Limited diagnostic value of serum inflammatory biomarkers in the diagnosis of fracture-related infections.

AIMS: The aim of this study was to evaluate the diagnostic value of preoperative serum CRP, white blood cell count (WBC), percentage of neutrophils (%N), and neutrophil to lymphocyte ratio (NLR) when using the fracture-related infection (FRI) consensus definition. METHODS: A cohort of 106 patients having surgery for suspected septic nonunion after failed fracture fixation were studied. Blood samples were collected preoperatively, and the concentration of serum CRP, WBC, and differential cell count were analyzed. The areas under the curve (AUCs) of diagnostic tests were compared using the z-test. Regression trees were constructed and internally cross-validated to derive a simple diagnostic decision tree. RESULTS: Using the FRI consensus definition, 46 patients (43%) were identified as infected. Sensitivity, specificity, and AUC of CRP were 67% (95% confidence interval (CI) 52% to 80%), 61% (95% CI 47% to 74%), and 0.64 (95% CI 0.54 to 0.74); of WBC count were 17% (95% CI 9% to 31%), 95% (95% CI 86% to 99%), and 0.57 (95% CI 0.50 to 0.62); of %N 13% (95% CI 6% to 26%), 87% (95% CI 76% to 93%), and 0.50 (95% CI 0.43 to 0.56); and of NLR 28% (95% CI 17% to 43%), 80% (95% CI 68% to 88%), and 0.54 (95% CI 0.46 to 0.63), respectively. A better performance of serum CRP was shown in comparison to the leucocyte count (p = 0.006), %N (p < 0.001), and NLR (p = 0.001). A statistically lower serum CRP level was shown in patients with an infection caused by a low virulence microorganism in comparison to high virulence bacteria (p = 0.008). We found that a simple decision tree approach using only low serum neutrophils (< 3.615 × 109/l) and low CRP (< 2.45 mg/l) may allow better identification of aseptic cases. CONCLUSION: The evaluated serum inflammatory markers showed limited diagnostic value in the preoperative diagnosis of FRI when using the uniform FRI Consensus Definition. Therefore, they should remain as suggestive criteria in diagnosing FRI. Although CRP showed a higher performance in comparison to the other serum markers, it is insufficiently accurate to diagnose a septic nonunion, especially when caused by low virulence microorganisms. Cite this article: Bone Joint J 2020;102-B(7):904-911.

Neuroendocrine Tumor Arising in a Tailgut Cyst: A Rare Presacral Tumor.

A tailgut cyst (retrorectal cystic hamartoma) is an uncommon lesion that develops in the presacral (retrorectal) space. Malignant change in a tailgut cyst is extremely rare and presents as a soft tissue (presacral) or bone (sacral) neoplasm. We report a case of tailgut cyst in which a neuroendocrine tumor developed in a 25-year-old female. Computed tomography and magnetic resonance imaging scans revealed a sacrococcygeal malformation with absent left S4 and S5 and a partly cystic lesion within the right presacral space. Histologically, the lesion contained cystic and solid elements. The cysts were lined by columnar and stratified squamous epithelial cells with underlying patchy smooth muscle. The solid element was a partly necrotic neuroendocrine tumor composed mainly of ribbons of tumor cells, which showed mitotic activity and expressed cytokeratin, chromogranin, and synaptophysin. Histologically, tailgut cysts are lined by epithelium and contain scattered smooth muscle bundles in the cyst wall. Although rare, the possibility of tailgut cyst with neuroendocrine tumor should be included in the differential diagnosis of an enlarging presacral tumor.

Giant cell tumour of the distal radius/ulna: response to pre-operative treatment with short-term denosumab.

BACKGROUND: Treatment of giant cell tumour of bone (GCTB) of the distal radius/ulna poses a surgical challenge, as complex reconstructive surgery may be required. This study evaluates the clinical, radiological and pathological findings in five cases of GCTB of the distal forearm where a 3 month course of denosumab was given prior to surgery. METHODS: Patients with biopsy proven distal forearm GCTB, treated for 3 months with denosumab, followed by salvage surgery (curettage and cementation) were included. Wrist pain and function were assessed using the modified Mayo Wrist Score (MMWS). Plain radiographs, MRI and PET/CT were performed pre-treatment and 2 months after initiation of denosumab therapy. Histological comparison was made between the original biopsy and surgical curettage specimens. RESULTS: Five patients with an average age of 25 years were included in the study. Improvement in wrist pain and function was seen in all patients with the average MMWS increasing from 30 pre-treatment to 85 at 3 months. Plain radiographs demonstrated marginal sclerosis in all cases with reconstitution of cortical and subarticular bone by 2 months; internal matrix sclerosis and osseous consolidation was more variable. Increased tumour heterogeneity and low signal were observed on T2-weighted MR images. PET/CT revealed a decrease in average SUV from 14.8 pre-treatment to 4.7 at 2 months. Histology showed disappearance of osteoclasts and increased fibro-osseous tissue. Denosumab treatment has the potential to facilitate salvage surgery, thus avoiding bone resection and graft reconstruction. A good outcome was achieved apart from local recurrence in one case. Follow up ranged from 17 to 54 months. CONCLUSION: Distal forearm GCTB responds clinically, radiologically and histologically to a short course of pre-operative denosumab therapy, which has the potential to facilitate salvage surgery.

Giant Cell-Containing Tumors of Bone.

Giant cell-containing tumors of bone are characterized morphologically by the presence of numerous osteoclastic giant cells. Correlation of clinical, radiologic, and laboratory findings is required for accurate histopathologic diagnosis and treatment of a giant cell-containing tumor of bone. In differential diagnosis, it is particularly important to note the age of the patient and the skeletal location of the lesion. This article considers the range of neoplastic and nonneoplastic lesions, which histologically contain numerous osteoclastic giant cells, and focuses on several lesions that frequently enter into the differential diagnosis.

Correlation of serum metal ion levels with pathological changes of ARMD in failed metal-on-metal-hip-resurfacing arthroplasties.

BACKGROUND: Metal-on-metal-hip-resurfacing arthroplasties (MoMHRAs) have been associated with an increased failure rates due to an adverse-response-to-metal-debris (ARMD) associated with a spectrum of pathological features. Serum levels of cobalt (Co) and chromium (Cr) are used to assess MoMHRAs, with regard to ARMD, but it is not certain whether ion levels correlate with pathological changes in periprosthetic tissues. METHODS: Serum Co and Cr levels were correlated with histological findings in 38 revised MoMHRAs (29 pseudotumour cases and 9 non-pseudotumour cases revised for pain). The extent of necrosis and macrophage infiltrate as well as the aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL) response was assessed semi-quantitatively; the prosthesis linear wear rate (PLWR) was also determined in ten cases. RESULTS: Cr levels were elevated in 82% and Co levels elevated in 53% of cases; the PLWR correlated with Cr level (rho = 0.8, p = 0.006). Tissue necrosis and macrophage infiltration were noted in all, most of which also exhibited significant ALVAL. Although a discrete correlation was not seen between Co and/or Cr ion levels and the extent of necrosis, degree of macrophage infiltration, or ALVAL score, it was noted that cases with acceptable metal ions levels had high ALVAL score. CONCLUSION: Histological features of both innate and adaptive immune response to metal wear are seen in periprosthetic tissues in cases with both elevated and non-elevated metal ion levels. MoMHRA failures with acceptable ion levels exhibited a pronounced ALVAL response. Although metal ion levels are elevated in most cases of MoMHRA failure due to ARMD, the finding of a normal metal ion level does not exclude this diagnosis.

Role of LIGHT in the pathogenesis of joint destruction in rheumatoid arthritis.

AIM: To characterise the role of substitutes for receptor-activator nuclear factor kappa-B ligand (RANKL) in rheumatoid arthritis (RA) joint destruction. METHODS: Synovial fluid (SF) macrophages isolated from the knee joint of RA patients were incubated with 25 ng/mL macrophage-colony stimulating factor (M-CSF) and 50 ng/mL LIGHT (lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) in the presence and absence of 25 ng/mL RANKL and 100 ng/mL osteoprotegerin (OPG) on glass coverslips and dentine slices. Osteoclastogenesis was assessed by the formation of multinucleated cells (MNCs) expressing tartrate-resistant acid phosphatase (TRAP) on coverslips and the extent of lacunar resorption pit formation on dentine slices. The concentration of LIGHT in RA and osteoarthritis (OA) synovial fluid was measured by an enzyme-linked immunosorbent assay (ELISA) and the expression of LIGHT in RA and OA synovium was determined by immunohistochemistry using an indirect immunoperoxidase technique. RESULTS: In cultures of RA SF macrophages treated with LIGHT and M-CSF, there was significant formation of TRAP + MNCs on coverslips and extensive lacunar resorption pit formation on dentine slices. SF-macrophage-osteoclast differentiation was not inhibited by the addition of OPG, a decoy receptor for RANKL. Resorption pits were smaller and less confluent than in RANKL-treated cultures but the overall percentage area of the dentine slice resorbed was comparable in LIGHT- and RANKL-treated cultures. LIGHT significantly stimulated RANKL-induced lacunar resorption compared with RA SF macrophages treated with either RANKL or LIGHT alone. LIGHT was strongly expressed by synovial lining cells, subintimal macrophages and endothelial cells in RA synovium and the concentration of LIGHT was much higher in RA compared with OA SF. CONCLUSION: LIGHT is highly expressed in RA synovium and SF, stimulates RANKL-independent/dependent osteoclastogenesis from SF macrophages and may contribute to marginal erosion formation.

Outcome Following Debridement, Antibiotics, and Implant Retention in Hip Periprosthetic Joint Infection-An 18-Year Experience.

BACKGROUND: Debridement-antibiotics-and-implant-retention (DAIR) may be considered a suitable surgical option in periprosthetic joint infections (PJIs) with soundly fixed prostheses, despite chronicity. This study aims to define the long-term outcome following DAIR in hip PJI. METHODS: We reviewed all hip DAIRs performed between 1997 and 2013 (n = 122) to define long-term outcome and identify factors influencing it. Data recorded included patient demographics, medical history, type of DAIR performed (+/- exchange of modular components), and organisms identified. Outcome measures included complications and/or mortality rate, implant survivorship, and functional outcome (Oxford Hip Score). RESULTS: Most DAIRs (67%) were of primary arthroplasties and 60% were performed within 6 weeks from the index arthroplasty. Infection eradication was achieved in 68% of the first DAIR procedure. In 32 cases, more than one DAIR was required. Infection eradication was achieved in 85% of the cases (104/122) with the (single or multiple) DAIR approach. The most common complication was PJI-persistence (15%), followed by dislocation (14%). Very good functional outcomes were obtained, especially in primary arthroplasties. All streptococcus infections were resolved with DAIR and had better outcome. Twenty-one hips have been revised (17%) to-date, 16 were for persistence of PJI. The 10-y implant survivorship was 77%. Early PJI and exchanging modular components at DAIR were independent factors for a 4-fold increased infection eradication and improved long-term implant survival. CONCLUSION: DAIR is, therefore, a valuable option in the treatment of hip PJI, especially in the early postoperative period (≤6 weeks), with good outcomes. However, DAIR is associated with increased morbidity; further surgery may be necessary and instability may occur. Where possible, exchange of modular implants should be undertaken.

The pathology of failed McKee-Farrar implants: correlation with modern metal-on-metal-implant failure.

The McKee-Farrar (MF) prosthesis was the first widely used total hip replacement (THR) to employ a metal-on-metal (MoM) articulation. These implants had a high rate of early aseptic loosening but a number achieved good long-term implant survival, stimulating the reintroduction of second and third generation implants of this type. In this study we analysed archival histopathology of periprosthetic tissues in twenty cases of MF aseptic implant failure to determine if there was evidence of an innate and adaptive immune response similar to that seen in modern MoM implants. The presence of macrophages, the extent of necrosis and the ALVAL response were graded semi-quantitatively. Variable but in most cases extensive tissue necrosis was associated with a heavy macrophage response to Cobalt-Chrome (Co-Cr) wear particles in periprosthetic tissues; most cases also contained evidence of a predominantly lymphocyte response which in eight cases was moderate or heavy (Oxford Grade 2/3). Our findings show that inflammatory and necrotic changes to deposition of Co-Cr wear particles are found in periprosthetic tissues of failed MF implants, indicating that there is an innate and adaptive response similar to that noted in second/third generation MoM implants; they also suggest that the pathobiological response to metal wear particles is likely to have contributed to MF implant failure in these cases.

The Diagnosis of Infection in Metal-on-Metal Hip Arthroplasties.

BACKGROUND: Distinction of aseptic from septic hip arthroplasty failure can be challenging. Some studies report an increased incidence of septic failure with metal-on-metal (MoM) hip arthroplasties. The Musculoskeletal Infection Society (MSIS) have formulated criteria to facilitate the diagnosis of periprosthetic joint infection (PJI). In this study, we determined the prevalence and histologic features of septic MoM hip failure. METHODS: Overall, 104 cases of failed MoM hip arthroplasty, classified as septic or aseptic by MSIS microbiological criteria, were analyzed. The overall prevalence of septic failure was determined and the nature of the causative organisms noted. The extent of the neutrophil polymorph (NP) infiltrate in periprosthetic tissue in all cases was analyzed by hematoxylin-eosin and chloroacetate esterase staining. RESULTS: The prevalence of septic MoM hip arthroplasty failure was 6.7%. Infective organisms were coagulase-negative Staphylococcus in 4 cases; Staphylococcus aureus, Streptococcus, and Propionibacterium species were isolated in the remaining cases. Chloroacetate esterase staining facilitated identification of NPs. All cases of PJI contained more than 5 NPs per high-power field (HPF) on average. Four cases of aseptic MoM implant failure contained scanty or scattered NPs (less than 5 per HPF on average). CONCLUSION: The prevalence of PJI as a cause of MoM hip arthroplasty failure was relatively high compared to other hip bearing combinations; however, the organisms responsible were similar. Histologically, a minority of aseptic MoM implant failures contained some NPs, but the MSIS criteria for the histologic diagnosis of PJI (>5 NPs/HPF) correctly identified all microbiologically confirmed cases of septic failure.

Quantification of neutrophil polymorphs in infected and noninfected second-stage revision hip arthroplasties.

AIMS: In the diagnosis of periprosthetic joint infection (PJI), a heavy neutrophil polymorph (NP) infiltrate (>5 per high-power field [HPF] by MSIS criteria) is characteristically seen in periprosthetic tissues. PJI is commonly treated by a two-stage procedure with surgical clearance of infected tissues followed by intensive antibiotic treatment before re-implantation; tissues are sampled at the time of second stage but whether MSIS histological criteria can be used to diagnose the presence or absence of infection in second stage samples has not been established. METHODS: Periprosthetic tissues from 31 cases of second-stage revision hip arthroplasty (including 3 cases fulfilling the microbiological MSIS criteria for PJI), were analysed histologically after haematoxylin-eosin and chloroacetate esterase (CAE) staining. The extent of the NP infiltrate was determined semiquantitatively and correlated with the microbiological diagnosis. RESULTS: CAE staining facilitated identification of NPs in arthroplasty tissues and showed that in those cases where an organism was cultured in 2 or more samples, meeting the MSIS microbiological criteria for definite diagnosis of PJI, there was a heavy polymorph infiltrate (>5 NP per HPF on average). It was noted that isolated or scattered NPs were seen in 42.8% of periprosthetic tissues from noninfected second-stage revisions. CONCLUSIONS: The MSIS histological criteria which support a diagnosis of PJI in specimens from a primary revision hip arthroplasty (i.e. >5 NPs per HPF on average) are also valid for the assessment of a second-stage specimens. NPs can be seen in samples of periprosthetic tissue from uninfected second-stage revisions, indicating that strict histological criteria should be used in evaluating their significance in this context.

Angiopoietin-like 4 is over-expressed in rheumatoid arthritis patients: association with pathological bone resorption.

INTRODUCTION: Osteoclasts are responsible for the bone loss associated with rheumatoid arthritis (RA). The secreted adipokine angiopoietin-like 4 (ANGPTL4) specifically increases osteoclast-mediated bone resorption. We have investigated expression of ANGPTL4 and its regulatory transcription factor, hypoxia-inducible factor-1 alpha (HIF-1α), in osteoclasts and other cells within rheumatoid synovium. We have also examined whether circulating levels of ANGPTL4 differ in RA patients compared with that in normal controls or patients with osteoarthritis (OA). RESULTS: Immunohistochemical analysis revealed that bone-apposing osteoclasts within the rheumatoid synovium express both ANGPTL4 and HIF-1α. ANGPTL4 was also strongly expressed in synovial lining cells, endothelial cells, stromal cells, CD68+ macrophages and plasma cells within RA synovium. Little ANGPTL4 was evident in normal synovial tissue. This reflected the over-expression of HIF-1α in rheumatoid versus normal synovial tissue. The concentration of ANGPTL4 was higher in both the serum and the synovial fluid of RA patients than in patients with OA or normal controls. High serum ANGPTL4 associated with elevated levels of the serum marker of bone resorption, receptor activator for nuclear factor κB ligand (RANKL). CONCLUSIONS: Over-expression of ANGPTL4 in multiple cell types within the rheumatoid synovium potentially provides a local pool of ANGPTL4 to stimulate osteoclast-mediated bone resorption in RA. Additionally, correlation of high serum ANGPTL4 with circulating RANKL suggests that ANGPTL4 may represent a novel marker for bone destruction in RA.

Quantification of the heterogeneity of prognostic cellular biomarkers in ewing sarcoma using automated image and random survival forest analysis.

Driven by genomic somatic variation, tumour tissues are typically heterogeneous, yet unbiased quantitative methods are rarely used to analyse heterogeneity at the protein level. Motivated by this problem, we developed automated image segmentation of images of multiple biomarkers in Ewing sarcoma to generate distributions of biomarkers between and within tumour cells. We further integrate high dimensional data with patient clinical outcomes utilising random survival forest (RSF) machine learning. Using material from cohorts of genetically diagnosed Ewing sarcoma with EWSR1 chromosomal translocations, confocal images of tissue microarrays were segmented with level sets and watershed algorithms. Each cell nucleus and cytoplasm were identified in relation to DAPI and CD99, respectively, and protein biomarkers (e.g. Ki67, pS6, Foxo3a, EGR1, MAPK) localised relative to nuclear and cytoplasmic regions of each cell in order to generate image feature distributions. The image distribution features were analysed with RSF in relation to known overall patient survival from three separate cohorts (185 informative cases). Variation in pre-analytical processing resulted in elimination of a high number of non-informative images that had poor DAPI localisation or biomarker preservation (67 cases, 36%). The distribution of image features for biomarkers in the remaining high quality material (118 cases, 104 features per case) were analysed by RSF with feature selection, and performance assessed using internal cross-validation, rather than a separate validation cohort. A prognostic classifier for Ewing sarcoma with low cross-validation error rates (0.36) was comprised of multiple features, including the Ki67 proliferative marker and a sub-population of cells with low cytoplasmic/nuclear ratio of CD99. Through elimination of bias, the evaluation of high-dimensionality biomarker distribution within cell populations of a tumour using random forest analysis in quality controlled tumour material could be achieved. Such an automated and integrated methodology has potential application in the identification of prognostic classifiers based on tumour cell heterogeneity.

Localized Ewing sarcoma of the tibia.

Ewing sarcoma (ES) is a high-grade malignant primary round cell tumour of bone in which there is commonly extension into extraosseous soft tissues at the time of diagnosis. This report details the clinical, radiological and pathological features of a case of ES of the tibia in which there was extensive osseous involvement but no infiltration beyond the periosteum into surrounding soft tissue. We also record the findings of one other ES case that exhibited similar behaviour. Both cases were male, involved the tibia and had the characteristic t (11;22) (q24;q12) translocation. No recurrence of tumour or metastasis has been seen in these two cases, both of which have had 6 years follow-up. Our findings indicate that there is heterogeneity in the behaviour of ES and show that localized ES is associated with a good prognosis.

VEGF, FLT3 ligand, PlGF and HGF can substitute for M-CSF to induce human osteoclast formation: implications for giant cell tumour pathobiology.

Giant cell tumour of bone (GCTB) is a primary bone tumour that contains numerous very large, hyper-nucleated osteoclastic giant cells. Osteoclasts form from CD14+ monocytes and macrophages in the presence of receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage-colony stimulating factor (M-CSF). GCTB contains numerous growth factors, some of which have been reported to influence osteoclastogenesis and resorption. We investigated whether these growth factors are capable of substituting for M-CSF to support osteoclast formation from cultured human monocytes and whether they influence osteoclast cytomorphology and resorption. Vascular endothelial growth factor-A (VEGF-A), VEGF-D, FLT3 ligand (FL), placental growth factor (PlGF) and hepatocyte growth factor (HGF) supported RANKL-induced osteoclastogenesis in the absence of M-CSF, resulting in the formation of numerous TRAP+ multinucleated cells capable of lacunar resorption. Monocytes cultured in the presence of M-CSF, HGF, VEGF-A and RANKL together resulted in the formation of very large, hyper-nucleated (GCTB-like) osteoclasts that were hyper-resorptive. M-CSF and M-CSF substitute growth factors were identified immunohistochemically in GCTB tissue sections and these factors stimulated the resorption of osteoclasts derived from a subset of GCTBs. Our findings indicate that there are growth factors that are capable of substituting for M-CSF to induce human osteoclast formation and that these factors are present in GCTB where they influence osteoclast cytomorphology and have a role in osteoclast formation and resorption activity.

Interobserver reliability in the histopathological diagnosis of cartilaginous tumors in patients with multiple osteochondromas.

The distinction between benign and malignant cartilaginous tumors located peripherally in the bone may be a challenging task in surgical pathology. The aim of this study was to investigate interobserver reliability in histological diagnosis of cartilaginous tumors in the setting of multiple osteochondromas and to evaluate possible histological parameters that could differentiate among osteochondroma, low- and high-grade secondary peripheral chondrosarcoma. Interobserver reliability was assessed by 12 specialized bone-tumor pathologists in a set of 38 cases. Substantial agreement on diagnosis among all the reviewers was observed (intraclass correlation coefficient=0.78). Our study confirmed that mitotic figures and nuclear pleomorphism are hallmarks of high-grade secondary peripheral chondrosarcoma. However, despite the substantial agreement, we demonstrated that histology alone cannot distinguish osteochondroma from low-grade secondary peripheral chondrosarcoma in the setting of multiple osteochondromas, as nodularity, the presence of binucleated cells, irregular calcification, cystic/mucoid changes and necrosis were not helpful to indicate malignant transformation of an osteochondroma. On the other hand, among the concordant cases, the cartilage cap in osteochondroma was significantly less thicker than in low- and high-grade secondary peripheral chondrosarcoma. Therefore, our study showed that a multidisciplinary approach integrating clinical and radiographical features and the size of the cartilaginous cap in combination with a histological assessment are crucial to the diagnosis of cartilaginous tumors.

CD14- mononuclear stromal cells support (CD14+) monocyte-osteoclast differentiation in aneurysmal bone cyst.

Aneurysmal bone cyst (ABC) is a benign osteolytic bone lesion in which there are blood-filled spaces separated by fibrous septa containing giant cells. The nature of the giant cells in this lesion and the mechanism of bone destruction in ABC is not certain. In this study, we have analysed several characteristics of mononuclear and multinucleated cells in the ABC and examined the cellular and molecular mechanisms of ABC osteolysis. The antigenic and functional phenotype of giant cells in ABC was determined by histochemistry/immunohistochemistry using antibodies to macrophage and osteoclast markers. Giant cells and CD14+ and CD14- mononuclear cells were isolated from ABC specimens and cultured on dentine slices and coverslips with receptor activator of nuclear factor κB ligand (RANKL)+/- macrophage-colony stimulating factor (M-CSF) and functional and cytochemical evidence of osteoclast differentiation sought. Giant cells in ABC expressed an osteoclast-like phenotype (CD51+, CD14-, cathepsin K+, TRAP+) and were capable of lacunar resorption, which was inhibited by zoledronate, calcitonin and osteoprotegerin (OPG). When cultured with RANKL±M-CSF, CD14+, but not CD14-, mononuclear cells differentiated into TRAP+ multinucleated cells that were capable of lacunar resorption. M-CSF was not necessary for osteoclast formation from CD14+ cell cultures. CD14- cells variably expressed RANKL, OPG and M-CSF but supported osteoclast differentiation. Our findings show that the giant cells in ABC express an osteoclast-like phenotype and are formed from CD14+ macrophage precursors. CD14- mononuclear stromal cells express osteoclastogenic factors and most likely interact with CD14+ cells to form osteoclast-like giant cells by a RANKL-dependent mechanism.

Analysis of stromal cells in osteofibrous dysplasia and adamantinoma of long bones.

Adamantinoma of long bones and osteofibrous dysplasia are rare, osteolytic primary bone tumours of uncertain origin containing areas of fibrous and fibro-osseous proliferation. We investigated the nature of the stromal cells in adamantinoma of long bones and osteofibrous dysplasia, and determined cellular and molecular mechanisms of osteolysis in these tumours. Cell culture, molecular (RT-PCR, western blot) and immunohistochemical studies on cases of adamantinoma of long bones and of osteofibrous dysplasia were undertaken to determine the expression of epithelial, osteoblast and osteoclast markers. Ultrastructural and immunophenotypic studies on cultured adamantinoma and osteofibrous dysplasia stromal cells showed that these cells were mainly fibroblast-like with few cells expressing epithelial markers. Osteofibrous dysplasia but not adamantinoma cells expressed alkaline phosphatase. Both osteofibrous dysplasia and adamantinoma cells expressed the ostoclastogenic factors M-CSF and RANKL. Adamantinoma and osteofibrous dysplasia cells also expressed messenger RNA for osteocalcin, osteonectin, osteopontin, osterix and collagen type 1. Adamantinoma and osteofibrous dysplasia cells cultured alone on dentine slices were not capable of lacunar resorption, but in co-cultures with monocytes induced formation of osteoclast-like cells was observered. Cultured osteofibrous dysplasia and adamantinoma stromal cells show similar ultrastructural and immunophenotypic characteristics, and differentially express osteoblast markers. Promotion of osteoclastogenesis by stromal cells may contribute to osteolysis in adamantinoma of long bones and osteofibrous dysplasia.

Osteoclast formation and function in pigmented villonodular synovitis.

Pigmented villonodular synovitis (PVNS) is a synovial tumour-like lesion that frequently causes osteolysis. PVNS contains numerous macrophages and osteoclast-like giant cells. In this study, we have analysed the cytochemical and functional characteristics of mononuclear and multinucleated cells in PVNS and determined the cellular and humoral mechanisms underlying giant cell formation and resorption in PVNS. Giant cells and CD14(+) and CD14(-) mononuclear cell populations were isolated from PVNS synovial tissue and cultured alone or in the presence and absence of the osteoclastogenic factors, RANKL and M-CSF. Osteoclast formation and activity was assessed by expression of TRAP and evidence of lacunar resorption. Giant cells in PVNS expressed an osteoclast-phenotype (CD51(+) , TRAP(+) , CD14(-) , HLA-DR(-) ) and were formed only in cultures of mononuclear cells that expressed the macrophage marker CD14. Osteoclast formation required RANKL and occurred in both the presence and absence of exogenous M-CSF. CD14(-) cells in PVNS expressed RANKL. Lacunar resorption by PVNS-derived giant cells was abolished by the addition of the bisphosphonate, zoledronate. Our findings indicate that osteoclasts form by a RANKL-dependent mechanism from CD14(+) mononuclear phagocytes in PVNS. Osteoclast formation occurred even in the absence of exogenous M-CSF, a finding which is in keeping with over-expression of M-CSF playing a pathogenic role in this condition. Anti-osteoclast resorptive treatment may be useful to control osteolysis in PVNS.

Ewing sarcoma cells express RANKL and support osteoclastogenesis.

Ewing sarcoma (ES) is a primary malignant round cell tumour of bone characterized by rapid and extensive osteolysis. Cellular mechanisms underlying the rapid bone resorption in ES have not been characterized. Osteoclasts are marrow-derived multinucleated cells that effect tumour osteolysis. The role of ES tumour cells in influencing osteoclast formation and/or directly contributing to the osteolysis in ES has not been determined. Using a tissue culture bioassay, we found that lacunar resorption is not carried out by (CD99(+) ) ES tumour cells, but by (CD68(+) ) macrophage/osteoclast-like cells; this resorption occurred in the absence of the osteoclastogenic factor, receptor activator of nuclear factor κB ligand (RANKL). ES cell lines cultured directly on dentine slices did not resorb the mineral or organic components of the bone matrix. Immunohistochemistry of ES tissue microarrays, western blotting, and RT-PCR studies showed that ES cells strongly expressed both RANKL and macrophage-colony stimulating factor (M-CSF), two major osteoclastogenic factors. When co-cultured with human monocytes, ES cells induced the formation of TRAP(+) osteoclastic cells. Conditioned medium from cultured ES cells did not result in osteoclast formation, indicating that cell-cell contact is required for ES-induced osteoclastogenesis. Our findings indicate that ES cells do not resorb bone directly but that they may support osteoclast formation by a RANKL-dependent mechanism.

Podoplanin expression in adamantinoma of long bones and osteofibrous dysplasia.

Adamantinoma of long bones (ALB) and osteofibrous dysplasia (OFD) are rare osteolytic bone tumours that principally arise in the tibia. Both ALB and OFD contain epithelial and stromal elements, as well as areas of fibro-osseous proliferation. We assessed expression of podoplanin, a glycoprotein found in osteocytes, in OFD and ALB as well as in fibrous dysplasia and metastatic cancer. Forty-two cases of ALB and OFD, 20 cases of fibrous dysplasia and 20 cases of metastatic carcinoma to bone were stained by immunohistochemistry for expression of podoplanin, epithelial (cytokeratin, epithelial membrane antigen) and vascular (CD34, LYVE-1) markers. Podoplanin was expressed in epithelial cells and tumour glands in ALB as well as in scattered intertrabecular stromal cells in both ALB and OFD. Podoplanin was not expressed by intertrabecular stromal cells in fibrous dysplasia or in metastatic adenocarcinoma. Podoplanin was expressed by osteocytes but not osteoblasts of woven and lamellar bone trabeculae in ALB, OFD, fibrous dysplasia and skeletal metastases. The finding of a common osteocyte marker in OFD/ALB stromal cells is in keeping with a close histogenetic relationship between OFD and ALB; this may reflect the prominence of fibro-osseous proliferation in these tumours. The expression of podoplanin in an osteolytic tumour of the tibia may be useful as a diagnostic discriminant in distinguishing OFD from fibrous dysplasia and ALB from metastatic adenocarcinoma.