Preoperative deltoid assessment by contrast-enhanced ultrasound (CEUS) as predictor for shoulder function after reverse shoulder arthroplasty: a prospective pilot study.

INTRODUCTION: Although the deltoid represents the main motor muscle after reverse shoulder arthroplasty (RSA), its standardized preoperative assessment regarding morphology and function is still not established. Its clinical relevance and interactions with major biomechanical parameters like the medialization of the center of rotation (COR) regarding shoulder function after RSA are yet unknown. We evaluated contrast-enhanced ultrasound (CEUS) of the deltoid as possible surrogate marker for individual deltoid properties of patients receiving an RSA, and its predictive value for postoperative shoulder function. MATERIALS AND METHODS: 35 patients were prospectively assessed. Before and 6 months after RSA, dynamic deltoid perfusion, caliber and a combination of both (PE*caliber, named DeltoidEfficacy) was quantified by CEUS. Changes of deltoid properties and the predictive value of preoperative CEUS-based deltoid properties for shoulder function after RSA were assessed. To analyze interrelating effects with deltoid properties, COR-medialization and deltoid lengthening were quantified. RESULTS: Deltoid caliber and perfusion significantly increased after RSA (p = 0.0004/p = 0.002). Preoperative deltoid caliber, perfusion and the combined value DeltoidEfficacy significantly correlated with shoulder function after RSA within the whole study cohort (caliber: r = 0.445, p = 0.009; perfusion: r = 0.593, p = 0.001; DeltoidEfficacy: r = 0.66; p = 0.0002). The predictive value of DeltoidEfficacy for shoulder function after RSA varied among patient subgroups: Multivariate regression analysis revealed the strongest prediction in patients with either very high or very low deltoid properties (Beta = 0.872, r = 0.84, p = 0.0004), independent from COR-medialization or deltoid lengthening. Contrary, in patients with intermediate deltoid properties, COR-medialization revealed the strongest predictive value for shoulder function after RSA (Beta = 0.660, r = 0.597; p = 0.024). CONCLUSION: Deltoid CEUS seems to allow an assessment of individual deltoid properties and deltoid adaptations after RSA. Deltoid CEUS seems to predict shoulder function after RSA and might support an identification of patients requiring special attention regarding COR positioning.

Dynamic contrast-enhanced ultrasound and elastography assess deltoid muscle integrity after reverse shoulder arthroplasty.

BACKGROUND: The outcome after reverse shoulder arthroplasty (RSA) depends on the condition of the deltoid muscle, which we assessed with new ultrasound modalities and electromyography (EMG). Contrast-enhanced ultrasound (CEUS) and acoustic radiation force impulse (ARFI) were applied to assess perfusion and elasticity of the deltoid muscle compared with the clinical and functional outcome. METHODS: The study recruited 64 patients (mean age, 72.9 years) treated with RSA between 2004 and 2013. The deltoid muscle was examined with EMG and ultrasound imaging. Functional scores such as Constant score and American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form score were assessed. Among other CEUS parameters, the wash-in perfusion index, time to peak, and rise time were compared between the operated-on and contralateral shoulders as well as between patients with above-average and below-average outcome. The stiffness of the deltoid muscle was analyzed with ARFI. RESULTS: After RSA, deltoid perfusion (wash-in perfusion index, Δ = -12% ± 22%, P = .0001) and shoulder function (Constant score, Δ = -14 ± 24, P < .0001) were both inferior compared with the contralateral side. This perfusion deficit was associated with a limited range of motion (time to peak and anteversion: r = -0.290, P = .022). Deltoid perfusion was higher in patients with above-average outcome (rise time, Δ = 33% ± 13%, P = .038). The operated-on deltoid muscles showed higher stiffness than the contralateral muscles (ARFI, Δ = 0.2 ± 0.9 m/s, P = .0545). EMG excluded functionally relevant axillary nerve injuries in the study population. CONCLUSIONS: CEUS revealed reduced mean perfusion of the deltoid muscle after RSA. Reduced perfusion was associated with limited range of motion and below-average outcome. Functional shoulder impairment after RSA might be predicted by noninvasive CEUS as a surrogate parameter for the integrity of the deltoid muscle.

Elevated ratio of MMP2/MMP9 activity is associated with poor response to chemotherapy in osteosarcoma.

BACKGROUND: Matrix metalloproteinases (MMPs) are crucially involved in the regulation of multiple stages of cancer progression. Elevated MMP levels have been associated with the development of metastases and poor prognosis in several types of cancer. However, the role of MMPs in osteosarcoma and their prognostic value is still unclear. Available data are conflicting, most likely due to different technical approaches. We hypothesized that in contrast to total mRNA or protein levels frequently analyzed in previous studies the enzymatic activities of MMPs and their inhibitors the tissue inhibitors of matrix metalloproteinases (TIMPs) are closer related to their biological functions. We therefore aimed to evaluate the reliability of different zymography techniques for the quantification of MMP and TIMP activities in osteosarcoma biopsies in order to investigate their distribution, possible regulation and prognostic value. METHODS: All analyses were done using cryo-conserved osteosarcoma pretreatment biopsies (n = 18). Gene and protein expression of MMPs and TIMPs were analyzed by RT-qPCR and western blot analysis, respectively. Overall MMP activity was analyzed by in situ zymography, individual MMP activities were analyzed by gelatin zymography. Reverse zymography was used to detect and quantify TIMP activities. RESULTS: Strong overall MMP activities could be detected in osteosarcoma pretreatment biopsies with MMP2 and MMP9 as predominant active MMPs. In contrast to total RNA or protein expression MMP2 and MMP9 activities showed significant quantitative differences between good and poor responders. While MMP9 activity was high in the good responder group and significantly decreased in the poor responder group, MMP2 activity showed a reverse distribution. Likewise, significant differences were detected concerning the activity of TIMPs resulting in a negative correlation of TIMP1 activity with MMP2 activity (p = 0.044) and negative correlations of TIMP2 and TIMP3 with MMP9 activity (p = 0.007 and p = 0.006). CONCLUSION: In contrast to mRNA or protein levels MMP and TIMP activities showed significant differences between the analyzed good and poor responder groups. A shift from MMP9 to predominant MMP2 activity is associated with poor response to chemotherapy suggesting that the ratio of MMP2/MMP9 activity might be a valuable and easily accessible marker to predict the response to chemotherapy in osteosarcoma.

Improved survival in osteosarcoma patients with atypical low vascularization.

BACKGROUND: Osteosarcoma is considered a highly vascularized bone tumor with early metastatic dissemination through intratumoral blood vessels mostly into the lung. Novel targets for therapy such as tumor vascularization are highly warranted since little progress has been achieved in the last 30 years. However, proof of relevance for vascularization as a major prognostic parameter has been hampered by tumor heterogeneity, difficulty in detecting microvessels by immunohistochemistry, and small study cohorts. Most recently, we demonstrated that highly standardized whole-slide imaging could overcome these limitations (Kunz et al., PloS One 9(3):e90727, 2014). In this study, we applied this method to a multicenter cohort of 131 osteosarcoma patients to test osteosarcoma vascularization as a prognostic determinant. METHODS: Computer-assisted whole-slide analysis, together with enzymatic epitope retrieval, was used for CD31-based microvessel quantification in 131 pretreatment formalin-fixed and paraffin-embedded biopsies from three bone tumor centers. Kaplan-Meier-estimated survival and chemoresponse were determined and multivariate analysis was performed. Conventional hot-spot-based microvessel density (MVD) determination was compared with whole-slide imaging. RESULTS: We detected high estimated overall (p ≤ 0.008) and relapse-free (p ≤ 0.004) survival in 25 % of osteosarcoma patients with low osteosarcoma vascularization in contrast to other patient groups. Furthermore, all patients with low osteosarcoma vascularization showed a good response to neoadjuvant chemotherapy. Comparison of conventional MVD determination with whole-slide imaging suggests false high quantification or even exclusion of samples with low osteosarcoma vascularization due to difficult CD31 detection in previous studies. CONCLUSION: Low intratumoral vascularization at the time of diagnosis is a strong predictor for prolonged survival and good response to neoadjuvant chemotherapy in osteosarcoma.

Epigenetic silencing of genes and microRNAs within the imprinted Dlk1-Dio3 region at human chromosome 14.32 in giant cell tumor of bone.

BACKGROUND: Growing evidence exists that the neoplastic stromal cell population (GCTSC) within giant cell tumors (GCT) originates from mesenchymal stem cells (MSC). In a previous study we identified a microRNA signature that differentiates between these cell types. Five differentially expressed microRNAs are located within the Dlk1-Dio3 region on chromosome 14. Aberrant regulation within this region is known to influence cell growth, differentiation and the development of cancer. The aim of this study was to elucidate the involvement of deregulations within the Dlk1-Dio3 region in GCT pathogenesis. METHODS: Quantitative gene and microRNA expression analyses were performed on GCTSCs and MSCs with or without treatment with epigenetic modifiers. Methylation analysis of differentially methylated regions was performed by bisulfite sequencing. RESULTS: In addition to microRNA silencing we detected a significant downregulation of Dlk1, Meg3 and Meg8 in GCTSCs compared to MSCs. DNA methylation analyses of the Meg3-DMR and IG-DMR revealed a frequent hypermethylation within the IG-DMR in GCTs. Epigenetic modification could restore expression of some but not all analyzed genes and microRNAs suggesting further regulatory mechanisms. CONCLUSION: Epigenetic silencing of genes and microRNAs within the Dlk1-Dio3 region is a common event in GCTSCs, in part mediated by hypermethylation within the IG-DMR. The identified genes, micro RNAs and microRNA target genes might be valuable targets for the development of improved strategies for GCT diagnosis and therapy.

CEUS-assessed supraspinatus muscle perfusion improves after tendon repair and predicts anatomical and functional outcome: A 1-year prospective pilot study.

Rotator cuff tear including SSP (Supraspinatus) tendon tears are a very common and often painful condition with several therapeutic options such as tendon repair. Reflected by the high retear rates, the preoperative selection of patients suitable for surgery or conservative treatment, which often yields comparable results, remains difficult. Using contrast-enhanced ultrasound (CEUS), it is possible to quantify the SSP muscle perfusion as a surrogate parameter for its vitality and healing capabilities. In this study, we enrolled 20 patients who underwent an SSP repair for a preoperative and two postoperative (6 months and 1 year) clinical and sonographic exams including CEUS. Along with functional improvement (p < 0.001, Constant score), we found a significant increase in CEUS-assessed muscle perfusion after tendon repair (p < 0.001). Furthermore, weak preoperative muscle perfusion was associated with a higher risk of a retear (χ2 = 0.045) and a moderate trend toward lower postoperative shoulder function that did not reach significance (r = 0.435; p = 0.055, DASH score). If confirmed in larger studies, CEUS might be a valuable additional diagnostic method for a precise selection of patients who most likely profit from a tendon repair and those who can be treated conservatively with an equally good outcome.

Safety Profile of Musculoskeletal Contrast-Enhanced Ultrasound with Sulfur Hexafluoride Contrast Agent.

PURPOSE: Muscle, bone and tendon regeneration depend on the microperfusion of the corresponding tissue which can be quantified with contrast-enhanced ultrasound (CEUS) using sulfur hexafluoride contrast agent (SonoVue®). This study investigated the incidence of adverse events (AEs) in musculoskeletal patients and gives an overview of musculoskeletal CEUS applications. PATIENTS AND METHODS: Based on 13 studies in a standardized monocentric setting, a total of 2268 CEUS examinations in 764 patients were performed and AEs due to the administration of sulfur hexafluoride contrast agent were classified as either mild, moderate or severe. RESULTS: No fatal events occurred. AEs were reported in three cases, of which only one was classified as severe and two as mild. The total rate of all AEs was 0.13% and 0.04% for severe AEs. CONCLUSION: The present analysis confirms the safety of musculoskeletal CEUS using sulfur hexafluoride contrast agent with a lower rate of AEs than that reported for other contrast agents even in elderly patients with concomitant diseases.

Intra-observer and Device-Dependent Inter-observer Reliability of Contrast-Enhanced Ultrasound for Muscle Perfusion Quantification.

Muscle perfusion quantification by contrast-enhanced ultrasound (CEUS) may facilitate treatment decisions in musculoskeletal disorders. Translation into clinical routine relies on high intra-observer and inter-observer reliability and transferability between ultrasound devices to enable validation and multicenter studies. This study evaluates these aspects for deltoid muscle perfusion quantification, including possible multicenter study setups. One hundred sixty-six CEUS quantifications were conducted on 42 shoulders. Intra-observer reliability revealed a high intra-class correlation coefficient (ICC, r = 0.91) and low coefficient of variation (CV, 10.28%). Inter-observer reliability revealed an ICC of .84 and a CV of 17.1%, but these values decreased when different ultrasound devices were used (ICC = .60, CV = 18.6%). Re-evaluating subgroups with high sectional plane concordance significantly increased reliability (intra-observer: ICC = .97, CV = 5.49%, inter-observer/same device: ICC = .98, CV = 5.83%, varying devices: ICC = .78, CV = 9.8%). CEUS perfusion quantification of the deltoid seems applicable for multicenter studies, yet pooling different ultrasound devices remains critical. Sectional plane concordance appears to be crucial for reliability and transferability of CEUS muscle perfusion quantifications.

Contrast-Enhanced Ultrasound (CEUS) as Predictor for Early Retear and Functional Outcome After Supraspinatus Tendon Repair.

Supraspinatus (SSP) tendon tears represent a common indication for shoulder surgery. Yet, prediction of postoperative function and tendon retear remains challenging and primarily relies on morphologic magnetic resonance imaging (MRI)-based parameters, supported by patients' demographic data like age, gender, and comorbidities. Considering continuously high retear rates, especially in patients with larger tears and negative prognostic factors, improved outcome prediction could be of high clinical value. Contrast-enhanced ultrasound (CEUS) enables an assessment of dynamic perfusion of the SSP muscle. As a potential surrogate for muscle vitality, CEUS might reflect functional properties of the SSP and support improved outcome prediction after tendon repair. Fifty patients with isolated SSP tendon tears were prospectively enrolled. Preoperatively, SSP muscle perfusion was quantified by CEUS and conventional morphologic parameters like tear size, fatty infiltration, and tendon retraction were assessed by MRI. At six months follow-up, shoulder function, tendon integrity, and muscle perfusion were reassessed. The predictive value of preoperative CEUS for postoperative shoulder function and tendon integrity was evaluated. 35 patients entered the statistical analysis. Preoperative CEUS-based assessment of SSP perfusion significantly correlated with early postoperative shoulder function (Constant, r = 0.48, p < 0.018) and tendon retear (r = 0.67, p < 0.001). CEUS-based subgroup analysis identified patients with exceptionally high, respectively low risk for tendon retear. CEUS-based assessment of the SSP seemed to predict early shoulder function and tendon retear after SSP repair and allowed to identify patient subgroups with exceptionally high or low risk for tendon retear. © 2019 The Authors. Journal of Orthopaedic Research® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 38:1150-1158, 2020.

Methods of biological reconstruction for bone sarcoma: indications and limits.

Therapy of bone sarcoma has dramatically changed over the past few decades. Several successful interdisciplinary treatment strategies have led to an increase of the survival rates from 20% to 60%-80%. Consequently new demands on the operative treatment of bone and soft tissue sarcoma have arisen. Nowadays limb salvage can be achieved in 80%-90% using tumour megaprostheses or biological reconstruction procedures. In this article we outline the indications and limitations of biological reconstruction procedures after bone tumour resection. We therefore introduce the different biological approaches such as free autologous bone grafting, reimplantation of extracorporeal devitalized autografts or distraction osteogenesis and summarize the currently available data on the individual procedures. Our analyses demonstrate a wide applicability of biological procedures in tumour situations. Although accompanied by considerable complications in the early postoperative phase, biological reconstructions clearly demonstrate the potential of having excellent long-term durability and functionality.

Regulatory T cells and tolerance induction.

Transplantation tolerance is the ultimate goal of organ transplantation. Natural regulatory T cells (Treg) have been expected to reach clinical applications for tolerance induction because their initial description. More than ten yr later, Treg have started moving from experimental animal models into clinical applications. Although the molecular mechanism of contact-dependent inhibition remains to be unraveled, alterations of Treg numbers have been shown in several human diseases: Whereas several autoimmune diseases have been reported to be associated with decreased Treg numbers, Treg are frequently accumulated in solid tumors and hematologic malignancies. Monitoring of Treg numbers could be instrumental in identifying patients with risk of graft failure and might help minimizing immunosuppressive therapy in transplant recipients. Molecular mechanisms of Treg proliferation and Treg elimination such as CD95 ligand (CD95L)-mediated apoptosis are currently explored for their clinical usability as therapeutical targets. Immunosuppressive drugs might modulate the number of Tregs. Expansion of the Treg numbers in vivo or in vitro resembles a novel therapeutical strategy to reach partial or even operational tolerance after organ transplantation.

Optimization of the chicken chorioallantoic membrane assay as reliable in vivo model for the analysis of osteosarcoma.

Survival rates of osteosarcoma patients could not be significantly improved by conventional chemotherapeutic treatment regimens since the introduction of high-dose chemotherapy 35 years ago. Therefore, there is a strong clinical need for new therapeutic targets and personalized treatment strategies, requiring reliable in vivo model systems for the identification and testing of potential new treatment approaches. Conventional in vivo rodent experiments face ethical issues, are time consuming and costly, being of particular relevance in orphan diseases like osteosarcoma. An attractive alternative to such animal experiments is the chicken chorioallantoic membrane (CAM) assay. The CAM is a highly vascularized, non-innervated extra-embryonic membrane that is perfectly suited for the engraftment of tumor cells. However, only few reports are available for osteosarcoma and reported data are inconsistent. Therefore, the aim of this study was the adaptation and optimization of the CAM assay for its application in osteosarcoma research. Tumor take rates and volumes of osteosarcoma that developed on the CAM were analyzed after modification of several experimental parameters, including egg windowing, CAM pretreatment, inoculation technique and many more. Eight osteosarcoma cell lines were investigated. Our optimized OS-CAM-assay was finally validated against a rat animal xenograft model. Using the cell line MNNG HOS as reference we could improve the tumor take rates from 51% to 94%, the viability of the embryos from initially 40% to >80% and achieved a threefold increase of the tumor volumes. We were able to generate solid tumors from all eight osteosarcoma cell lines used in this study and could reproduce results that were obtained using an osteosarcoma rat animal model. The CAM assay can bridge the gap between in vitro cell culture and in vivo animal experiments. As reliable in vivo model for osteosarcoma research the optimized CAM assay may speed up preclinical data collection and simplifies research on potential new agents towards personalized treatment strategies. Further, in accordance with Russell's and Burch's "Principles of Humane Experimental Technique" the reasonable use of this model provides a refinement by minimizing pain and suffering of animals and supports a considerable reduction and/or replacement of animal experiments.

Tumor Suppressor Function of miR-127-3p and miR-376a-3p in Osteosarcoma Cells.

Since the introduction of high-dose chemotherapy about 35 years ago, survival rates of osteosarcoma patients have not been significantly improved. New therapeutic strategies replacing or complementing conventional chemotherapy are therefore urgently required. MicroRNAs represent promising targets for such new therapies, as they are involved in the pathology of multiple types of cancer, and aberrant expression of several miRNAs has already been shown in osteosarcoma. In this study, we identified silencing of miR-127-3p and miR-376a-3p in osteosarcoma cell lines and tissues and investigated their role as potential tumor suppressors in vitro and in vivo. Transfection of osteosarcoma cells (n = 6) with miR-127-3p and miR-376a-3p mimics significantly inhibited proliferation and reduced the colony formation capacity of these cells. In contrast, we could not detect any influence of miRNA restoration on cell cycle and apoptosis induction. The effects of candidate miRNA restoration on tumor engraftment and growth in vivo were analyzed using a chicken chorioallantoic membrane (CAM) assay. Cells transfected with mir-127-3p and miR-376a-3p showed reduced tumor take rates and tumor volumes and a significant decrease of the cumulative tumor volumes to 41% and 54% compared to wildtype cells. The observed tumor suppressor function of both analyzed miRNAs indicates these miRNAs as potentially valuable targets for the development of new therapeutic strategies for the treatment of osteosarcoma.

Contrast-Enhanced Ultrasound Determines Supraspinatus Muscle Atrophy After Cuff Repair and Correlates to Functional Shoulder Outcome.

BACKGROUND: Muscle degeneration as a consequence of rotator cuff tears is mainly assessed by magnetic resonance imaging. Contrast-enhanced ultrasound (CEUS) is a new functional imaging method to assess microvascular perfusion as a fundamental parameter of muscle tissue vitality. In this cross-sectional study, the authors evaluated supraspinatus muscle perfusion after cuff repair and analyzed its association with functional shoulder outcome and the grade of echogenicity in B-mode ultrasound indicating fatty infiltration. HYPOTHESIS: The authors expected reduced microperfusion of the operated versus the contralateral supraspinatus muscle and a correlation of the muscular microperfusion with functional shoulder outcome. STUDY DESIGN: Cross-sectional study; Level of evidence, 3. METHODS: Patients who received unilateral repair of the supraspinatus tendon between 2009 and 2014 were invited for a single follow-up examination. Functional scores were assessed, including the Constant-Murley score and American Shoulder and Elbow Surgeons score. CEUS examination was performed bilaterally in an oblique sagittal plane of the supraspinatus fossa. Perfusion was quantified by the parameters wash-in perfusion index (WiPI) and peak enhancement via VueBox quantification software. The results of the Constant-Murley score, American Shoulder and Elbow Surgeons score, and perfusion parameters were referenced to the contralateral shoulder. Echogenicity of the supraspinatus muscle was classified with a 3-point scale as compared with the trapezius muscle. RESULTS: Sixty-seven patients were available, with a mean follow-up of 38.0 ± 18.5 months. Functional assessment showed impaired shoulder function on the operated shoulder as compared with the contralateral side (relative Constant Score [CS], 80% ± 19%). CEUS revealed diminished perfusion on the operated shoulder (WiPI, 55.1% ± 40.2%, P < .001). A strong correlation could be demonstrated between the perfusion deficit and functional impairment (relative WiPI and CS: rs = .644, P < .001). Higher grade of echogenicity in B-mode ultrasound was associated with reduced perfusion. CONCLUSION: CEUS could visualize impaired supraspinatus muscle perfusion after rotator cuff repair as compared with the contralateral, healthy shoulder. With its ability to quantify microvascular perfusion as a surrogate parameter for muscle vitality and function, CEUS may serve as a quantitative method to evaluate rotator cuff muscles.

Angiopoietin-2 overexpression in morris hepatoma results in increased tumor perfusion and induction of critical angiogenesis-promoting genes.

UNLABELLED: Monitoring of angiogenesis-relevant approaches with functional imaging and histomorphometric analyses is desirable to evaluate the biologic effects. In this study we wished to examine the complex effects of angiopoietin-2 (Ang-2) gene transfer in a rat hepatoma model. METHODS: Using a bicistronic retroviral vector for Ang-2, Morris hepatoma (MH3924A) cell lines with Ang-2 expression were generated (Ang-2-MH3924A). In human umbilical vein endothelial cells (HUVECs) cocultured with Ang-2-MH3924A, the proliferative action with or without growth factors were determined. Furthermore, animal experiments were performed to measure effects on tumor growth and perfusion. Finally, tumors were examined by immunohistochemistry and DNA chip analysis. RESULTS: Ang-2-expressing MH3924A enhanced basic fibroblast growth factor-mediated endothelial cell proliferation. Perfusion, as measured by H(2)(15)O PET, was increased in genetically modified tumors. Consistent with the increased perfusion, micro- and macrovascularization were increased. However, tumor growth was similar to wild-type MH3924A (WT-MH3924A). Proliferating cell nuclear antigen and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) staining revealed an increased number of positive cells, indicating a compensation of increased proliferation by enhanced apoptosis. DNA chip analysis showed an induction of angiogenesis-promoting genes, including crucial vascular growth factor receptors, as well as genes related to extracellular matrix (ECM), apoptosis, signal transduction, and oxidative stress. CONCLUSION: Our results suggest that Ang-2 expression increases perfusion or vascularization, especially in interaction with the vascular growth factor system, without affecting tumor growth. Simultaneous, enhanced expression of genes for ECM, apoptosis, and signal transduction indicates Ang-2's versatile role in angiogenesis including its destabilizing function on ECM and endothelium.

Restoration of miR-127-3p and miR-376a-3p counteracts the neoplastic phenotype of giant cell tumor of bone derived stromal cells by targeting COA1, GLE1 and PDIA6.

Although generally benign, giant cell tumors of bone (GCTB) display an aggressive behavior associated with significant bone destruction and lung metastasis in rare cases. This and the very high recurrence rate observed after surgical resection ranging from 20 to 55% necessitates the development of more effective treatment strategies. To identify valuable therapeutic targets, we screened a previously identified microRNA signature consisting of 23 microRNAs predominantly down-regulated in GCTB. We preselected eight candidate microRNAs and analyzed the impact of their restored expression on the neoplastic phenotype of GCTB stromal cells (GCTSC). A consistent and significant inhibition of cell proliferation, migration, colony formation and spheroid formation could be induced by transfection of primary GCTSC cell lines with miR-127-3p and miR-376a-3p, respectively. Genome wide expression analysis of miR-127-3p and miR-376a-3p transfected cells revealed four novel target genes for each microRNA. Luciferase reporter assays demonstrated direct interactions of miR-127-3p with COA1 and direct interaction of miR-376a-3p with GLE1 and PDIA6, suggesting a pivotal role of these genes in the molecular etiology of GTCB. Interestingly, both microRNAs are located within a chromosomal region frequently silenced in GCTB and many other types of cancers, indicating that these microRNAs and their target genes are valuable therapeutic targets for the treatment of GCTB and possibly other tumor entities.

Dynamic contrast-enhanced ultrasound (CEUS) after open and minimally invasive locked plating of proximal humerus fractures.

INTRODUCTION: Closed reduction and locked plate fixation of proximal humerus fractures with the minimally invasive deltoid-splitting approach intends to minimize soft tissue damage although axillary nerve injury has been reported. The aim of this study was to assess the deltoid muscle perfusion with dynamic contrast-enhanced ultrasound (CEUS) as novel technique and evaluate its relation to the functional and neurologic outcome after open (ORIF) and minimally invasive (MIPO) fracture fixation. PATIENTS AND METHODS: 50 patients, 30 with deltopectoral ORIF and 20 with deltoid-splitting MIPO approach were examined 6-49 months after surgery. Only patients with a healthy, contralateral shoulder were selected. Shoulder function, satisfaction as well as psychosocial outcome were assessed with established scores (Constant, DASH, Simple Shoulder Test, ASES, SF-12). Electromyography (EMG) of the deltoid muscle was performed to determine axillary nerve damage. Ultrasound of both shoulders included CEUS and Power Doppler after deltoid muscle activation via active abduction for two minutes. RESULTS: None of the examinations and scores showed significant differences between ORIF and MIPO patients, the psychosocial outcome was similar. The fracture types were equally distributed in both groups. The normalized Constant Score was 76.3±18.6 in the ORIF and 81.6±16.1 in the MIPO group (p=0.373). Deltoid muscle perfusion in CEUS and Power Doppler revealed no differences between both approaches. EMG excluded functionally relevant axillary nerve injuries. Compared with the contralateral shoulder, Constant- and ASES-Scores (p≤0.001 for both ORIF and MIPO) as well as the deltoid CEUS perfusion (ORIF p=0.035; MIPO p=0.030) were significantly worse for both approaches. CONCLUSIONS: Convincing consensus of functional, ultrasonographic and neurologic examinations demonstrated comparable outcomes after deltopectoral and deltoid-splitting approach. The quantification of the deltoid muscle perfusion with CEUS indicates that the proclaimed benefits of the MIPO approach on soft tissue might not be as great as expected.

CD8+/FOXP3+-ratio in osteosarcoma microenvironment separates survivors from non-survivors: a multicenter validated retrospective study.

Osteosarcoma is the most common primary bone tumor characterized by juvenile onset, tumor heterogeneity, and early pulmonary metastasis. Therapeutic improvement stagnates since more than two decades. Unlike major malignancies, biomarkers as prognostic factors at time of diagnosis are missing. Disease rareness hampers study recruitment of patient numbers sufficient to outweigh tumor heterogeneity. Here, we analyzed in a multicenter cohort the osteosarcoma microenvironment to reduce effects of tumor cell heterogeneity. We hypothesized that quantitative ratios of intratumoral CD8+T-cells to FOXP3+T-cells (CD8+/FOXP3+-ratios) provide strong prognostic information when analyzed by whole-slide imaging in diagnostic biopsies. We followed recommendations-for-tumor-marker-prognostic-studies (REMARK). From 150 included cases, patients with complete treatment were identified and assigned to the discovery (diagnosis before 2004) or the validation cohort (diagnosis 2004-2012). Highly standardized immunohistochemistry of CD8+ and FOXP3+, which was validated by methylation-specific gene analysis, was performed followed by whole-slide analysis and clinical outcome correlations. We observed improved estimated survival in patients with CD8+/FOXP3+-ratios above the median (3.08) compared to patients with lower CD8+/FOXP3+-ratios (p = 0.000001). No patients with a CD8+/FOXP3+-ratio above the third quartile died within the observation period (median follow-up 69 mo). Multivariate analysis demonstrated independence from current prognostic factors including metastasis and response to neoadjuvant chemotherapy. Data from an independent validation cohort confirmed improved survival (p = 0.001) in patients with CD8+/FOXP3+-ratios above 3.08. Multivariate analysis proofed that this observation was also independent from prognostic factors at diagnosis within the validation cohort. Intratumoral CD8+/FOXP3+-ratio in pretreatment biopsies separates patients with prolonged survival from non-survivors in osteosarcoma.

Osteosarcoma microenvironment: whole-slide imaging and optimized antigen detection overcome major limitations in immunohistochemical quantification.

BACKGROUND: In osteosarcoma survival rates could not be improved over the last 30 years. Novel biomarkers are warranted to allow risk stratification of patients for more individual treatment following initial diagnosis. Although previous studies of the tumor microenvironment have identified promising candidates, novel biomarkers have not been translated into routine histopathology. Substantial difficulties regarding immunohistochemical detection and quantification of antigens in decalcified and heterogeneous osteosarcoma might largely explain this translational short-coming. Furthermore, we hypothesized that conventional hot spot analysis is often not representative for the whole section when applied to heterogeneous tissues like osteosarcoma. We aimed to overcome these difficulties for major biomarkers of the immunovascular microenvironment. METHODS: Immunohistochemistry was systematically optimized for cell surface (CD31, CD8) and intracellular antigens (FOXP3) including evaluation of 200 different antigen retrieval conditions. Distribution patterns of these antigens were analyzed in formalin-fixed and paraffin-embedded samples from 120 high-grade central osteosarcoma biopsies and computer-assisted whole-slide analysis was compared with conventional quantification methods including hot spot analysis. RESULTS: More than 96% of osteosarcoma samples were positive for all antigens after optimization of immunohistochemistry. In contrast, standard immunohistochemistry retrieved false negative results in 35-65% of decalcified osteosarcoma specimens. Standard hot spot analysis was applicable for homogeneous distributed FOXP3+ and CD8+ cells. However, heterogeneous distribution of vascular CD31 did not allow reliable quantification with hot spot analysis in 85% of all samples. Computer-assisted whole-slide analysis of total CD31- immunoreactive area proved as the most appropriate quantification method. CONCLUSION: Standard staining and quantification procedures are not applicable in decalcified formalin-fixed and paraffin-embedded samples for major parameters of the immunovascular microenvironment in osteosarcoma. Whole-slide imaging and optimized antigen retrieval overcome these limitations.

Enhanced reconstruction of long bone architecture by a growth factor mutant combining positive features of GDF-5 and BMP-2.

Non healing bone defects remain a worldwide health problem and still only few osteoinductive growth factors are available for clinical use in bone regeneration. By introducing BMP-2 residues into growth and differentiation factor (GDF)-5 we recently produced a mutant GDF-5 protein BB-1 which enhanced heterotopic bone formation in mice. Designed to combine positive features of GDF-5 and BMP-2, we suspected that this new growth factor variant may improve long bone healing compared to the parent molecules and intended to unravel functional mechanisms behind its action. BB-1 acquired an increased binding affinity to the BMP-IA receptor, mediated enhanced osteogenic induction of human mesenchymal stem cells versus GDF-5 and higher VEGF secretion than BMP-2 in vitro. Rabbit radius defects treated with a BB-1-coated collagen carrier healed earlier and with increased bone volume compared to BMP-2 and GDF-5 according to in vivo micro-CT follow-up. While BMP-2 callus often remained spongy, BB-1 supported earlier corticalis and marrow cavity formation, showing no pseudojoint persistence like with GDF-5. Thus, by combining positive angiogenic and osteogenic features of GDF-5 and BMP-2, only BB-1 restored a natural bone architecture within 12 weeks, rendering this promising growth factor variant especially promising for long bone regeneration.