Florian Sevelda

EGFR is not a major driver for osteosarcoma cell growth in vitro but contributes to starvation and chemotherapy resistance

BackgroundEnhanced signalling via the epidermal growth factor receptor (EGFR) is a hallmark of multiple human carcinomas. However, in recent years data have accumulated that EGFR might also be hyperactivated in human sarcomas. Aim of this study was to investigate the influence of EGFR inhibition on cell viability and its interaction with chemotherapy response in osteosarcoma cell lines.MethodsWe have investigated a panel of human osteosarcoma cell lines regarding EGFR expression and downstream signalling. To test its potential applicability as therapeutic target, inhibition of EGFR by gefitinib was combined with osteosarcoma chemotherapeutics and cell viability, migration, and cell death assays were performed.ResultsOsteosarcoma cells expressed distinctly differing levels of functional EGFR reaching in some cases high amounts. Functionality of EGFR in osteosarcoma cells was proven by EGF-mediated activation of both MAPK and PI3K/AKT pathway (determined by phosphorylation of ERK1/2, AKT, S6, and GSK3β). The EGFR-specific inhibitor gefitinib blocked EGF-mediated downstream signal activation. At standard in vitro culture conditions, clinically achievable gefitinib doses demonstrated only limited cytotoxic activity, however, significantly reduced long-term colony formation and cell migration. In contrast, under serum-starvation conditions active gefitinib doses were distinctly reduced while EGF promoted starvation survival. Importantly, gefitinib significantly supported the anti-osteosarcoma activities of doxorubicin and methotrexate regarding cell survival and migratory potential.ConclusionOur data suggest that EGFR is not a major driver for osteosarcoma cell growth but contributes to starvation- and chemotherapy-induced stress survival. Consequently, combination approaches including EGFR inhibitors should be evaluated for treatment of high-grade osteosarcoma patients.

Extendible Prostheses for Children After Resection of Primary Malignant Bone Tumor: Twenty-seven Years of Experience

BACKGROUND Endoprosthetic replacement in children following resection of a malignant bone tumor still is controversial because of the high number of reoperations. The aim of this study was to evaluate the long-term outcome with respect to limb-lengthening potential, satisfaction rate, and complications after implantation of extendible devices. METHODS Seventy-one patients with a sarcoma in an extremity who had been followed for more than twenty-four months (mean, 131.6 months; range, 27.2 to 281.8 months) after tumor resection and prosthetic reconstruction with an extendible device were analyzed. The mean age at the time of the operation was ten years (range, four to sixteen years). The complication-free survival rate was evaluated with competing-risk analysis. Clinical outcomes and complications were rated with use of the Musculoskeletal Tumor Society (MSTS) score and a failure mode classification for segmental tumor endoprostheses, respectively. RESULTS Twelve of seventy-one patients died of their disease. The overall MSTS score averaged 87.8% (range, 23.3% to 100%). The most common mode of failure was soft-tissue failure (46%), followed by structural failure (28%), infection (17%), and aseptic loosening (8%); only 2% of the children had local recurrence. An average of 4.4 lengthening operations per patient were required for an average limb elongation of 70.8 mm (range, 0 to 224 mm). An average of 2.5 operations (range, zero to eleven) per patient were performed for complications. CONCLUSIONS Although limb lengthening with an extendible endoprosthesis seems to be effective, many children have related complications. These data will be a source of preoperative information for children and parents, and will provide a benchmark for further clinical improvements.

AB0075 MTOR: An Unexpected Role on the TNF-Regulated MRNA Transcriptome in Rheumatoid Fibroblast-Like Synoviocytes

Background Accumulating evidence supports the concept that resident mesenchymal cells, namely fibroblast-like synoviocytes (FLS) actively participate in the destructive, inflammatory process of rheumatoid arthritis (RA). FLS maintain a synovial microenvironment that helps to recruit, retain, and activate immune cells, resulting in chronic inflammation with attendant joint destruction. Here, we provide evidence that the mechanistic target of rapamycin (mTOR), which has evolved as a major factor for the maintainance of tissue homeostasis and function, is a critical component of the inflammatory process in synovitis. Results Immunohistochemical studies revealed that mTOR is activated in rheumatoid synovitis, particularly in FLS of the hyperplastic synovial lining. By using western blot or advanced cell biological methods, such as a recently described synovial organ culture system, we demonstrate that the proinflammatory cytokine TNF activates the mTOR pathway in FLS. To further define the biological significance of these findings, we made use of genome-wide expression profiling. Remarkably, this analysis revealed a so far unknown modulatory role for mTOR in the gene expression programs induced by TNF. Specific inhibition of mTOR by Torin-1 or PP242 increased the TNF-induced expression of genes, known to be regulated by the transcription complex NFkB (e.g. PTGS2, IL8, CCL20). By contrast, FLS treatment with Torin-1 or PP242 diminished the TNF-mediated induction of interferon regulated genes (IRGs), including TNFSF10, CXCL11 and TNFSF13B (BAFF). Conclusions In conclusion, these studies provide insight into determinants of the synovial tissue response to inflammation and suggest a multifaceted regulatory role for mTOR in synovial inflammatory processes. Disclosure of Interest None declared

Targeted inhibition of Janus kinases abates interfon gamma-induced invasive behaviour of fibroblast-like synoviocytes

Objectives The aim was to explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodelling in RA and to determine whether IFNγ signalling controls the invasive potential of fibroblast-like synoviocytes (FLS). Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analysed in migration and invasion assays. Signalling events relevant to cellular motility were defined by western blots. Baricitinib and small interfering RNA pools were used to suppress Janus kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration upon IFNγ stimulation. Given that cell shape and cell motility are under the control of the focal adhesion kinase (FAK), we next analysed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. Small interfering RNA knockdown of JAK2, but not JAK1, substantially abrogated FAK activation by IFNγ. Correspondingly, IFNγ-induced FAK activation and invasion of FLS was abrogated by the JAK inhibitor, baricitinib. Conclusion Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA.

C-reactive protein: An independent predictor for dedifferentiated chondrosarcoma: CRP PREDICTS THE DEDIFFERENTIATED CHONDROSARCOMA

Dedifferentiated chondrosarcoma is a rare primary bone malignancy with a very poor prognosis. The aim of the study was to identify pretreatment serum markers as prognostic factors for the overall survival (OS) of patients with dedifferentiated chondrosarcoma. We retrospectively reviewed 33 patients with histologically confirmed dedifferentiated chondrosarcoma treated at our department from 1977 to 2015. Kaplan‐Meier estimation, uni‐ and multivariable Cox proportional hazard model were performed to evaluate the association between serum markers such as the C‐reactive protein and OS. In univariable analysis, CRP was strongly associated with OS (HR 1.35; 95%CI 1.13–1.61; p = 0.001). This association prevailed after adjustment for AJCC tumor stage (HR 1.31; 95%CI 1.02–1.57; p = 0.031) in multivariable analysis. In conclusion, our data gave evidence that baseline CRP is an independent predictor for OS in patients with dedifferentiated chondrosarcoma. CRP could be exploited for the clinical prediction of this disease in the future.

Multiplex PCR Unyvero i60 ITI application improves detection of low-virulent microorganisms in periprosthetic joint infections

PurposeThe aim of this study was to evaluate the pre-operative performance of an automated multiplex PCR (mPCR) system in patients with suspected periprosthetic joint infection (PJI).MethodsUnder sterile conditions, synovial fluid samples from patients with a suspected PJI were collected pre-operatively. One hundred eighty microliter of the aspirate was used for analysis in the mPCR. The remaining joint fluid was sent for microbiological analysis. PJI was diagnosed by using the Musculoskeletal Infection Society (MSIS) criteria. Total percentage agreement and Cohen’s kappa coefficient were calculated to measure overall agreement.ResultsOverall, 90 patients with a suspected PJI were included. Using MSIS criteria, 38 (42%) patients were classified as septic. Total percent agreement between mPCR and synovial fluid culture was 86% with a Cohen’s kappa of 0.68. The mPCR and synovial fluid culture showed sensitivities of 71% and 84%, respectively. Combined evaluation provided an even higher sensitivity of 92%. While Cutibacterium spp. were detected five times by mPCR, it could only be cultured once. A higher detection rate of CoNS by mPCR (n = 7) compared to conventional culture (n = 5) was also demonstrated. In comparison to synovial fluid culture, the mPCR missed Staphylococcus aureus five times.ConclusionWith a moderate agreement between synovial fluid mPCR and culture, the mPCR system could be a useful adjunct in diagnosing a PJI pre-operatively. Due to faster availability of results and a higher detection rate of low-virulent microorganisms, it can complement conventional culture.

Is Total Femur Replacement a Reliable Treatment Option for Patients With Metastatic Carcinoma of the Femur

Background The majority of metastatic bone lesions to the femoral bone can be treated without surgery or with minimally invasive intramedullary nailing. In rare patients with extensive metastatic disease to the femur, total femur replacement may be the only surgical alternative to amputation; however, little is known about this approach. Questions/purposes In a highly selected small group of patients with metastatic carcinoma of the femur, we asked: (1) What was the patient survivorship after this treatment? (2) What was the implant survivorship free from all-cause revision and amputation, and what complications were associated with this treatment? (3) What functional outcomes were achieved by patients after total femur replacement for this indication? Methods Eleven patients (three men, eight women) with a mean age of 64 years (range, 41-78 years) received total femur replacements between 1986 and 2016; none were lost to followup. The most common primary disease was breast cancer. In general, during this period, our indications for this procedure were extensive metastatic disease precluding internal fixation or isolated proximal or distal femur replacement, and an anticipated lifespan exceeding 6 months. Our contraindication for this procedure during this time was expected lifespan less than 6 months. Patient survival was assessed by Kaplan-Meier analysis; implant survival free from revision surgery and amputation were assessed by competing risk analysis. Function was determined preoperatively and 6 to 12 weeks postoperatively with the Musculoskeletal Tumor Society (MSTS) score normalized to a 100-point scale, with higher scores representing better function from a longitudinally maintained institutional database. Results Eleven patients died at a median of 5 months (range, 1-31 months) after surgery. One-year revision-free and limb survival were 82% (95% CI, 51%-98%) and 91% (95% CI, 61%-99%), respectively. Reasons for reoperation were hip dislocation, infection and local recurrence in one patient each. The latter two complications resulted in amputation in two patients. The median MSTS score was 32 (range, 13-57). Conclusions Despite attempts to select patients who might have anticipated greater life expectancy, eight of 11 patients died by 6 months after surgery, and an additional two patients had undergone an amputation at 8 and at 17 months postoperatively. Most patients undergoing total femur replacement in this series did not recover from the procedure by the time they died, despite our best attempts to perform the procedure in patients whom we thought would live at least 6 months. Based on this, we believe that most patients with extensive metastatic disease to the femur should be offered palliative care, rather than major reconstruction. Level of Evidence Level IV, therapeutic study

mTOR Senses Environmental Cues to Shape the Fibroblast-like Synoviocyte Response to Inflammation

Summary Accumulating evidence suggests that metabolic master regulators, including mTOR, regulate adaptive and innate immune responses. Resident mesenchymal tissue components are increasingly recognized as key effector cells in inflammation. Whether mTOR also controls the inflammatory response in fibroblasts is insufficiently studied. Here, we show that TNF signaling co-opts the mTOR pathway to shift synovial fibroblast (FLS) inflammation toward an IFN response. mTOR pathway activation is associated with decreased NF-κB-mediated gene expression (e.g., PTGS2, IL-6, and IL-8) but increased STAT1-dependent gene expression (e.g., CXCL11 and TNFSF13B). We further demonstrate how metabolic inputs, such as amino acids, impinge on TNF-mTORC1 signaling to differentially regulate pro-inflammatory signaling circuits. Our results define a critical role for mTOR in the regulation of the pro-inflammatory response in FLSs and unfold its pathogenic involvement in TNF-driven diseases, such as rheumatoid arthritis (RA).

FRI0018 Targeted inhibition of janus kinases abates IFN-GAMMA-INDUCED invasive behavior of fibroblast-like synoviocytes

Background Emerging evidence suggests that fibroblast-like synoviocytes (FLS) are key effector cells in rheumatoid arthritis (RA) and research into the mechanisms defining FLS activity in RA indicate that cytokines secreted by leukocytes play a crucial role. Nevertheless, the contribution of IFNγ, which is increased in rheumatoid synovitis, to the inflammatory synovial tissue reaction is not known. Objectives To explore the function of the T-cell cytokine IFNγ for mesenchymal tissue remodeling in RA, and to determine whether IFNγ-signaling controls the invasive potential of FLS. Methods To assess architectural responses, FLS were cultured in three-dimensional micromasses. FLS motility was analyzed in migration-, spreading- and invasion assays. Signaling events relevant to cellular motility were defined by western blots. Baricitinb and siRNA pools were used to suppress Janus Kinase (JAK) functions. Results Histological analyses of micromasses revealed unique effects of IFNγ on FLS shape and tissue organization. This was consistent with accelerated migration, pronounced actin and focal adhesion (FA) re-organization upon IFNγ stimulation. Since actin and FA dynamics and, thus, cell motility are integrated by the focal adhesion kinase (FAK), we next analyzed its activity. Indeed, IFNγ stimulation induced the phosphorylation of FAK-Y925, a phosphosite implicated in FAK-mediated cell migration. siRNA knockdown of JAK2, but not JAK1, abrogated FAK activation by IFNγ. Correspondingly, IFNγ-inudced FAK activation and invasion of FLS was abrogated by the JAK-inhibitor baricitinib. Conclusions Our study contributes insight into the synovial response to IFNγ and reveals JAK2 as a potential therapeutic target for FLS-mediated joint destruction in arthritis, especially in RA. Disclosure of Interest None declared

04.05 The metabolic checkpoint kinase mtor regulates the rheumatoid mesenchymal tissue response to inflammation

Background By producing cytokines, chemokines and other inflammatory factors fibroblast-like synoviocytes (FLS) frame a microenvironemt that significantly contributes to persistent synovial inflammation in rheumatoid arthritis (RA). The molecular mechanisms and pathways that regulate these responses within FLS are, however, insufficiently explored. Since mTOR is emerging as a regulator of tissue homeostasis that coordinates the cellular reponse to environmental stress, such as inflammation, we hypothesised that this kinase might also determine the FLS response to inflammation. Material and methods mTOR activation was assessed by immunhistochemistry and western blots. For functional in-vitro studies RA-FLS were stimulated with TNF and specific mTOR inhibitors (Torin-1, PP242 and rapamycin) were applied. Affymetrix microarrays were used for gene expression profiling. NF-κB and STAT1 pathway activation was assessed by western-blots and EMSA. Results Immunohistochemistry revealed increased mTOR activity in RA synovitis when compared to osteoarthritis. TNF stimulation of FLS ex vivo promoted the phosphorylation of mTOR in an AKT-dependent manner indicating that the pro-inflammatory synovial milieu may drive mTOR activation in RA. Gene expression profiling revealed a so far unknown role for mTOR in the regulation of TNF-induced gene programs: mTOR activation limits the TNF-induced expression of NF-κB-regulated genes (eg, IL6, IL8, PTGS2) by promoting the re-appearence of the NF-κB-inhibitor IκB-α. Contrary, mTOR activation augments the TNF-mediated induction of interferon regulated genes (IRGs; eg, CXCL11, TNFSF13B) by increasing the activity of the transcription factor STAT1. Conclusions The metabolic checkpoint kinase mTOR fine-tunes the induced gene expression program in FLS. Thus, mTOR emerges as crucial factor for the development of the RA-typical synovial, inflammatory microenvironment.