Regulation of osteosarcoma cell lung metastasis by the c-Fos/AP-1 target FGFR1.

Osteosarcoma is the most common primary malignancy of the skeleton and is prevalent in children and adolescents. Survival rates are poor and have remained stagnant owing to chemoresistance and the high propensity to form lung metastases. In this study, we used in vivo transgenic models of c-fos oncogene-induced osteosarcoma and chondrosarcoma in addition to c-Fos-inducible systems in vitro to investigate downstream signalling pathways that regulate osteosarcoma growth and metastasis. Fgfr1 (fibroblast growth factor receptor 1) was identified as a novel c-Fos/activator protein-1(AP-1)-regulated gene. Induction of c-Fos in vitro in osteoblasts and chondroblasts caused an increase in Fgfr1 RNA and FGFR1 protein expression levels that resulted in increased and sustained activation of mitogen-activated protein kinases (MAPKs), morphological transformation and increased anchorage-independent growth in response to FGF2 ligand treatment. High levels of FGFR1 protein and activated pFRS2α signalling were observed in murine and human osteosarcomas. Pharmacological inhibition of FGFR1 signalling blocked MAPK activation and colony growth of osteosarcoma cells in vitro. Orthotopic injection in vivo of FGFR1-silenced osteosarcoma cells caused a marked twofold to fivefold decrease in spontaneous lung metastases. Similarly, inhibition of FGFR signalling in vivo with the small-molecule inhibitor AZD4547 markedly reduced the number and size of metastatic nodules. Thus deregulated FGFR signalling has an important role in osteoblast transformation and osteosarcoma formation and regulates the development of lung metastases. Our findings support the development of anti-FGFR inhibitors as potential antimetastatic therapy.

Use of chloroacetate esterase staining for the histological diagnosis of prosthetic joint infection.

A heavy neutrophil polymorph infiltrate [>5 per high-power field (HPF) after examination of at least 5 HPF by Musculoskeletal Infection Society (MSIS) criteria] is characteristically seen in peri-implant tissues of infected prosthetic hip and knee joints. We determined whether chloroacetate esterase (CAE) staining facilitated the identification of neutrophil polymorphs in peri-implant tissues in cases of hip and knee arthroplasty infection and reassessed MSIS criteria in the light of our findings. Frozen and paraffin sections of peri-prosthetic tissues of 76 cases of failed hip and knee arthroplasties classified as septic or aseptic loosening microbiologically were analysed histologically by both haematoxylin-eosin and CAE staining. The extent of the neutrophil polymorph infiltrate was determined semiquantitatively and correlated with the microbiological and clinical diagnosis. CAE staining facilitated identification of neutrophil polymorphs in arthroplasty tissues. All cases of aseptic loosening contained fewer than two neutrophil polymorphs per HPF. CAE staining showed that in some cases of septic loosening, fewer than five neutrophil polymorphs per HPF (on average) are present in peri-prosthetic tissues. The histological criterion of more than two neutrophil polymorphs per HPF showed increased sensitivity and accuracy for the diagnosis of septic loosening. CAE is a useful stain that facilitates the identification of neutrophil polymorphs in both frozen and paraffin sections of peri-implant tissues. CAE staining shows that some microbiologically confirmed cases of septic loosening contain relatively few neutrophil polymorphs, indicating that the MSIS histological criterion of more than five neutrophil polymorphs per HPF is too high an index figure for the diagnosis of all cases of hip and knee arthroplasty infection.

Dentine matrix protein 1 (DMP-1) is a marker of bone formation and mineralisation in soft tissue tumours.

Dentine matrix protein 1 (DMP-1) is a non-collagenous matrix protein found in dentine and bone. It is highly expressed by osteocytes and has been identified in primary benign and malignant osteogenic bone tumours. Bone formation and matrix mineralisation are seen in a variety of benign and malignant soft tissue tumours and tumour-like lesions, and in this study, we analysed immunohistochemically the DMP-1 expression in a wide range of soft tissue lesions (n = 254) in order to assess whether DMP-1 expression is useful in the histological diagnosis of soft tissue tumours. Matrix staining of DMP-1 was seen in all cases of myositis ossificans, fibro-osseous tumour of the digits, extraskeletal soft tissue osteosarcoma and in most cases of ossifying fibromyxoid tumour. DMP-1 was also noted in dense collagenous connective tissue of mineralising soft tissue lesions such as tumoural calcinosis, dermatomyositis and calcific tendinitis. DMP-1 was expressed in areas of focal ossification and calcification in synovial sarcoma and other soft tissue tumours. With few exceptions, DMP-1 was not expressed in other benign and malignant soft tissue tumours. Our findings indicate that DMP-1 is a matrix marker of bone formation and mineralisation in soft tissue tumours. DMP-1 expression should be particularly useful in distinguishing extraskeletal osteosarcoma and ossifying fibromyxoid tumour from other sarcomas and in identifying areas of osteoid/bone formation and mineralisation in soft tissue tumours.

The role of calcium and nicotinic acid adenine dinucleotide phosphate (NAADP) in human osteoclast formation and resorption.

Osteoclasts are specialised bone resorbing cells which form by fusion of circulating mononuclear phagocyte precursors. Bone resorption results in the release of large amounts of calcium into the extracellular fluid (ECF), but it is not certain whether changes in extracellular calcium concentration [Ca(2+)]e influence osteoclast formation and resorption. In this study, we sought to determine the effect of [Ca(2+)]e and NAADP, a potent calcium mobilising messenger that induces calcium uptake, on human osteoclast formation and resorption. CD14+ human monocytes were cultured with M-CSF and RANKL in the presence of different concentrations of calcium and NAADP and the effect on osteoclast formation and resorption evaluated. We found that the number of TRAP+ multinucleated cells and the extent of lacunar resorption were reduced when there was an increase in extracellular calcium and NAADP. This was associated with a decrease in RANK mRNA expression by CD14+ cells. At high concentrations (20 mM) of [Ca(2+)]e mature osteoclast resorption activity remained unaltered relative to control cultures. Our findings indicate that osteoclast formation is inhibited by a rise in [Ca(2+)]e and that RANK expression by mononuclear phagocyte osteoclast precursors is also [Ca(2+)]e dependent. Changes in NAADP also influence osteoclast formation, suggesting a role for this molecule in calcium handling. Osteoclasts remained capable of lacunar resorption, even at high ECF [Ca(2+)]e, in keeping with their role in physiological and pathological bone resorption.

Intraosseous schwannoma in schwannomatosis.

This study investigates the clinical, radiological, and pathological features of two cases of intraosseous schwannoma that arose in patients with multiple soft tissue schwannomas. In both cases, the patients were adult females and the tibial bone was affected. Vestibular schwannomas were not identified, indicating that these were not cases of neurofibromatosis 2 (NF2). Radiographs showed a well-defined lytic lesion in the proximal tibia; in one case, this was associated with a pathological fracture. Histologically, both cases showed typical features of benign schwannoma. Molecular analysis of one of the excised tumors showed different alterations in the NF2 gene in keeping with a diagnosis of schwannomatosis. Our findings show for the first time that intraosseous schwannomas can occur in schwannomatosis.

Locally aggressive fibrous dysplasia.

Although fibrous dysplasia (FD) is a benign fibro-osseous lesion, locally aggressive behaviour has rarely been described but is poorly characterised. In this study, we document clinical, radiological and pathological (including molecular genetics) findings in three cases of locally aggressive FD, two of which involved the ribs. Lesions in these cases, one of which was a recurrent lesion, were followed up for 2-7 years. All of the lesions showed typical histological features of FD but were characterised by extension through the bone cortex into the extra-osseous soft tissue. The lesions did not exhibit overexpression/amplification of CDK4 and MDM2; in two of the cases, a GNAS mutation was identified. Our findings confirm that FD can rarely exhibit locally aggressive behaviour with extension beyond the bone compartment into the surrounding soft tissue; these lesions can be distinguished from low-grade intramedullary osteosarcoma by lack of amplification/overexpression of CDK4 and MDM2 and the presence of a GNAS mutation.

Dentine matrix protein 1 (DMP-1) is a marker of bone-forming tumours.

Dentin matrix protein 1 (DMP-1) is highly expressed by osteocytes and is a non-collagenous matrix protein found in dentin and bone. In this study, we determined the expression of DMP-1 in mature and immature human bone and examined whether DMP-1 is useful in distinguishing osteoid/bone-forming tumours from other primary and secondary bone tumours. DMP-1 expression was immunohistochemically determined in paraffin sections of a wide range of benign and malignant primary bone tumours and tumour-like lesions (n = 353). DMP-1 mRNA expression was also examined in osteosarcoma and fibrosarcoma cell lines as well as bone tumour specimens (n = 5) using real-time PCR. In lamellar and woven bone, DMP-1 was expressed in the matrix around osteocyte lacunae and canaliculi; osteoblasts and other cell types in the bone were negative. Matrix staining of the osteoid and bone was seen in bone-forming tumours including osteoma, osteoid osteoma, osteoblastoma and osteosarcoma. DMP-1 staining was also seen in fibrous dysplasia, osteofibrous dysplasia and chondroblastoma and in reactive bone in solitary bone cysts and aneurysmal bone cysts. DMP-1 was not expressed in the tumour component of other bone neoplasms including Ewing sarcoma, chondrosarcoma, leiomyosarcoma, fibrosarcoma, giant cell tumour of bone and metastatic carcinoma. DMP-1 mRNA was expressed in osteosarcoma cell lines and tumour samples. DMP-1 is a matrix marker expressed around osteocytes in human woven and lamellar bone and is useful in identifying osteosarcoma and other bone-forming tumours.

Diagnostic utility of aP2/FABP4 expression in soft tissue tumours.

Adipocyte P2 (aP2), also known as fatty acid-binding protein 4 (FABP4), is a fatty acid-binding protein found in the cytoplasm of cells of adipocyte differentiation. In this study, we examined a large number of soft tissue tumours with a commercial polyclonal anti-aP2/FABP4 antibody and a newly developed mouse monoclonal antibody raised against this protein to determine the diagnostic utility of aP2/FABP4 as a marker of tumours of adipose differentiation. A mouse monoclonal antibody, clone 175d, was raised against a mixture of synthetic peptides corresponding to the amino acid sequence of residues 10-28 and 121-132 of the human aP2/FABP4 protein. Antigen expression with polyclonal and monoclonal antibodies was immunohistochemically determined in paraffin sections of normal adipose tissue and a wide range of benign and malignant primary soft tissue tumours (n = 200). aP2/FABP4 was expressed around the cytoplasmic lipid vacuole in white and brown fat cells in benign lipomas and hibernomas. Immature fat cells and lipoblasts in spindle cell/pleomorphic lipoma, atypical lipomatous tumour/well-differentiated liposarcoma, myxoid/round cell liposarcoma and pleomorphic liposarcoma also reacted strongly for aP2/FABP4. No specific staining was seen in non-adipose benign and malignant mesenchymal and non-mesenchymal tumours. aP2/FABP4 is expressed by mature and immature fat cells and is a marker of tumours of adipose differentiation. Immunophenotypic aP2/FABP4 expression is useful in identifying lipoblasts, and immunohistochemistry with polyclonal/monoclonal anti-aP2/FABP4 antibodies should be useful in distinguishing liposarcoma from other malignancies, particularly round cell, myxoid and pleomorphic soft tissue sarcomas.

Investigation of osteoclastogenic signalling of the RANKL substitute LIGHT.

LIGHT (TNFSF14) is a member of the TNF superfamily and is known to substitute for RANKL to induce osteoclast differentiation. LIGHT binds HVEM and LTßR, but it is not known whether these receptors play a role in osteoclast formation or whether LIGHT acts via RANKL signalling pathways. We found that both RANKL and LIGHT strongly induced phosphorylation of Akt and NFκB but not JNK in mouse osteoclast precursor cells. The addition of an Akt inhibitor showed decreased osteoclast differentiation and resorption mediated by both RANKL and LIGHT. RT-PCR and FACS analysis showed that CD14(+) human osteoclast precursors expressed HVEM and LTßR; expression levels of HVEM increased in the course of osteoclastogenesis and a decrease in LIGHT expression was associated with an increase in HVEM suggesting that there is a feedback loop related to this receptor. Our findings show that LIGHT is not inhibited by the soluble RANKL receptor OPG and that LIGHT is a potent osteoclastogenesis factor that activates the Akt, NFκB and JNK pathways.

Giant-cell-rich pseudotumour in Paget's disease.

Paget's disease (PD) of the bone is a disorder of bone remodelling that may be polyostotic or monostotic. Although development of a sarcoma in PD is well-recognised, it is less well recognised that pseudosarcomas in bone and soft tissue can also arise in this condition. In this report we document the case of a large giant-cell-rich pseudotumour that developed in the tibia and overlying soft tissues in a case of polyostotic PD. Bone and soft tissues were highly vascular and contained abundant haemorrhage with focal areas of new bone formation and a diffuse infiltrate of osteoclastic giant cells. The lesion has not recurred or produced metastases 3 years after removal. Clinicians should be aware that a benign giant-cell-rich pseudotumour can develop in PD and that it needs to be distinguished from other giant-cell-rich tumours.

Chondroclasts are mature osteoclasts which are capable of cartilage matrix resorption.

Multinucleated cells termed chondroclasts have been observed on the deep surface of resorbed hyaline cartilage but the relationship of these cells to macrophages and osteoclasts and their role in rheumatoid arthritis (RA) and other arthritic conditions is uncertain. Multinucleated cells in RA and other arthritic conditions showing evidence of cartilage resorption were characterised immunohistochemically for expression of macrophage/osteoclast markers. Mature human osteoclasts formed from circulating monocytes and tissue macrophages were cultured for up to 4 days on slices of human cartilage and glycosaminoglycan (GAG) release was measured. Multinucleated cells resorbing unmineralised cartilage were seen in osteoarthritis, RA, septic arthritis, avascular necrosis and in four cases of giant cell tumour of bone that had extended through the subchondral bone plate. Chondroclasts expressed an osteoclast-like phenotype (TRAP+, cathepsin K+, MMP9+, CD14-, HLA-DR-, CD45+, CD51+ and CD68+). Both macrophages and osteoclasts cultured on cartilage released GAG. These findings indicate that chondroclasts have an osteoclast-like phenotype and that mature human osteoclasts are capable of cartilage matrix resorption. Resorption of unmineralised subchondral cartilage by chondroclasts and macrophages can be a feature of joint destruction in inflammatory and non-inflammatory arthropathies as well as inflammatory and neoplastic subchondral bone lesions.

Smooth muscle actin expression in primary bone tumours.

Alpha isoform of smooth muscle actin (SMA) expression has been reported in giant cell tumour of bone (GCTB) and other benign and malignant bone tumours, but the pattern of SMA expression and the precise nature of SMA-expressing cells in these lesions is uncertain. We determined by immunohistochemistry the expression of SMA and other muscle and vascular markers in normal bone, GCTB and a wide range of primary benign and malignant bone tumours. Cultured stromal cells of GCTB, chondroblastoma (CB), and aneurysmal bone cyst (ABC) were also analysed for SMA expression. SMA was only noted in blood vessels in normal bone. SMA was expressed by mononuclear stromal cells (MSC) cultured from GCTB, ABC and CB. SMA was strongly and diffusely expressed by MSC in non-ossifying fibroma, fibrous dysplasia, and "brown tumour" of hyperparathyroidism. SMA expression was also noted in GCTB, ABC, CB, chondromyxoid fibroma, malignant fibrous histiocytoma of bone and osteosarcoma. Little or no SMA was noted in Langerhans cell histiocytosis, simple bone cyst, Ewing's sarcoma, osteoblastoma, osteoid osteoma, enchondroma, osteochondroma, chondrosarcoma, myeloma, lymphoma, chordoma and adamantinoma. Our findings show that there is differential SMA expression in primary bone tumours and that identifying the presence or absence of SMA is useful in the differential diagnosis of these lesions. The nature of SMA-expressing cells in bone tumours is uncertain but they are negative for desmin and caldesmon and could represent either myofibroblasts or perivascular cells, such as pericytes.

Chronic expanding hematoma in combination with a pseudoaneurysm: a case report.

After bleeding from trauma or surgery, most of the hematomas undergo spontaneous reabsorption. But, in some rare cases, hematomas persist for long periods as slowly expanding masses for months or years. These hematomas were termed as chronic expanding hematomas. In this report, we describe a case of chronic expanding hematoma with a pseudoaneurysm that underwent surgical biopsy, which led to an increase in its expansion speed.

Hepatocellular oncofetal protein, glypican 3 is a sensitive marker for alpha-fetoprotein-producing gastric carcinoma.

AIMS: Glypican 3 (GPC3) is a cell surface heparan sulphate proteoglycan expressed specifically in the fetal liver and malignant neoplasms of hepatocyte lineage. The aim was to evaluate the significance of GPC3 in alpha-fetoprotein (AFP)-producing gastric carcinoma (GC) and other forms of GC. METHODS AND RESULTS: We immunohistochemically evaluated GPC3 expression in representative cases of AFP-producing GC and in a tissue microarray of a consecutive series of GCs with other markers of hepatocyte lineage (AFP, PIVKA-II and hepatocyte antigen, HEP). In a series of 10 cases of AFP-producing GC, we observed immunohistochemical positivity for GPC3, PIVKA-II and HEP in 10, three and three cases in components with a hepatoid pattern and in nine, two and five cases in components with a non-hepatoid pattern, respectively. In a series of 118 cases of GC, we observed positivity for AFP, GPC3, PIVKA-II and HEP in one (0.8%), four (3.4%), six (5.1%) and 26 cases (22%), respectively. GPC3 was observed concurrently with AFP and discordantly with PIVKA-II and HEP. GPC3 positivity was clearly stronger in a larger area compared with immunoreactivity for AFP. CONCLUSIONS: GPC3 is a sensitive marker for AFP-producing GC and its hepatoid component and is therefore useful to identify this aggressive subgroup of GC.

Sonographic features of hepatic artery calcification in chronic renal failure.

PURPOSE: Little is known about radiological aspects of hepatic artery calcification. For this reason, the sonographic features of calcified hepatic arteries were studied in patients with chronic renal failure who frequently develop tissue calcification due to hyperparathyroidism. MATERIAL AND METHODS: Ultrasound was performed in 314 patients on dialysis who underwent examination of the liver, spleen and kidney twice in the past 4 years and were reevaluated with particular attention to acoustic shadowing. Abdominal CT was carried out in 219 of these 314 patients, and ultrasound findings were reevaluated with reference to CT findings. RESULTS: A large acoustic shadow was seen in the hilum that corresponded to the calcified hepatic artery on CT in 8 patients, and in another 9, a similar large shadow was seen in the hilum without CT confirmation. Acoustic shadows of various sizes were definitely or most likely due to arterial calcification within the liver in 36 patients. The strength of shadow depended on the angle at which the ultrasound beam hit the artery. Probable calcification of small arteries accompanying portal veins was seen in 70 patients. These changes are different from those due to pneumobilia and biliary tract diseases. CONCLUSION: Calcification of the hepatic artery can be identified by its acoustic shadow.

Clinical significance of extra-pulmonary involvement of invasive aspergillosis: a retrospective autopsy-based study of 107 patients.

Disseminated aspergillus infection has a poor prognosis, but few reports have been published on extra-pulmonary involvement in aspergillosis. We reviewed 107 autopsy records of patients with invasive aspergillosis. Fifty-five patients had extra-pulmonary aspergillosis. Organs involved included heart, kidney, central nervous system, gastrointestinal tract, spleen, liver, thyroid gland and pancreas. Extra-pulmonary aspergillosis produces different manifestations according to involved organs. Risk factors associated with dissemination included cytotoxic chemotherapy within a month of death (P=0.0087). Lack of response to empiric or preemptive treatment of amphotericin B predicted IA dissemination (P=0.0328). To improve prognosis of IA, it is important to recognize clinical features of extra-pulmonary aspergillosis and to institute the aggressive anti-fungal treatment.

The value of the chest computed tomography halo sign in the diagnosis of invasive pulmonary aspergillosis. An autopsy-based retrospective study of 48 patients.

To evaluate the diagnostic value of a halo on computed tomography (CT) in the diagnosis of invasive pulmonary aspergillosis (IPA), we retrospectively reviewed chest CT scans and autopsy reports for patients who had been admitted to our hospitals for the treatment of hematological malignancy. Pulmonary complications were suspected in all patients and chest CT scans were taken within a month of death. We examined the association between autopsy and CT findings in 48 patients who were diagnosed as IPA (n = 17), candidosis (n = 4), zygomycosis (n = 2), infiltration of hematological malignancy (n = 12), bacterial pneumonia (n = 6), cytomegalovirus pneumonia (n = 2), pulmonary hemorrhage (n = 2), or pulmonary congestion (n = 1). Patients with IPA showed a variety of CT findings, including halo (n = 13), nodules (n = 14), granular shadows (n = 3), masses (n = 6), consolidations (n = 9), wedge-shaped consolidations (n = 1), and cavitation (n = 2). In contrast, 0, 11 and two of the 31 patients without IPA showed halo, nodules and masses, respectively. These signs were more frequently observed in IPA patients than in non-IPA patients. The CT halo, especially, seemed to be specific for IPA in hospitalized neutropenic patients with hematological malignancies who developed antibiotic-resistant fever. For CT findings other than these three signs, there were no significant differences between IPA- and non-IPA patients.