases were found positive for smooth muscle actin (SMA) and/or muscle specific actin (MSA), most in 1-33% of the MSCs. Ultrastructurally, most MSCs were fibroblasts, and a significant number of cells displayed myofibroblastic differentiation. Myofibroblasts are an important component of MSCs in GCTB. The myofibroblastic population may be responsible in part for the production of matrix metalloproteinases (MMPs), which probably play a role in bone destruction, tumor aggression, and recurrence.

Cytochemical and ultrastructural changes in the osteoclast-like giant cells of giant cell tumor of bone following bisphosphonate administration.

Giant cell tumor of bone (GCT) is a local aggressive neoplasm of bone characterized by expansive osteolytic lesions at the epiphysis of long bones. Bisphosphonates have been used to prevent bone resorption in secondary osteolytic tumors because of their strong anti-osteoclastic action. The authors studied the apoptosis and ultrastructural changes induced in osteoclast-like giant cells of GCT, following treatment with the aminobisphosphonate pamidronate in 16 patients with GCT of bone. Transmission electron microscopy (TEM) was used to identify ultrastructural changes, indicative of apoptosis, in the cytoplasm and the nucleus of the giant cells. Significant changes were observed in tumor samples from all 16 patients. In the cytoplasm these changes were characterized by abundant large tubular vesicles containing a central electrodense core scattered through the cytoplasm. In addition, mitochondria in the sections from pamidronate-treated patients appeared to be edematous when compared with sections from untreated patients. Nuclear changes in the giants cells were characterized by the formation of dense chromatin material scattered throughout the nucleus. The TUNEL labeling assay indicated that the mean pretreatment apoptotic index of 7.8% increased to 53% following pamidronate treatment. This was statistically significant (p<.001) and correlated well with the ultrastructural changes noted on TEM. The formation of abundant tubular vesicles in giant cells following bisphosphonate treatment may reflect disturbed vesicular trafficking and may affect the bone resorbing activity of giant cells.

Ultrastructural features of giant cell tumors in Paget's disease.

Giant cell tumor is a rare complication of Paget's disease. This association is especially notable in patients originating from Avellino, Italy. Many types of evidence point to a viral etiology for Paget's disease and giant cell tumors arising in it. Three patients who had giant cell tumors and Paget's disease were studied. Two of the patients have a connection to Avellino (one was born in Avellino, and one descended from natives of Avellino). Distinctive light microscopic and ultrastructural features common in these three patients were identified. In all three patients, the giant cell tumors had peculiar irregular aggregates of microfilaments of uncertain genesis. The possibility that these reflect viral infection is discussed.

In vitro studies of human and rat osteoclast activity on hydroxyapatite, beta-tricalcium phosphate, calcium carbonate.

Investigations on the ceramic degradation caused by osteoclasts are designed to assess osteoclast-ceramic interactions and to determine which ceramics are more suitable for use as bone substitute. This study investigated the resorptive activity of osteoclasts on ceramics presenting different solubility rates. Osteoclasts isolated from new-born rat and from human giant cell tumour were cultured on different bioceramics: hydroxyapatite (HA), beta-tricalcium phosphate (TCP) and calcium carbonate (calcite). Cytoskeletal was revealed by actin labelling and ceramic surfaces were observed by scanning electron microscopy (SEM). On all materials, the distribution of actin in typical ring was revealed. SEM examinations showed a clear difference in the shape and the depth of resorption lacunae on different ceramics. On pure HA, a superficial attack, clearly visible but very little extended. Numerous resorption lacunae, deep and well-delimited were observed on pure beta-TCP, but attacks less punctually were detected too. On pure calcite, an attack with form of spikes, very widespread but superficial was revealed. Degradation measurements revealed a significant increase of P release from the phosphocalcic ceramics and of Ca from all ceramics in the presence of osteoclasts. The both cell models found these characteristics, the rat osteoclasts were also an excellent model to study the ceramic resorption.

Giant-cell tumor of bone arising from the falx cerebri. A case report.

The histological and ultrastructural features of a giant-cell tumor of bone arising in the falx cerebri of a 27 year-old man are described. The tumor was embedded in the medial aspect of the left frontal lobe and was not attached to any of the bones of the skull. At surgery, the tumor was lightly adherent to the falx and was easily extracted. Histologically, the tumor was composed of mononuclear spindle-shaped and ovoid stromal cells, multinuclear giant cells containing 20-30 nuclei, and foci of osteoid and bone production. Hemorrhagic and cystic areas were also present within the tumor. Ultrastructurally, the spindle-shaped cells resembled fibroblasts and were surrounded by small bundles of collagen fibrils. The ovoid cells contained numerous mitochondria, abundant rough endoplasmic reticulum, vesicles, lysosomes, phagosomes and osseous material in the cytoplasm suggesting their monocyte-macrophage lineage. These cells were closely apposed and displayed evidence of fusion in the form of focal and linear subplasmalemmal densities to form multinucleated giant cells with similar organelles and multiple nuclei. It is suggested that the primary giant cell tumor of the one arose from the metaplastic ossification of the falx. To our knowledge, a giant-cell tumor of bone arising from the falx cerebri has not been previously described.

Osteoprotegerin inhibits osteoclast formation and bone resorbing activity in giant cell tumors of bone.

Osteolysis is a common complication of tumors that arise in, or metastasize to, bone. The recent discovery of key regulators of osteoclast formation and activity, including receptor activator of nuclear factor of kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG), may facilitate new treatment regimes for certain tumors associated with excessive bone loss. We recently showed that the stromal cells of osteolytic giant cell tumors (GCT) of bone express high levels of mRNA encoding RANKL, relative to mRNA for the RANKL antagonist, OPG, compared with the expression patterns of other lytic and nonlytic bone tumors. In this study, we found that expression of RANKL and OPG mRNA continued by the stromal element of these tumors in a constitutive manner for at least 9 days in the absence of giant cells. Immunostaining of unfractionated GCT cultured in vitro revealed punctate cytoplasmic/membranous staining for RANKL and both cytoplasmic and extracellular matrix staining for OPG in stromal cells. Giant cells (osteoclasts) were negative for RANKL staining, but stained brightly for cytoplasmic OPG protein. We also investigated the functional relevance of these molecules for GCT osteolysis by adding recombinant OPG and RANKL to cultured GCT cells. We found that OPG treatment potently and dose-dependently inhibited resorption of bone slices by GCT, and could also inhibit the formation of multinucleated osteoclasts from precursors within the GCT. These effects of OPG were reversed by stoichiometric concentrations of exogenous RANKL. These data indicate that both the processes of osteoclast formation and activation in GCT are promoted by RANKL. Therefore, GCT represent a paradigm for the direct stimulation of osteoclast formation and activity by tumor stromal cells, in contrast to the mechanisms described for osteolytic breast tumors and multiple myeloma. The demonstration of these relationships is important in developing approaches to limit tumor-induced osteolysis.

Presence of leukaemia inhibitory factor (LIF) and LIF-receptor chain (gp190) in osteoclast-like cells cultured from human giant cell tumour of bone. Ultrastructural distribution.

The behaviour of multinucleated giant cells (MNC) obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized dentin slices was investigated using light and electron microscopy. Cells were studied in the presence or absence of LIF a cytokine known to be involved in bone turnover and to act as a growth factor in some solid tumours. The direct effect of LIF on MNC was examined by a post-embedding colloidal gold immunocytochemistry process using human anti-LIF and anti-LIF-receptor (chain gp190) antibodies. After 7 days of culture, the MNC obtained displayed osteoclast immunocytochemical features. Moreover, these MNC were able to resorb large amounts of dentin and presented typical features of active osteoclasts. Immunolocalization of LIF and LIF-receptor revealed the presence of this cytokine and its receptor within the cytoplasm and nucleus of active resorbing MNC. LIF upmodulated MNC number and nucleation but decreased their ability to resorb dentin. The present study suggests that MNC obtained from human GCT, currently considered as osteoclast-like cells, are targets for LIF and may be a source of LIF production in this pathological condition.

Expression of regulatory apoptotic proteins in peripheral giant cell granulomas and lesions containing osteoclast-like giant cells.

Peripheral giant cell granuloma consists of mononuclear cells and osteoclast-like giant cells. The proliferative ability of peripheral giant cell granuloma is restricted to the mononuclear cell compartment, whereas multinucleated giant cells lack mitotic activity. Although the proliferative compartment of peripheral giant cell granuloma has been investigated in detail, the expression and distribution of proteins regulating apoptosis is unknown. The present study demonstrates strong expression of bak and bax in the majority of giant cells. In contrast, giant cells show only weak positivity for bcl-2 and moderate positivity for bcl-x. Mononuclear cells were negative to weakly positive for bcl-x. Only scattered mononuclear cells were positive for bak, bax and bcl-2. The frequency of apoptotic nuclei detected by TUNEL-staining compared to regular nuclei was 18 times higher in giant cells than in mononuclear cells. In summary, our findings support the presumption that giant cells of bone and soft tissue tumors are reactive cell forms and not of neoplastic origin.

PP60c-src expression in osteoclasts from osteopetrotic children and in giant tumor cells.

Malignant infantile osteopetrosis is a severe congenital disease characterized by impaired osteoclast activity. Among the multiple factors that influence bone resorption, the c-src protooncogene product pp60c-src plays an essential role, since mice which lack pp60c-src develop osteopetrosis. To gain insight into the possible role of pp60c-csrc in the pathogenesis of infantile osteopetrosis, we examined the osteoclasts of three children displaying the typical features of the disease, aged respectively one, four and seven months. pp60c-csrc expression and localization, together with the expression of a 80/85-kilodalton pp60c-src substrate, cortactin, were examined by immunoelectron microscopy. Osteoclasts from two giant cell tumors were used as controls. Bone and tumor samples were fixed in 4% paraformaldehyde, included in LR-White resin at -30 degrees C and the sections processed with mAb 327 or mAb anti p80/85 by an immunogold technique. pp60c-src was expressed in the cytoplasm, in nuclear membranes and in nuclei of the osteoclasts of the three osteopetrotic children. The subcellular localization of the kinase was not different from the localization in giant tumor cells. In both cases cortactin was abundant. In conclusion, in three children with malignant osteopetrosis, pp60c-src expression in osteoclasts does not appear to be involved in the pathogenesis of the disease. The presence of this protein, however, does not necessarily reflect normal c-src tyrosine kinase activity, nor normal c-src-dependent intracellular signaling pathways. Moreover the presence of the protein in nuclear membranes, and especially around nuclear pores supports the hypothesis that in osteoclasts, c-src may participate in the regulation of RNA processing.

Giant cell tumor of the occipital bone in a case of von Recklinghausen neurofibromatosis.

Von Recklinghausen neurofibromatosis (NF1) is the most common hereditary syndrome predisposing to neoplasia. The most common symptomatic manifestation of NF1 is the plexiform neurofibroma. We describe the case of a patient with classical von Recklinghausen neurofibromatosis presenting with a giant cell tumor (GCT) of the occipital bone infiltrating a surrounding plexiform neurofibroma.

Detection of mRNAs for urokinase-type plasminogen activator, its receptor, and type 1 inhibitor in giant cell tumors of bone with in situ hybridization.

Although giant cell tumor of bone (GCT) is generally considered to be an uncommon benign neoplasm, it can pursue an aggressive course with local recurrence and metastasis. Attempts to predict the biological behavior of GCT with histopathological parameters, however, have not been successful. The urokinase-type plasminogen activation system has been implicated in tumor invasion and metastasis and abnormalities of the components of this system have been found in several malignancies. In this study we postulated that the urokinase-type plasminogen activation system associated with bone destruction and local invasion is present in GCT. We therefore evaluated the mRNA levels for urokinase-type plasminogen activator (u-PA), urokinase-type plasminogen activator receptor (u-PAR), and plasminogen activator inhibitor type 1 (PAI-1) by using Northern blot analysis and in situ hybridization in four cases of GCT and spindle-shaped mononuclear cells at the 35th passage from a GCT. Our results showed that giant cell tumors of bone contained variable levels of u-PA, u-PAR, and PAI-1 mRNA, respectively, 2.3, 1.4, and 3.2 kb in size. In situ hybridization showed that u-PA, u-PAR, and PAI-1 mRNA were expressed in both the mononuclear cells and the osteoclast-like giant cells; the signal for u-PA mRNA in the spindle-shaped mononuclear cells was more intense than that in the osteoclast-like multinuclear giant cells. Some spherical mononuclear cells (macrophage-like cells) expressed high levels of PAI-1 mRNA in comparison with the spindle-shaped mononuclear cells. In addition, the 35th passaged spindle-shaped mononuclear cells were used to study the gene expression of u-PA during cell proliferation. The results showed that the level of u-PA mRNA increases after adding 10% fetal calf serum to quiescent cells. The induction was maximal at 16 hours and remained high during 48 hours of treatment. In conclusion, even though osteoclast-like cells are ultimately responsible for the bone resorption of GCT, the mononuclear neoplastic cells of GCT may also be involved in degradation of the extracellular matrix during invasive growth by facilitating the urokinase plasminogen activation system. In addition, our observation of upregulation of u-PA mRNA in spindle-shaped mononuclear cells after serum stimulation indicated that u-PA production may be linked to tumor growth.

[Osteoclastoma of temporal and zygomatic bone]. / Guz olbrzymiokomórkowy kosci skroniowej i jarzmowej.

A twelve year boy was admitted to the Otolaryngological Clinic of the Medical Academy in Cracow with a paralysis of the peripheral facial nerve and a tumor on the right zygomatic arc. A CT investigation revealed an expansive process in the central fossa of the skull, which continuously passed into the squama of the temporal bone and the are of the zygomatic bone on the right side, causing a paralysis of the nerve VII. A neurosurgical intervention was carried out as well as a subsequent treatment by radiotherapy. A remission of the tumor was achieved. Osteoclastoma was diagnosed histologically.

Relationship between microvascular structure and biological characteristics of giant cell tumour of bone.

The vascular architecture of bone giant cell tumor was observed histologically with resin cast technique and scanning electron microscopy. Three types of capillaries were noted in the tumor tissue: extruding club-like capillary in the outer zone of the tumor; sinusoid capillary running disorderly in the intermediate zone; cecum capillary in the central zone. The pattern of vascular structure was believed to be correlated with tumor growth.

Calcitonin receptors on neoplastic mononuclear cells cultured from a human giant-cell tumor of the sacrum.

Saturable, specific, high-affinity calcitonin receptors were demonstrated in cultured neoplastic mononuclear spindle cells from a giant-cell tumor of the sacrum of a 38-year-old woman. The receptor was analyzed by autoradiography and 125I-calcitonin binding assay. Binding reversibility of 125I-calcitonin to the cells was not complete and the structural specificity was indicated by the inability of unrelated hormones to compete with calcitonin. The 24,000 receptors/cell and dissociation constant (Kd) of 8.0 x 10(-10) M, calculated from linear Scatchard plots, suggested the existence of a single class of calcitonin binding sites in the neoplastic mononuclear cells. Flow-cytometric analysis in the primary culture showed that mononuclear cells consisted of mononuclear round cells of monocyte/macrophage lineage, which express more calcitonin receptors than neoplastic mononuclear spindle cells. Administration of calcitonin caused morphological and physiological alterations, resulting in involutional or irregular cytoplasmic shapes and inhibition of DNA synthesis in neoplastic mononuclear cells accompanied by the escape phenomenon. Cells preincubated with calcitonin showed a decrease in 125I-calcitonin binding activity, which could account for the escape phenomenon. The decrease in 125I-calcitonin binding was rapid, but the recovery was not observed for 24 h after elimination of calcitonin. This decrease may be caused by the disappearance of residual receptors or by a decrease in calcitonin affinity. The calcitonin-induced morphological changes and the inhibition of DNA synthesis of cells were revealed to be mediated by calcitonin receptors.

Tumor de células gigantes de los huesos (osteoclastoma): revisión anatomopatológica y clínica de 362 casos / Giant cells tumor of the bones (osteoclastoma): anatomopathologic and clinical revision of 362 cases

Se realizó un estudio anatomoclínico sobre 362 casos de T.C.G. óseo, correspondientes al Registro Latinoamericano de Patología Osea, entre 1940 y 1978. El T.C.G. es una neoplasia agresiva, potencialmente maligna, que recidiva en aproximadamente un 50 por ciento de los casos sometidos a curetaje, sufre una transformación sarcomatosa en aproximadamente un 10 por ciento y puede producir metástasis sin que exista una transformación maligna observable histológicamente. En los pocos casos que inicialmente el tumor posee una apariencia histológica de malignidad, ésta no se correlaciona con sus características clínicas y radiológicas. Por lo tanto la gradación histológica de esta neoplasia es considerada actualmente, como carente de valor. Estudios histoquímicos y ultraestructurales demuestran semejanza entre la célula gigante tumoral y el osteoclasto y condroclasto normales. Como asi mismo con las células gigantes multinucleadas presentes en las llamadas variantes. Es posible que avances en el conocimiento de la relación inmune huésped-tumor sirvan para esclarecer la patogenia y pronóstico de esta neoplasia. Es necesario establecer el diagnóstico clínico e histopatológico correcto de T.C.G. genuino y diferenciarlo de las llamadas variantes, dado que estas últimas poseen un pronóstico más favorable y son curables con simple curetaje. Dado las características agresivas del T.C.G. genuino, está indicada la resección tumoral como método de elección. La radioterapia mantiene su indicación en las localizaciones inaccesibles a la cirugía.

Tumor de células gigantes de los huesos (osteoclastoma): revisión anatomopatológica y clínica de 362 casos / Giant cells tumor of the bones (osteoclastoma): anatomopathologic and clinical revision of 362 cases

Se realizó un estudio anatomoclínico sobre 362 casos de T.C.G. óseo, correspondientes al Registro Latinoamericano de Patología Osea, entre 1940 y 1978. El T.C.G. es una neoplasia agresiva, potencialmente maligna, que recidiva en aproximadamente un 50 por ciento de los casos sometidos a curetaje, sufre una transformación sarcomatosa en aproximadamente un 10 por ciento y puede producir metástasis sin que exista una transformación maligna observable histológicamente. En los pocos casos que inicialmente el tumor posee una apariencia histológica de malignidad, ésta no se correlaciona con sus características clínicas y radiológicas. Por lo tanto la gradación histológica de esta neoplasia es considerada actualmente, como carente de valor. Estudios histoquímicos y ultraestructurales demuestran semejanza entre la célula gigante tumoral y el osteoclasto y condroclasto normales. Como asi mismo con las células gigantes multinucleadas presentes en las llamadas variantes. Es posible que avances en el conocimiento de la relación inmune huésped-tumor sirvan para esclarecer la patogenia y pronóstico de esta neoplasia. Es necesario establecer el diagnóstico clínico e histopatológico correcto de T.C.G. genuino y diferenciarlo de las llamadas variantes, dado que estas últimas poseen un pronóstico más favorable y son curables con simple curetaje. Dado las características agresivas del T.C.G. genuino, está indicada la resección tumoral como método de elección. La radioterapia mantiene su indicación en las localizaciones inaccesibles a la cirugía. (AU)