Cell Biology of Giant Cell Tumour of Bone: Crosstalk between m/wt Nucleosome H3.3, Telomeres and Osteoclastogenesis.

Giant cell tumour of bone (GCTB) is a rare and intriguing primary bone neoplasm. Worrisome clinical features are its local destructive behaviour, its high tendency to recur after surgical therapy and its ability to create so-called benign lung metastases (lung 'plugs'). GCTB displays a complex and difficult-to-understand cell biological behaviour because of its heterogenous morphology. Recently, a driver mutation in histone H3.3 was found. This mutation is highly conserved in GCTB but can also be detected in glioblastoma. Denosumab was recently introduced as an extra option of medical treatment next to traditional surgical and in rare cases, radiotherapy. Despite these new insights, many 'old' questions about the key features of GCTB remain unanswered, such as the presence of telomeric associations (TAs), the reactivation of hTERT, and its slight genomic instability. This review summarises the recent relevant literature of histone H3.3 in relation to the GCTB-specific G34W mutation and pays specific attention to the G34W mutation in relation to the development of TAs, genomic instability, and the characteristic morphology of GCTB. As pieces of an etiogenetic puzzle, this review tries fitting all these molecular features and the unique H3.3 G34W mutation together in GCTB.

The articularis genu muscle and its relevance in oncological surgical margins.

AIMS: The aticularis genu (AG) is the least substantial and deepest muscle of the anterior compartment of the thigh and of uncertain significance. The aim of the study was to describe the anatomy of AG in cadaveric specimens, to characterize the relevance of AG in pathological distal femur specimens, and to correlate the anatomy and pathology with preoperative magnetic resonance imaging (MRI) of AG. METHODS: In 24 cadaveric specimens, AG was identified, photographed, measured, and dissected including neurovascular supply. In all, 35 resected distal femur specimens were examined. AG was photographed and measured and its utility as a surgical margin examined. Preoperative MRIs of these cases were retrospectively analyzed and assessed and its utility assessed as an anterior soft tissue margin in surgery. In all cadaveric specimens, AG was identified as a substantial structure, deep and separate to vastus itermedius (VI) and separated by a clear fascial plane with a discrete neurovascular supply. Mean length of AG was 16.1 cm ( ± 1.6 cm) origin anterior aspect distal third femur and insertion into suprapatellar bursa. In 32 of 35 pathological specimens, AG was identified (mean length 12.8 cm ( ± 0.6 cm)). Where AG was used as anterior cover in pathological specimens all surgical margins were clear of disease. Of these cases, preoperative MRI identified AG in 34 of 35 cases (mean length 8.8 cm ( ± 0.4 cm)). RESULTS: AG was best visualized with T1-weighted axial images providing sufficient cover in 25 cases confirmed by pathological findings.These results demonstrate AG as a discrete and substantial muscle of the anterior compartment of the thigh, deep to VI and useful in providing anterior soft tissue margin in distal femoral resection in bone tumours. CONCLUSION: Preoperative assessment of cover by AG may be useful in predicting cases where AG can be dissected, sparing the remaining quadriceps muscle, and therefore function.Cite this article: Bone Joint Open 2020;1-9:585-593.

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.

Radiographic and Histological Analysis of a Synthetic Bone Graft Substitute Eluting Gentamicin in the Treatment of Chronic Osteomyelitis.

Introduction: Managing chronic osteomyelitis can be challenging and attention to the osseous dead-space left following resection is an important part of successful treatment. We assess radiographic bone healing following implantation of a gentamicin-eluting synthetic bone graft substitute (gBGS) used at chronic osteomyelitis (cOM) resection. We also describe histological carrier changes from biopsies in nine cases at various time points. Methods: This was a retrospective review of a prospectively collected consecutive series of 163 patients with Cierny-Mader Type III or IV cOM who underwent single-stage excision, insertion of gBGS and definitive soft-tissue closure or coverage. Bone defect filling was assessed radiographically using serial radiographs. Nine patients had subsequent surgery, not related to infection recurrence, allowing opportunistic biopsy between 19 days and two years after implantation. Results: Infection was eradicated in 95.7% with a single procedure. 138 patients had adequate radiographs for assessment with minimum one-year follow-up (mean 1.7 years, range 1.0-4.7 years). Mean void-filling at final follow-up was 73.8%. There was significantly higher void-filling in metaphyseal compared to diaphyseal voids (mean 79.0% versus 65.6%; p=0.017) and in cases with good initial interdigitation of the carrier (mean 77.3% versus 68.7%; p=0.021). Bone formation continued for more than two years in almost two-thirds of patients studied (24/38; 63.2%). Histology revealed active biomaterial remodelling. It was osteoconductive with osteoblast recruitment, leading to the formation of osteoid, then woven and lamellar bone on the substrate's surface. Immunohistochemistry demonstrated osteocyte specific markers, dentine matrix protein-1 and podoplanin within the newly formed bone. Conclusion: This antibiotic-loaded biomaterial is effective in managing dead-space in surgically treated cOM with a low infection recurrence rate (4.3%) and good mean bone void-filling (73.8%). The radiographic resolution of the bone defect is associated with bone formation, as supported by histological analysis.

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.

Histological Findings in Infected and Noninfected Second Stage Revision Knee Arthroplasties.

Tissues from a periprosthetic joint infection (PJI) of the knee contain a heavy neutrophil polymorph (NP) infiltrate (> 5 NPs per high-powered field [HPF] by Musculoskeletal Infection Society [MSIS] criteria). PJI of the knee can be treated by a two-stage procedure and our aim was to determine whether the MSIS histological criteria for PJI diagnosis are valid in a second-stage revision knee arthroplasty. Periprosthetic tissues from 45 second-stage revision knee cases were analyzed histologically by hematoxylin-eosin and chloroacetate esterase (CAE) staining for the identification of NPs. The number of NPs was determined semiquantitatively and results correlated with the microbiological and clinical findings. In 9 of the 45 cases, an organism was cultured in two or more samples, meeting MSIS microbiological criteria for a definite diagnosis of PJI; histologically, seven of these cases contained > 5 per NPs per HPF on average, with the remaining two cases containing 1 NP and 2 NPs per HPF. In noninfected second-stage revisions, NPs were not seen in 30 cases with 6 cases showing less than 1 NP per HPF on average. The sensitivity, specificity, accuracy, and positive and negative predictive values of MSIS histological criteria (> 5 NPs per HPF) to diagnose PJI were 78%, 100%, 96%, 100%, and 95%, respectively. MSIS histological criteria for the diagnosis of PJI are valid for most but not all infected second-stage revision knee arthroplasties. Correlation of histology with clinical, microbiology and other laboratory findings is required to establish a diagnosis of PJI in second-stage revision knee arthroplasties.

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.

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.

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.

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.

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.

Durable Response of Spinal Chordoma to Combined Inhibition of IGF-1R and EGFR.

Chordomas are rare primary malignant bone tumors arising from embryonal notochord remnants of the axial skeleton. Chordomas commonly recur following surgery and radiotherapy, and there is no effective systemic therapy. Previous studies implicated receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGF-1R), in chordoma biology. We report an adult female patient who presented in 2003 with spinal chordoma, treated with surgery and radiotherapy. She underwent further surgery for recurrent chordoma in 2008, with subsequent progression in pelvic deposits. In June 2009, she was recruited onto the Phase I OSI-906-103 trial of EGFR inhibitor erlotinib with linsitinib, a novel inhibitor of IGF-1R/insulin receptor (INSR). Treatment with 100 mg QD erlotinib and 50 mg QD linsitinib was well-tolerated, and after 18 months a partial response was achieved by RECIST criteria. From 43 months, a protocol modification allowed intra-patient linsitinib dose escalation to 50 mg BID. The patient remained stable on trial treatment for a total of 5 years, discontinuing treatment in August 2014. She subsequently experienced further disease progression for which she underwent pelvic surgery in April 2015. Analysis of DNA extracted from 2008 (pre-trial) tissue showed that the tumor harbored wild-type EGFR, and a PIK3CA mutation was detected in plasma, but not tumor DNA. The 2015 (post-trial) tumor harbored a mutation of uncertain significance in ATM, with no detectable mutations in other components of a 50 gene panel, including EGFR, PIK3CA, and TP53. By immunohistochemistry, the tumor was positive for brachyury, the molecular hallmark of chordoma, and showed weak-moderate membrane and cytoplasmic EGFR. IGF-1R was detected in the plasma membrane and cytoplasm and was expressed more strongly in recurrent tumor than the primary. We also noted heterogeneous nuclear IGF-1R, which has been linked with sensitivity to IGF-1R inhibition. Similar variation in IGF-1R expression and subcellular localization was noted in 15 further cases of chordoma. In summary, this exceptionally durable response suggests that there may be merit in evaluating combined IGF-1R/INSR and EGFR inhibition in patients with chordomas that recur following failure of local treatment.

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.

Prognostic impact of reduced connexin43 expression and gap junction coupling of neoplastic stromal cells in giant cell tumor of bone.

Missense mutations of the GJA1 gene encoding the gap junction channel protein connexin43 (Cx43) cause bone malformations resulting in oculodentodigital dysplasia (ODDD), while GJA1 null and ODDD mutant mice develop osteopenia. In this study we investigated Cx43 expression and channel functions in giant cell tumor of bone (GCTB), a locally aggressive osteolytic lesion with uncertain progression. Cx43 protein levels assessed by immunohistochemistry were correlated with GCTB cell types, clinico-radiological stages and progression free survival in tissue microarrays of 89 primary and 34 recurrent GCTB cases. Cx43 expression, phosphorylation, subcellular distribution and gap junction coupling was also investigated and compared between cultured neoplastic GCTB stromal cells and bone marow stromal cells or HDFa fibroblasts as a control. In GCTB tissues, most Cx43 was produced by CD163 negative neoplastic stromal cells and less by CD163 positive reactive monocytes/macrophages or by giant cells. Significantly less Cx43 was detected in α-smooth muscle actin positive than α-smooth muscle actin negative stromal cells and in osteoclast-rich tumor nests than in the adjacent reactive stroma. Progressively reduced Cx43 production in GCTB was significantly linked to advanced clinico-radiological stages and worse progression free survival. In neoplastic GCTB stromal cell cultures most Cx43 protein was localized in the paranuclear-Golgi region, while it was concentrated in the cell membranes both in bone marrow stromal cells and HDFa fibroblasts. In Western blots, alkaline phosphatase sensitive bands, linked to serine residues (Ser369, Ser372 or Ser373) detected in control cells, were missing in GCTB stromal cells. Defective cell membrane localization of Cx43 channels was in line with the significantly reduced transfer of the 622 Da fluorescing calcein dye between GCTB stromal cells. Our results show that significant downregulation of Cx43 expression and gap junction coupling in neoplastic stromal cells are associated with the clinical progression and worse prognosis in GCTB.

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.

Dsh homolog DVL3 mediates resistance to IGFIR inhibition by regulating IGF-RAS signaling.

Drugs that inhibit insulin-like growth factor 1 (IGFI) receptor IGFIR were encouraging in early trials, but predictive biomarkers were lacking and the drugs provided insufficient benefit in unselected patients. In this study, we used genetic screening and downstream validation to identify the WNT pathway element DVL3 as a mediator of resistance to IGFIR inhibition. Sensitivity to IGFIR inhibition was enhanced specifically in vitro and in vivo by genetic or pharmacologic blockade of DVL3. In breast and prostate cancer cells, sensitization tracked with enhanced MEK-ERK activation and relied upon MEK activity and DVL3 expression. Mechanistic investigations showed that DVL3 is present in an adaptor complex that links IGFIR to RAS, which includes Shc, growth factor receptor-bound-2 (Grb2), son-of-sevenless (SOS), and the tumor suppressor DAB2. Dual DVL and DAB2 blockade synergized in activating ERKs and sensitizing cells to IGFIR inhibition, suggesting a nonredundant role for DVL3 in the Shc-Grb2-SOS complex. Clinically, tumors that responded to IGFIR inhibition contained relatively lower levels of DVL3 protein than resistant tumors, and DVL3 levels in tumors correlated inversely with progression-free survival in patients treated with IGFIR antibodies. Because IGFIR does not contain activating mutations analogous to EGFR variants associated with response to EGFR inhibitors, we suggest that IGF signaling achieves an equivalent integration at the postreceptor level through adaptor protein complexes, influencing cellular dependence on the IGF axis and identifying a patient population with potential to benefit from IGFIR inhibition.

The correlation of wear with histological features after failed hip resurfacing arthroplasty.

BACKGROUND: Tissue necrosis and a macrophage and perivascular lymphocytic infiltrate are commonly seen in periprosthetic tissues around metal-on-metal hip resurfacing implants, including pseudotumors associated with these implants. The purpose of the present study was to correlate pathological changes in periprosthetic tissues with clinical findings and the amount of implant-derived metal wear. METHODS: We analyzed morphological changes in the periprosthetic soft tissues around fifty-six failed metal-on-metal hip resurfacing implants. The most common reason for failure was the presence of a symptomatic pseudotumor (n = 45). The extent of necrosis and the nature of the inflammatory cell infiltrate, including aseptic lymphocyte-dominated vasculitis-associated lesion (ALVAL), was evaluated semiquantitatively. Bearing surface wear was determined for all patients. Prostheses were considered to be highly worn if the total linear wear rate was ≥4 µm/yr. RESULTS: Substantial necrosis and a heavy macrophage infiltrate were noted in most periprosthetic tissues, including all pseudotumors, many of which contained a prominent ALVAL infiltrate. Most pseudotumors (80%) were associated with highly worn prostheses. It was noted that the extent of necrosis and macrophage infiltration correlated with the volume of generated metal wear. Although increased wear volume moderately correlated with a high ALVAL response, all pseudotumors associated with low wear had a strong ALVAL response. CONCLUSIONS: The majority of pseudotumors are associated with increased implant wear. This increased wear is associated with soft-tissue necrosis and a heavy nonspecific foreign-body macrophage response coupled with a variable adaptive or specific immune response (ALVAL). A minority of pseudotumors are associated with low wear and a prominent immune response. These findings confirm that minimizing wear from metal-on-metal hip resurfacing arthroplasty prostheses would lead to a reduction in the incidence of pseudotumor. However, a small number of pseudotumors are still likely to occur, which may be due to an exacerbated adaptive immune response.