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Four primary extraskeletal osteosarcomas of the pleura are presented. The patients were men between the ages of 63 and 78 years (average: 70.5 years) who presented with symptoms of chest pain, cough, and shortness of breath. Diagnostic imaging showed variably calcified, pleural-based masses and/or diffuse pleural thickening, clinically mimicking mesothelioma. Thoracoscopic surgical material was obtained for histopathological diagnosis. Histological findings showed the presence of a neoplastic spindle cell proliferation composed of fusiform cells with scant cytoplasm, elongated nuclei and inconspicuous nucleoli. Mitotic activity was easily identified. Additionally, all tumors showed extensive osseous differentiation in the form of osteoid matrix production; one tumor demonstrated additional chondrosarcomatous elements and another showed focal myxoid changes. Immunohistochemical analysis revealed that the tumor cells were uniformly negative for a wide variety of antibodies, including keratin AE1/AE3, keratin 5/6, D2-40, EMA, calretinin, WT-1, BerEP4, and HEG1; BAP1 was retained in all cases. In addition, fluorescence in situ hybridization for CDKN2A (p16) was negative for homozygous deletion in all tumors. Clinical follow-up showed that one patient had died 8 months after diagnosis and one had remained alive with short post-diagnostic follow-up. The tumors herein described highlight a challenging issue in the separation of mesothelioma with heterologous elements from true osteosarcomas of pleural origin. We propose that a diagnosis of extraskeletal osteosarcoma of the pleura is rendered for tumors with malignant osseous and/or cartilaginous differentiation in which comprehensive immunohistochemical studies and FISH analysis have failed to provide support for a diagnosis of mesothelioma with heterologous elements., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Telangiectatic osteosarcoma (TOS) is a rare variant of osteosarcoma that typically affects young individuals and long bones. The case under discussion was seen in the mandible of a 57-year-old female and had rapidly grown in size within a week. Microscopically, the tumour was characterised by large vascular cavities surrounded by anaplastic cells. Thin lacy tumour osteoid was observed at various foci. Abundant multinucleated osteoclastic giant cells along with areas of necrosis were also noted. The tumour cells were positive for SATB2, while negative for Cytokeratin AE1/3, CD 34. Ki-67 positivity was observed in more than 50% of tumour cells. A diagnosis of high grade telangiectatic osteosarcoma was thus made., (Copyright © 2024 Sociedad Española de Anatomía Patológica. Publicado por Elsevier España, S.L.U. All rights reserved.)

Environmentally, bisphenol A (BPA) is a well-known pollutant caused human health risk, including osteosarcoma (OS). OS, a deadly bone neoplasia, may occur in children and adults. However, the anti-OS pharmacotherapy prescribes limitedly in clinical practice. Interestingly, previous experimental evidences indicate calycosin-exerting potential anti-OS actions. Thus, in this report, we aimed to further characterize and detail the therapeutic targets and molecular mechanisms of calycosin-anti-BPA-related OS by using network pharmacology and molecular docking analyses. In results, the bioinformatics data disclosed all mapped, core targets, biological functions, molecular pathways of calycosin to treat BPA-related OS. The computational analysis using molecular docking indicated that potential binding ability of core targets in calycosin to treat BPA-related OS was identified. Moreover, detailed biological functions and optimal pathways of calycosin-anti-BPA-related OS were revealed, as shown in integrated network maps. Taken together, these network pharmacology and structural biology findings illustrate the core biotargets, pharmacological functions and pathways of calycosin-anti-BPA-related OS. Potentially, these core targets identified by molecular docking may attribute to the potential clinical application of calycosin against BPA-related OS.[Formula: see text].

The aim of this study is to assess the usefulness of beta-catenin immunohistochemical expression in the differential diagnosis of osteoid-producing primary tumors of bone. Seventy cases of osteoid-producing tumors of bone (24 conventional osteosarcomas, 18 osteoblastomas, 13 osteoblastoma-like osteosarcomas, 10 chondroblastomas, and 5 chondroblastoma-like osteosarcomas) diagnosed at Istituto Ortopedico Rizzoli were reviewed and evaluated for the intensity, extension, and subcellular distribution of immunohistochemical expression of beta-catenin. A majority of cases (73%, 51 cases) exhibited cytoplasmic and/or membranous positivity in varied degrees of intensity and proportion of positive cells, in the absence of nuclear staining. Fifteen cases (21%) were completely negative, including two osteoblastomas, five chondroblastomas, three conventional osteosarcomas, four osteoblastoma-like osteosarcomas, and one chondroblastoma-like osteosarcoma. A minority of cases (6%) including three osteoblastoma-like osteosarcomas and one osteoblastoma showed focal nuclear beta-catenin positivity with or without concomitant cytoplasmic staining. In the current series, beta-catenin showed not to be useful in the differential diagnosis of osteoid-producing primary bone tumors., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Purpose: To detect the expression level of hsa_circ_0005721 in osteosarcoma specimen and plasma of osteosarcoma patients, and to analyze the clinical significance of hsa_circ_0005721 as a diagnostic marker for osteosarcoma., Methods: Expression levels of hsa_circ_0005721 in osteosarcoma specimen and osteosarcoma cell lines were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relationship between hsa_circ_0005721 expression difference and overall survival in osteosarcoma was analyzed by Kaplan-Meier method. Expression level of hsa_circ_0005721 was stably downregulated in U-2OS and HOS cells by shRNA transfection. Proliferative potential in osteosarcoma cells regulated by hsa_circ_0005721 was assessed by colony formation and 5-Ethynyl-2'- deoxyuridine (EdU) assay. Differentially expressed hsa_circ_0005721 in the plasma of healthy controls, benign bone tumor patients and osteosarcoma patients was determined by qRT-PCR. The diagnostic capacity of hsa_circ_0005721 in osteosarcoma was examined by receiver operating characteristic (ROC) curves., Results: hsa_circ_0005721 was upregulated in osteosarcoma specimen and osteosarcoma cell lines. High level of hsa_circ_0005721 predicted poor prognosis in osteosarcoma patients. In vitro experiments showed that knockdown of hsa_circ_0005721 suppressed proliferative ability in osteosarcoma cells. Compared with that in healthy controls and benign bone tumor patients, plasma level of hsa_circ_0005721 was higher in osteosarcoma patients. ROC curves demonstrated the diagnostic potential of hsa_circ_0005721 in osteosarcoma., Conclusions: hsa_circ_0005721 is upregulated in osteosarcoma samples, which acts as an oncogene responsible for aggravating the progression. hsa_circ_0005721 can be a promising diagnostic marker for osteosarcoma.

There is a growing interest in immunotherapy in childhood cancers. Osteosarcoma is a compelling potential target as there are few targeted options available for this aggressive cancer. We provide a description of the landscape of programmed cell death-1 (PD-1), programmed cell death-ligand 1 (PD-L1) and relevant immune markers in serial samples from 15 osteosarcoma patients. PD-1 and PD-L1 expression was present in biopsy samples (47% and 53%, respectively), absent in resections, and present in metastases (40% and 47%). Both decalcified and nondecalcified specimens demonstrated expression of PD-1 and PD-L1. The results suggest that biopsy or metastatic specimens maybe most valuable in assessing expression of PD-1 and PD-L1.

Cellular adaptation to a hypoxic microenvironment is essential for tumour progression and is largely mediated by HIF-1α and hypoxia-regulated factors, including CXCR4, VEGF-A and GLUT-1. In human osteosarcoma, hypoxia is associated with resistance to chemotherapy as well as with metastasis and poor survival, whereas little is known about its role in canine osteosarcoma (cOSA). This study aimed primarily to evaluate the prognostic value of several known hypoxic markers in cOSA. Immunohistochemical analysis for HIF-1α, CXCR4, VEGF-A and GLUT-1 was performed on 56 appendicular OSA samples; correlations with clinicopathological features and outcome was investigated. The second aim was to investigate the in vitro regulation of markers under chemically induced hypoxia (CoCl 2 ). Two primary canine osteosarcoma cell lines were selected, and Western blotting, immunofluorescence and qRT-PCR were used to study protein and gene expression. Dogs with high-grade OSA (35.7%) were more susceptible to the development of metastases (P = 0.047) and showed high HIF-1α protein expression (P = 0.007). Moreover, HIF-1α overexpression (56%) was correlated with a shorter disease-free interval (DFI; P = 0.01), indicating that it is a reliable negative prognostic marker. The in vitro experiments identified an accumulation of HIF-1α in cOSA cells after chemically induced hypoxia, leading to a significant increase in GLUT-1 transcript (P = 0.02). HIF-1α might be a promising prognostic marker, highlighting opportunities for the use of therapeutic strategies targeting the hypoxic microenvironment in cOSA. These results reinforce the role of the dog as a comparative animal model since similar hypoxic mechanisms are reported in human osteosarcoma., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Changes in lipid metabolism are involved in several pathological conditions, such as cancer. Among lipids, eicosanoids are potent inflammatory mediators, synthesized from polyunsaturated fatty acids (PUFAs), which coexist with other lipid-derived ones, including endocannabinoids (ECs) and N -acylethanolamides (NAEs). In this work, a bioanalytical assay for 12 PUFAs/eicosanoids and 20 ECs/NAEs in cell culture medium and human biofluids was validated over a linear range of 0.1-2.5 ng/mL. A fast pretreatment method consisting of protein precipitation with acetonitrile followed by a double step liquid-liquid extraction was developed. The final extracts were injected onto a Kinetex ultra-high-performance liquid chromatography (UHPLC) XB-C18 column with a gradient elution of 0.1% formic acid in water and methanol/acetonitrile (5:1; v / v ) mobile phase. Chromatographic separation was followed by detection with a triple-quadrupole mass spectrometer operating both in positive and negative ion-mode. A full validation was carried out in a small amount of cell culture medium and then applied to osteosarcoma cell-derived products. To the best of our knowledge, this is the first lipid profiling of bone tumor cell lines (SaOS-2 and MG-63) and their secretome. Our method was also partially validated in other biological matrices, such as serum and urine, ensuring its broad applicability as a powerful tool for lipidomic translational research.

Confocal Raman microspectral imaging was adopted to elucidate the cellular drug responses of osteosarcoma cells (OC) to N-[N-(3, 5-difluorophenyl acetyl)-L-alanyl]-sphenylglycine butyl ester (DAPT), a γ-secretase inhibitor, by identifying the drug induced subcellular compositional and structural changes. Methods : Spectral information were acquired from cultured osteosarcoma cells treated with 0 (Untreated Group, UT), 10 (10 μM DAPT treated, 10T), 20 μM (20 μM DAPT treated, 20T) DAPT for 24 hours. A one-way ANOVA and Tukey's honest significant difference (HSD) post hoc multiple test were sequentially applied to address spectral features among three groups. Multivariate algorithms such as K-means clustering analysis (KCA) and Principal component analysis (PCA) were used to highlight the structural and compositional differences, while, univariate imaging was applied to illustrate the distribution pattern of certain cellular components after drug treatment. Results : Major biochemical changes in DAPT-induced apoptosis came from changes in the content and structure of proteins, lipids, and nucleic acids. By adopted multivariate algorithms, the drug induced cellular changes was identified by the morphology and spectral characteristics between untreated cells and treated cells, testified that DAPT mainly acted in the nuclear region. With the increase of the drug concentration, the content of main subcellular compositions, such nucleic acid, protein, and lipid decreased. In an addition, DAPT-induced nuclear fragmentation and apoptosis was depicted by the univariate Raman image of major cellular components (nucleic acids, proteins and lipids). Conclusions : The achieved Raman spectral and imaging results illustrated detailed DAPT-induced subcellular compositional and structural variations as a function of drug dose. Such observations can not only explain drug therapeutic mechanisms of OC DAPT treatment, and also provide new insights for accessing the medicine curative efficacy and predicting prognosis., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Biological reconstruction of allografts and recycled autografts have been widely implemented in high-grade osteogenic sarcoma. For treating tumor-bearing autografts, extracorporeal irradiation (ECIR) and liquid nitrogen (LN) freezing techniques are being used worldwide as a gold standard treatment procedure. Both the methods aim to eradicate the tumor cells from the local recurrence and restore the limb function. Therefore, it is essential and crucial to find, and compare the alterations at molecular and physiological levels of the treated and untreated OGS recycled autografts to obtain valuable clinical information for better clinical practice. Thus, we aimed to investigate the significantly expressed altered proteins from ECIR-and cryotherapy/freezing- treated OGS (n = 12) were compared to untreated OGS (n = 12) samples using LC-ESI-MS/MS analysis, and the selected proteins from this protein panel were verified using immunoblot analysis. From our comparative proteomic analysis identified a total of 131 differentially expressed proteins (DEPs) from OGS. Among these, 91 proteins were up-regulated (2.5 to 3.5-folds), and 40 proteins were down-regulated (0.2 to 0.5 folds) (p < 0.01 and 0.05). The functional enrichment analysis revealed that the identified DEPs have belonged to more than 10 different protein categories include cytoskeletal, extracellular matrix, immune, enzyme modulators, and cell signaling molecules. Among these, we have confirmed two potential candidates' expressions levels such as Fibronectin and Protein S100 A4 using western blot analysis. Our proteomic study revealed that LN-freezing and ECIR treatments are effectively eradicating tumor cells, and reducing the higher expressions of DEPs at molecular levels which may help in restoring the limb functions of OGS autografts effectively. To the best of our knowledge, this is the first proteomic study that compared proteomic profiles among freezing, ECIR treated with untreated OGS in recycled autografts. Moreover, the verified proteins could be used as prognostic or diagnostic markers that reveal valuable scientific information which may open various therapeutic avenues in clinical practice to improve patient outcomes.

Osteosarcoma (OS) is the most common malignant primary bone tumour in humans and dogs. Several studies have established the vital role of parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) in bone formation and remodeling. In addition, these molecules play a role in the progression and metastasis of many human tumour types. This study investigated the expression of PTHR1 and PTHrP in canine OS tissues and assessed their prognostic value. Formalin-fixed, paraffin-embedded tissue samples from 50 dogs diagnosed with primary OS were immunolabeled with antibodies specific for PTHR1 and PTHrP. The immunostaining intensity of tumours from patients with OS was correlated with survival time. Both PTHR1 and PTHrP were detected in all OS samples (n = 50). Dogs with OS tumours showing high immunostaining intensity for PTHR1 (n = 36) had significantly shorter survival times (p = 0.028, Log Rank; p = 0.04, Cox regression) when compared with OS that had low immunostaining intensity for PTHR1 (n = 14).PTHrP immunostaining intensity did not correlate with survival time (p > 0.05). The results of this study indicate that increased expression of PTHR1 antigen in canine OS is associated with poor prognosis. This suggests that PTHR1 may be useful as a prognostic indicator in canine OS.

Quantifying the chemical composition of unstained intact tissue and cellular samples with high spatio-temporal resolution in three dimensions would provide a step change in cell and tissue analytics critical to progress the field of cell biology. Label-free optical microscopy offers the required resolution and noninvasiveness, yet quantitative imaging with chemical specificity is a challenging endeavor. In this work, we show that hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy can be used to provide quantitative volumetric imaging of human osteosarcoma cells at various stages through cell division, a fundamental component of the cell cycle progress resulting in the segregation of cellular content to produce two progeny. We have developed and applied a quantitative data analysis method to produce volumetric three-dimensional images of the chemical composition of the dividing cell in terms of water, proteins, DNAP (a mixture of proteins and DNA, similar to chromatin), and lipids. We then used these images to determine the dry masses of the corresponding organic components. The attribution of proteins and DNAP components was validated using specific well-characterized fluorescent probes, by comparison with correlative two-photon fluorescence microscopy of DNA and mitochondria. Furthermore, we map the same chemical components under perturbed conditions, employing a drug that interferes directly with cell division (Taxol), showing its influence on cell organization and the masses of proteins, DNAP, and lipids.

Background: Osteosarcoma is a malignant bone tumor with a high potential for lung metastasis, and the prognosis for patients with metastatic disease is very poor. The interaction between fibronectin (FN) and integrin αvβ3 in soft-tissue sarcoma promotes cell migration, invasion, and lung metastasis. This study aimed to investigate the prognostic significance of FN and αvβ3 in osteosarcoma., Methods: Immunohistochemistry and western blotting were used to detect the expression of FN and αvβ3 in 60 osteosarcoma specimens and in 30 osteochondroma specimens. Furthermore, correlations of FN and αvβ3 with the clinicopathological features of osteosarcoma patients were analyzed using the χ 2 test and Fisher's exact test. Disease-free survival and overall survival of osteosarcoma patients were assessed using the Kaplan-Meier method and Cox proportional hazards model. The predictive accuracy of the model was determined by the Harrell concordance index., Results: FN (P < 0.05) and αvβ3 (P < 0.05) were overexpressed in osteosarcoma specimens compared with osteochondroma specimens. High FN expression was associated with a poor response to chemotherapy (P = 0.001) and poor disease-free (P < 0.001) and overall (P < 0.001) survival. High expression of αvβ3 was linked to an advanced surgical stage (P = 0.028), a poor response to chemotherapy (P = 0.002), and both poor disease-free survival (P < 0.001) and overall survival (P < 0.001). FN and αvβ3 co-expression were associated with sex (P = 0.011), an advanced surgical stage (P = 0.013), and a poor response to chemotherapy (P = 0.002). Moreover, high expression of both proteins can serve as an independent prognostic value for reduced survival time in osteosarcoma patients., Conclusions: The results of this study suggest that FN and αvβ3 expression is associated with an unfavorable clinical outcome of osteosarcoma, and these molecules may constitute attractive therapeutic targets for osteosarcoma treatment. To improve the survival of osteosarcoma patients, further investigations are required to clarify their prognostic values in a larger population.

How cancer cells and their anchorage-dependent normal counterparts respond to the adhesion ligand density and stiffness of the same extracellular matrix (ECM) is still not very clear. Here we investigated the effects of ECM adhesion ligand density and stiffness on bone tumor cells (osteosarcoma cells) and bone forming cells (osteoblasts) by using poly (ethylene glycol) diacrylate (PEGDA) and methacrylated gelatin (GelMA) hydrogels. By independently changing the PEGDA and GelMA content in the hydrogels, we achieved crosslinked hydrogel matrix with independently tunable stiffness (1.6, 6 and 25 kPa for 5%, 10%, 15% PEDGA, respectively) and adhesion ligand density (low, medium and high for 0.05%, 0.2%, 0.5% GelMA respectively). By using a series of biochemical and cell biological characterizations as well as in vivo studies, we confirmed that osteosarcoma and osteoblastic cells responded differently to the stiffness and adhesion ligand density within 3D ECM. When cultured within the 3D PEGDA/GelMA hydrogel matrix, osteosarcoma cells are highly dependent on the matrix stiffness via regulating the integrin-mediated focal adhesion (FA) pathway, whereas osteoblasts are highly sensitive to the matrix adhesion ligand density through regulating the integrin-mediated adherens junction (AJ) pathway. However, when seeded on the 2D surface of the hydrogels, osteosarcoma cells behaved differently and became sensitive to the matrix adhesion ligand density because they were "forced" to attach to the substrate, similar to anchorage-dependent osteoblasts. This study might provide new insights into rational design of scaffolds for generating in vitro tumor models to test anticancer therapeutics and for regenerating tissue to repair defects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Osteosarcoma is the most common primary malignant bone tumor. In the last years, several studies have demonstrated that the increase of Hydroxyapatite (HA) and Interleukin-6 (IL-6) syntheses compared to those expressed by normal osteoblasts could be used to detect the degree of malignancy of osteosarcoma cells. Conventional biochemical methods widely employed to evaluate bone cell differentiation, including normal and cancerous phenotypes, are time consuming and may require a large amount of cells. HA is a mineral form of calcium phosphate whose presence increases with maturation of osteosarcoma cells. Analogously, IL-6 is a fundamental cytokine whose production is highly increased in osteosarcoma cells. In this study, we employ Raman spectroscopy to the identification and discrimination of osteosarcoma cells from osteo-differentiated mesenchymal stromal cells (MSCs) by detecting the presence of HA and IL-6. However, while the identification of HA is facilitated by the characteristic peak at 960 cm -1 , corresponding to symmetric stretching (P-O) mode, the quantification of IL-6 it is much more elusive, being its Raman signal characterized by cysteine, but also by phenylalanine, amide I II and III whose signals are common to other proteins. Supported by an accurate multivariate analysis, the results show that Raman spectroscopy is a high sensitivity technique dealing out a direct and quantitative measurement of specific mineralization levels of osteosarcoma cells. In turn, by exploiting the Surface-Enhanced Raman Scattering stimulated by internalized Gold Nanoshells (AuNSs) and combined with scanning probe microscopies, we were able to employ Raman spectroscopy to study subcellular components locally.

Background: Transforming acidic coiled-coil containing protein 3 (TACC3) is expressed during the mitotic phase of nuclear division and regulates microtubules. Recently, high TACC3 expression in tumor cells of various cancers including soft tissue sarcoma has been reported. However, its role in osteosarcoma remains unknown. Because we have few prognostic markers for survival in osteosarcoma, we wanted to investigate the potential role of TACC3 in human osteosarcoma and determine if it is associated with survival., Questions/purposes: (1) Is there a relationship between TACC3 expression and clinicopathologic characteristics such as sex, age (< 20 or ≥ 20 years), histologic type (osteoblastic or others), tumor location (femur or others), American Joint Committee on Cancer staging system (AJCC stage IIA or IIB), tumor necrosis percentage after chemotherapy (< 90% or ≥ 90%), p53 expression (low or high), and Ki-67 expression (low or high)? (2) Is TACC3 expression associated with event-free and overall survival in patients with osteosarcoma?, Methods: Forty-six conventional patients with osteosarcoma were treated at our institution from 1989 to 2013. Patients were excluded because of unresectable primary site (two patients) and no chemotherapy (two patients). Patients with metastasis at the initial visit (five patients), without pretreatment biopsy samples (two patients), or clinical charts (two patients) were also excluded. The left 33 patients who received neoadjuvant and adjuvant chemotherapy, which consisted of cisplatin/doxorubicin/methotrexate or cisplatin/doxorubicin/methotrexate/ifosfamide, and completed surgical resection with histologic wide tumor margins. Primary tumor samples before chemotherapy were used in this study. We investigated TACC3 expression using immunohistochemical staining and statistically analyzed the TACC3 expression, clinicopathologic characteristics, and event-free and overall survival in patients with osteosarcoma., Results: High TACC3 expression was observed in 19 of 33 osteosarcoma specimens (58%), and this was associated with larger tumor size (ie, AJCC stage IIB in this study; p = 0.002), higher p53 expression (p = 0.007), and higher Ki-67 expression (p = 0.002). The estimated metastasis-free survival at 5 years was 21% (95% confidence interval [CI], 7%-41%) in patients with high TACC3 expression and 79% (95% CI, 47%-93%) in patients with low TACC3 expression (p < 0.001), and the estimated overall survival at 5 years was 34% (95% CI, 13%-56%) in patients with high TACC3 expression and 86% (95% CI, 54%-96%) in patients with low TACC3 expression (p < 0.001). Furthermore, high TACC3 expression was an independent poor prognostic factor for metastasis-free survival with a hazard ratio of 3.89 (95% CI, 1.07-19.78; p = 0.039) as well as overall survival with 4.41 (95% CI, 1.01-32.97; p = 0.049)., Conclusions: High TACC3 expression was associated with aggressive clinicopathologic features and unfavorable prognosis in these patients with osteosarcoma. Our preliminary results suggest that further analysis about mutation or an inactive form of TACC3 would be useful to understand the mechanism of abnormal TACC3 expression in patients with osteosarcoma. If these findings are substantiated in larger studies, TACC3 might be useful for predicting survival and a potential therapeutic target for osteosarcoma., Level of Evidence: Level III, therapeutic study.

Osteogenic sarcoma (OS) is the most common malignant bone tumor in children and adolescents. Despite advances in molecular genetic characterization of pediatric and adult tumors, the diagnosis of OS still depends almost entirely on light microscopy. The lack of consistent genetic changes in OS has greatly hindered the development of any diagnostic molecular test. Recently, whole-genome sequencing has shown that ~50% of cases of OS have a translocation involving the TP53 gene with breakpoints confined to the first intron. We developed a 2 color break-apart fluorescent in situ hybridization (FISH) probe for intron 1 of TP53 and applied it to an archived series to assess its diagnostic utility. The study group included 37 cases of OS (including osteoblastic, chondroblastic, and fibroblastic), as well as 53 cases of non-OS pediatric sarcomas (including Ewing sarcoma, rhabdomyosarcoma, undifferentiated small cell sarcoma, CCNB3-BCOR sarcoma, CIC-DUX sarcoma, synovial sarcoma, and malignant peripheral nerve sheath tumor) and 27 cases of benign bone lesions (including osteoblastoma, chondromyxoid fibroma, fibrous dysplasia, and fibro-osseous dysplasia). A rearranged signal was found in 20/37 cases (54%) of OS and in none of the other sarcomas or benign bone lesions, giving the FISH test 100% specificity for a diagnosis of OS. p53 immunostaining was generally not predictive of the results obtained by FISH and could not substitute for this test. This FISH probe offers a simple and specific genetic test to aid in the diagnosis of OS, despite the genetic complexity of this tumor.

Background: Programmed death 1 (PD-1) signaling in the tumor microenvironment dampens immune responses to cancer, and blocking this axis induces antitumor effects in several malignancies. Clinical studies of PD-1 blockade are only now being initiated in pediatric patients, and little is known regarding programmed death-ligand 1 (PD-L1) expression in common childhood cancers. The authors characterized PD-L1 expression and tumor-associated immune cells (TAICs) (lymphocytes and macrophages) in common pediatric cancers., Methods: Whole slide sections and tissue microarrays were evaluated by immunohistochemistry for PD-L1 expression and for the presence of TAICs. TAICs were also screened for PD-L1 expression., Results: Thirty-nine of 451 evaluable tumors (9%) expressed PD-L1 in at least 1% of tumor cells. The highest frequency histotypes comprised Burkitt lymphoma (80%; 8 of 10 tumors), glioblastoma multiforme (36%; 5 of 14 tumors), and neuroblastoma (14%; 17 of 118 tumors). PD-L1 staining was associated with inferior survival among patients with neuroblastoma (P = .004). Seventy-four percent of tumors contained lymphocytes and/or macrophages. Macrophages were significantly more likely to be identified in PD-L1-positive versus PD-L1-negative tumors (P < .001)., Conclusions: A subset of diagnostic pediatric cancers exhibit PD-L1 expression, whereas a much larger fraction demonstrates infiltration with tumor-associated lymphocytes. PD-L1 expression may be a biomarker for poor outcome in neuroblastoma. Further preclinical and clinical investigation will define the predictive nature of PD-L1 expression in childhood cancers both at diagnosis and after exposure to chemoradiotherapy. Cancer 2017;123:3807-3815. © 2017 American Cancer Society., (© 2017 American Cancer Society.)

Objective: To investigate the clinical significance of detection of circulating tumor cells (CTCs) in peripheral blood from patients with osteosarcoma (OS) using the iFISH (immunofluorescence and fluorescence in situ hybridization) method. Methods: The live cells recovery rate of immune-magnetic beads was evaluated by live-cell fluorescent tracer technology. The expression of CD45 and CK18 on the cell surface of HOS and HepG2 cells was measured by flow cytometry. And the chromosome aneuploidy was detected by centromeric FISH probe CEP8. Subsequently, 23 OS patients were enrolled and divided into two groups, relapse or metastasis group and primary group. And the prognostic significance of CTCs numbers was analyzed. Results: The live cells recovery rate of immune-magnetic beads was higher than 90%. The flow cytometry results showed that HOS cells were double negative for the surface biomarkers of CD45 and CK18. In addition, the FISH-CEP8 signal abnormality rate were 96.5% in HOS cells. Thus, CTC was identified using the criteria as follows: the cells with CEP8-positive signal >2 accounted for more than 96.5% of the total cells, of which the cells with >3 positive signal were more than 65.0%. Among the enrolled patients, 19 patients had detectable CTCs in the peripheral blood. The CTCs numbers in the relapse or metastasis group and primary group were 2.846±1.281 and 1.400±1.506, respectively. The results showed that the CTCs in patients with recurrence or metastasis were significantly higher than those in primary patients ( P =0.021). Conclusions: To our knowledge, this is the first evidence of existence of CTCs in OS patients. The CTCs numbers were positively associated with disease progression and poor prognosis. These results may provide a potential prognostic tool for monitoring metastasis and recurrence in OS patients. Trial registration: Chinese Clinical Trial Registry, ChiCTR-OOC-15005925.

Distinguishing hyalinized stroma from osteoid production by a heterologous osteosarcomatous component can be challenging in gynecologic tract carcinosarcomas. As heterologous components in a carcinosarcoma may have prognostic and therapeutic implications, it is important that these are recognized. This study examines interobserver reproducibility among gynecologic pathologists in the diagnosis of osteosarcomatous components, and its correlation with expression of the novel antibody SATB2 (marker of osteoblastic differentiation) in these osteosarcomatous foci. Digital H&E images from 20 gynecologic tract carcinosarcomas were reviewed by 22 gynecologic pathologists with a request to determine the presence or absence of an osteosarcomatous component. The 20 preselected cases included areas of classic heterologous osteosarcoma (malignant cells producing osteoid; n=10) and osteosarcoma mimics (malignant cells with admixed nonosteoid matrix; n=10). Interobserver agreement was evaluated and SATB2 scored on all 20 cases and compared with the original diagnoses. Moderate agreement (Fleiss' κ=0.483) was identified for the 22 raters scoring the 20 cases with a median sensitivity of 7/10 and a median specificity of 9/10 for the diagnosis of osteosarcoma. SATB2 showed 100% sensitivity (10/10) and 60% (6/10) specificity in discriminating classic osteosarcoma from osteosarcoma mimics. Utilizing negative SATB2 as a surrogate marker to exclude osteosarcoma, 73% (16/22) of the reviewers would have downgraded at least 1 case to not contain an osteosarcomatous component (range, 1-6 cases, median 1 case). Gynecologic pathologists demonstrate only a moderate level of agreement in the diagnosis of heterologous osteosarcoma based on morphologic grounds. In such instances, a negative SATB2 staining may assist in increasing accuracy in the diagnosis of an osteosarcomatous component.

Osteoblastoma is a benign bone tumor that can often be difficult to distinguish from malignant osteosarcoma. Because misdiagnosis can result in unfavorable clinical outcomes, we have investigated microRNAs as potential diagnostic biomarkers for distinguishing between these two tumor types. Next generation RNA sequencing was used as an expression screen to evaluate >2,000 microRNAs present in tissue derived from rare formalin fixed paraffin embedded (FFPE) archival tumor specimens. MicroRNAs displaying the greatest ability to discriminate between these two tumors were validated on an independent tumor set, using qPCR assays. Initial screening by RNA-seq identified four microRNA biomarker candidates. Expression of three miRNAs (miR-451a, miR-144-3p, miR-486-5p) was higher in osteoblastoma, while the miR-210 was elevated in osteosarcoma. Validation of these microRNAs on an independent data set of 22 tumor specimens by qPCR revealed that miR-210 is the most discriminating marker. This microRNA displays low levels of expression across all of the osteoblastoma specimens and robust expression in the majority of the osteosarcoma specimens. Application of these biomarkers to a clinical test case showed that these microRNA biomarkers permit re-classification of a misdiagnosed FFPE tumor sample from osteoblastoma to osteosarcoma. Our findings establish that the hypoxia-related miR-210 is a discriminatory marker that distinguishes between osteoblastoma and osteosarcoma. This discovery provides a complementary molecular approach to support pathological classification of two diagnostically challenging musculoskeletal tumors. Because miR-210 is linked to the cellular hypoxia response, its detection may be linked to well-established pro-angiogenic and metastatic roles of hypoxia in osteosarcomas and other tumor cell types. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1137-1146, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Extraskeletal osteosarcoma (ESOS) is an uncommon malignant neoplasm. Most ESOSs are high grade, although some low-grade cases have been reported. A few cases of ESOS with MDM2 amplification have also been reported, suggesting some similarity to skeletal low-grade osteosarcoma such as parosteal osteosarcoma, where MDM2 is often amplified. However, the frequency of low-grade cases and cases with MDM2 amplification among ESOSs remains unknown, and their relationship is unclear. To clarify this, we examined 18 primary ESOS cases clinically, pathologically, and genetically, focusing on their MDM2 amplification status. Our cases comprised 10 men and 8 women whose mean age was 58.6 years; the most common site of the lesion was the thigh and buttock. There were one histologically low-grade case evaluated by biopsy specimen with an aggressive course and 2 relatively low-grade cases whose lesions were of low grade for the most part. MDM2 amplification status was revealed by fluorescence in situ hybridization in all 18 cases; 2 patients-histologically intermediate- and high-grade cases-were found to have MDM2 amplification. In conclusion, this study indicates that histologically low-grade and relatively low-grade cases of ESOS are not always associated with MDM2 amplification. The ESOS case with MDM2 amplification could be high grade, although MDM2-amplified dedifferentiated liposarcoma with osteogenic differentiation should be ruled out in making the diagnosis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The potential prognostic and predictive value of p16 in high-grade localized osteosarcoma of the extremities has been recently investigated in small series of cases, and the results from different studies were somewhat controversial. A retrospective immunohistochemical analysis of p16 expression was performed in a series of 357 patients, included in different neoadjuvant chemotherapy protocols from 1986 to 2010, to explore its potential prognostic and predictive value. Immunohistochemistry was performed with a commercially available p16 monoclonal mouse antibody. Follow-up data were available in all cases with a median of 120 months. Positivity for p16 was detected in 70.6% (252/357) of cases. The p16 expression did not differ by age, sex, tumor site, histologic subtype, tumor volume, surgical margin, serum alkaline phosphatase levels, and lactate dehydrogenase levels. In the different chemotherapy protocols included, the incidence of p16 expression was similar. The absence of p16 expression was significantly associated with an adverse disease-free survival (P=.04) and overall survival (P=.05) when compared with the presence of p16 expression. At the multivariate Cox regression analysis, p16 expression lost its prognostic significance. Multivariate logistic regression analysis showed that as p16 expression was the only statistically significant parameter to predict the pathological response to neoadjuvant chemotherapy treatment with an odds ratio of 3.025 (P<.001) for "good" chemotherapy response. Our data indicate that the negative expression of p16 is associated with a reduced rate of good response to primary chemotherapy and to a worse probability of survival, although it was not confirmed as an independent prognostic biomarker after multivariate analysis., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Osteosarcoma (OS) is the most common bone sarcoma in adolescents, and has poor prognosis. A vicious cycle is established between OS cells and their microenvironment in order to facilitate the tumor growth and cell spreading. The present work aims to better characterize the tumor microenvironment in OS in order to identify new therapeutic targets relating to metastatic process. Tissue microarrays of pre-chemotherapy OS biopsies were used for characterizing the tumor niche by immunohistochemistry. Parameters studies included: immune cells (M1, M2-subtypes of tumor-associated macrophages (TAM); T, B lymphocytes; mast cells), vascularization (endothelial, perivascular cells), OPG, RANKL, and mitotic index. Two groups of patients were defined, 22 localized OS (OS Meta-) and 28 metastatic OS (OS Meta+). The OS Meta- group was characterized by a higher infiltration of INOS+ M1-polarizedmacrophages and upregulated OPG immunostaining. OS Meta+ tumors showed a significant increase in CD146+ cells. INOS+ M1-macrophages were correlated with OPG staining, and negatively with the presence of metastases. CD163+ M2-macrophages were positively correlated with CD146+ cells. In multivariate analysis, INOS and OPG were predictive factors for metastasis. An older age, non-metastatic tumor, good response to chemotherapy, and higher macrophage infiltration were significantly associated with better overall survival. TAMs are associated with better overall survival and a dysregulation of M1/M2 polarized-macrophages in favor of M1 subtype was observed in non-metastatic OS.

The present study examines the heating efficiency of a combination of manganese or cobalt ferrites in a binary (Co- or Mn-) ferrite nanoparticle form with magnetite, covered with citric acid to improve biocompatibility. The nanoparticle synthesis is based on the aqueous co-precipitation of proper salts, a facile, low-cost, environmentally friendly and high yield synthetic approach. By detailed structural and magnetic characterisation, the direct influence of structural and magnetic features on magnetic hyperthermia concludes to optimum heating efficiency. At a second stage, best performing magnetic nanoparticles undergo in vitro testing in three cell lines: one cancer cell line and two reference healthy cell lines. Both binary ferrite (MnFe2O4/Fe3O4 and CoFe2O4/Fe3O4) appear to be internalised and well tolerated by the cells while a versatile hyperthermia protocol is attempted in an effort to further improve their in vitro performance. Within this protocol, hyperthermia sequences are split in two runs with an intermediate 48 h time interval cell incubation stage while in each run a variable field mode (single or multiple pulses) is applied. Single-pulse field mode represents a typical hyperthermia application scheme where cells undergo the thermal shock continuously. On the other hand multiple-pulses mode refers to multiple, much shorter in duration AC field changes (field ON/OFFs), at each hyperthermia run, resulting eventually in high heating rate and much more harmful cell treatment. Consequently, we propose a novel series of improved performance heat mediators based on ferrite structures which show maximum efficiency at cancer cells when combined with a versatile multiple-pulse hyperthermia module.

We report 2 cases of primary dermal osteosarcoma. The patients were an 88-year-old man and a 72-year-old man complaining of masses occurring in the ear pavilion and the palm, deemed suspicious for basal cell carcinoma and metastatic colonic carcinoma, and were treated by resection. Microscopically, both featured a dermal lesion mostly composed of atypical spindle cells within a fibrous to hyaline matrix often showing mineralization. Osteoid material was rimmed by atypical tumor cells and was also associated with osteoclast-like giant cells. Tumor cells were positive for SATB2 and negative for markers of epithelial (low-molecular and high-molecular weight cytokeratins, epithelial membrane antigen, p63), melanocytic (S100 protein, HMB45, Melan A), and skeletal/smooth muscle differentiation (desmin, myogenin). No further therapy has been administered. Follow-up at 6 (case 1) and 8 months (case 2) was uneventful. A brief differential diagnosis discussing cutaneous tumors capable of showing osseous differentiation is summarized, along with a review of the pertinent literature. The specificity and sensitivity of SATB2 is also shortly addressed.

Primary cutaneous sweat gland carcinomas (SGCs) are rare tumors that commonly involve axillae, have a high local recurrence rate, and rarely show sarcomatoid transformation. A 68-year-old man presented with rapid enlargement of a previously stable, asymptomatic pea-sized nodule in the left axilla. Initial excision (with positive surgical margins) at another institution showed characteristic histologic features of a high-grade osteosarcoma and molecular analysis using a 92-gene real-time quantitative reverse transcription-polymerase chain reaction assay confirmed a diagnosis of osteosarcoma with 96% certainty. Notably, the molecular assay demonstrated consistently high relative expression of pannexin 3 (PANX3), a gene involved in normal osteoblast differentiation which, when highly expressed, strongly predicts osteosarcoma per the assay's algorithm. However, on further histologic review, the tumor also contained focal cystic areas, nests, and ducts composed of malignant epithelial cells reminiscent of SGC; these areas directly transitioned into the osteosarcomatous component and were strongly positive for pancytokeratin, CK7, and p63. Within 2 weeks, the lesion recurred and grew rapidly, prompting complete resection, histologic sections of which showed high-grade osteosarcoma without residual epithelial elements. This is the fifth report, to our knowledge, of osteosarcomatous transformation in a SGC, and the only report to date including molecular data. This case demonstrates that osteosarcoma arising from a SGC has a similar molecular profile to de novo primary osteosarcoma of bone. It also emphasizes the importance of histopathologic findings as the established diagnostic gold standard and the need to interpret molecular results within the clinical context.

Objective: To investigate the role of SATB2 in the pathological diagnosis and differential diagnosis of osteosarcoma., Methods: Immunostaining of SATB2 was performed in 47 cases of osteosarcomas, 5 osteoblastomas, 4 fibrous dysplasias, 5 myositis ossificans, 10 chondroblastomas, 8 chondrosarcomas, 5 Ewing sarcomas, 5 undifferentiated pleomorphic sarcomas, 6 fibrosarcomas and 2 leiomyosarcomas., Results: All osteoblastomas (5/5) and myositis ossificans (5/5), 83.0%(39/47) of osteosarcomas and 2/10 of chondroblastomas showed nuclear immunoreactivity for SATB2. SATB2 staining was negative in all cases of fibrous dysplasia, chondrosarcomas, Ewing sarcomas and all bone primary spindle cell sarcomas(undifferentiated pleomorphic sarcoma, fibrosarcoma and leiomyosarcoma)., Conclusion: SATB2 is a reliable osteoblastic marker for differential diagnosis of osteosarcoma and non-osteoid sarcoma, although with a limited role in separating osteosarcoma from non-malignant osteoblastic lesions.

Ezrin is a protein that functions as a cross-linker between actin cytoskeleton and plasma membrane. Its clinical role in osteosarcoma is unclear. The aim of this study was to investigate, in osteosarcoma, the prognostic value of ezrin expression at biopsy and changes in expression levels after preoperative chemotherapy. Thirty-eight newly diagnosed osteosarcoma patients aged 6-23 years were included. At diagnosis, 20 patients had localized disease, the others had distant metastases. Median follow-up was 75 months (range 13-135). Ezrin expression was assessed immunohistochemically in biopsy tissue and primary tumour specimens resected after chemotherapy. The influence on survival of changes in ezrin expression after chemotherapy was analysed. Ezrin expression was significantly higher after preoperative chemotherapy and changes compared to biopsy tissue were significantly lower in patients with early progression than in patients with relapse or no further evidence of disease (p = 0.006 and p = 0.002, respectively). Similarly, ezrin expression was higher after preoperative chemotherapy and exhibited less change in expression in deceased patients compared to patients surviving more than 5 years (both p = 0.001). Ezrin expression at biopsy was significantly associated with both histopathological aggressiveness (p < 0.001) and tumour size (p = 0.037). The results of this study provide evidence that changes in overexpression of ezrin due to preoperative chemotherapy could be a useful predictive and prognostic marker in patients with osteosarcoma.

The presence of greater than or equal to 90% necrosis after neoadjuvant chemotherapy is a favorable prognostic factor in osteosarcomas. A recent study using tissue microarrays of 40 conventional osteosarcomas showed that p16 expression independently predicted the necrotic response to neoadjuvant chemotherapy. In this study, we investigated this finding using whole sections in a larger group of osteosarcomas. Cases of 83 patients who had pretreatment biopsies and received neoadjuvant chemotherapy and surgical resection were collected from 3 reference hospital archives. Age, sex, tumor size, tumor subtype, location, and percentage of tumor necrosis were recorded; 4-μm sections from pretreatment biopsies were stained for p16. More than 30% strong nuclear staining was regarded as positive. The median age was 17 years (5-68 years), and male/female ratio was 2.3. The mean tumor diameter was 9.9 cm (2-30 cm). Tumors were most commonly of the osteoblastic type (60%) and located at the femur (47%). p16 positivity was seen in 66% of the patients. The median pathologic necrosis was 65%, and 39% of the patients responded favorably (≥%90 necrosis) to neoadjuvant therapy. In univariate analysis, p16 expression significantly correlated with greater than or equal to 90% response (P = .022). On multivariate analysis, p16 expression (odds ratio [OR], 7.71; P = .008), female sex (OR, 8.62; P = .006), and smaller tumor size (OR, 0.86; P = .023) were independent predictors of favorable response to neoadjuvant chemotherapy. We confirmed the finding that p16 expression predicts postchemotherapy necrotic response in conventional osteosarcomas., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In contrast to long bone osteosarcoma, mandibular osteosarcoma is highly heterogeneous and morphologically overlaps with benign tumors, obscuring diagnosis and treatment selection. Molecular characterization is difficult due to the paucity of available specimens of this rare disease. We aimed to characterize the spectrum of mandibular osteosarcoma using immunohistochemistry and molecular techniques (quantitative polymerase chain reaction and sequencing) and compare them with benign fibro-osseous lesions. Forty-nine paraffin-embedded mandible osteosarcoma tissue samples were collected retrospectively and compared with 10 fibrous dysplasia and 15 ossifying fibroma cases. These were analyzed for molecular markers thought to differ between the different diseases and subtypes: MDM2 (murine double-minute type 2) overexpression, GNAS (guanine nucleotide-binding protein/α subunit) mutations, and amplification of MDM2 and/or RASAL1 (RAS protein activator like 1). Five fibroblastic high-grade osteosarcoma subtypes showed MDM2 amplification, including 2 with a microscopic appearance of high-grade osteosarcoma with part low-grade osteosarcoma (differentiated/dedifferentiated osteosarcoma) and MDM2 overexpression. The other 3 contained a coamplification of MDM2 and RASAL1, a signature also described for juvenile ossifying fibroma, with no overexpression of MDM2. These were of the giant cell-rich high-grade osteosarcoma, with areas mimicking juvenile ossifying fibroma (ossifying fibroma-like osteosarcoma). Our results show that some diagnosed high-grade osteosarcomas are differentiated/dedifferentiated osteosarcomas and harbor an overexpression and amplification of MDM2. In addition, juvenile ossifying fibromas can potentially evolve into giant cell-rich high-grade osteosarcomas and are characterized by a RASAL1 amplification (osteosarcoma with juvenile ossifying fibroma-like genotype). Thus, the presence of a RASAL1 amplification in ossifying fibroma may indicate a requirement for closer follow-up and more aggressive management., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Although the function of hypoxia-inducible factor 1 (HIF1) in many kinds of solid tumor has been revealed, the significance of HIF1 in osteosarcoma is still controversial and not well understood., Methods: Immunohistochemistry was used to detect HIF1 expression. The correlation between HIF1 and clinicopathology factors was analyzed by use of chi-squared tests. The prognostic value of HIF1 was evaluated by univariate and multivariate analysis. Moreover, the function of HIF1 in osteosarcoma cells was further investigated in in-vitro experiments by regulating HIF1 and vascular endothelial growth factor-A (VEGF-A) expression., Results: Expression of HIF1 was high for 56.82 % of the samples in our investigation. HIF1 expression was significantly associated with positive metastasis (P = 0.037). By use of the Kaplan-Meier method, high expression of HIF1 was proved to be related to poorer overall survival (P = 0.007). By use of a Cox-regression model, HIF1 was identified as an independent prognostic biomarker (P = 0.019). We also proved that HIF1 can promote osteosarcoma invasion in hypoxia by inducing VEGF-A expression., Conclusions: HIF1 was identified as an independent prognostic biomarker in osteosarcoma. It can promote osteosarcoma cell invasion by inducing VEGF-A expression, indicating that HIF1 is a potential drug target in osteosarcoma.

Background: Trichorhinophalangeal syndrome 1 (Trps1) gene is a member of GATA transcription factor family and has an important function in tumorigenesis and progression. However, there are rare studies on its roles in carcinogenesis and prognostic significance in human osteosarcoma., Methods: The expression of Trps1 was detected by immunohistochemistry, and MVD was evaluated to determine the amounts of microvessels by counting CD31-positive endothelial cells., Results: Of the 74 cases that underwent study, Trps1-positive cases were 24. And it was associated with MVD significantly (P = 0.008). The data also exhibited more cases of remote metastasis (P = 0.013) and higher Enneking stage (P = 0.017) in Trps1-positive group compared to Trps1-negative group. Univariate analysis revealed that distant metastasis, MVD and Trps1 expression were associated with a lower 3-year overall survival rate and disease-free survival rate (P = 0.003, and P = 0.012 respectively). Furthermore, Trps1 and distant metastasis retained their significant prognostic effects on patients survival rate by multivariate analysis (P < 0.05)., Conclusions: Trps1 plays a crucial role in osteosarcoma angiogenesis, metastasis and clinical surgical stage. Trps1 can be a novel promising prognostic marker and therapeutic target, and antiangiogenic therapy which targets Trps1 molecule in patients with osteosarcoma may lead to improved prognosis and longer-term survival.

Vitronectin (Vn), a multifunctional adhesive protein, is found in association with tumor progression, angiogenesis and metastasis in a variety of (human) tumors. But no studies concerning its correlation to osteosarcoma prognosis were found. Hence, we aimed to investigate the prognostic value of Vitronectin (Vn) in osteosarcoma. Here, we studied the expression of VN in the tumor tissues from 67 patients with osteosarcoma and 20 patients with osteochondroma using immunohistochemistry and estimated the effects of VN expression in osteosarcoma on progression-free survival (PFS) and overall survival (OS) using the Kaplan-Meier curve and COX proportional hazards regression model. Increased expression of VN in osteosarcoma tissue compared to no VN expression in osteochondroma tissue was shown in immunohistochemical assay. No associations were observed between VN expression and osteosarcoma patients' gender (P = 0.675), age (P = 0.813), tumor size (P = 0.436), histologic subtype (P = 0.0.543) or tumor location (P = 0.456). Univariate survival analysis demonstrated significant correlations of high VN expression with shorter PFS (P = 0.002) and OS (P = 0.001); multivariate survival analysis revealed high VN expression as a significant independent prognostic indicator for shorter PFS (HR 2.788, P = 0.003) and OS (HR2.817, P = 0.003). In conclusion, the high expression of VN in tumor cells independently indicated poor clinical prognosis in patients with osteosarcoma, other than large tumor size and non-neoadjuvant chemoradiotherapy, suggesting that VN may serve as a potential therapeutic target in osteosarcoma.

Recently, we read the article "P16(INK4a) overexpression and survival in osteosarcoma patients: a meta analysis" by Jie Bu and his colleagues, published in the recent issue of International Journal Of Experimental Pathology. This research performed a meta-analysis to uncover the role of P16(INK4a) expression in overall survival rate in patients with osteosarcoma. The investigators concluded as follows: (i) the pateints with overexpression of P16(INK4a) had a longer overall survival rate than that with loss expression of P16(INK4a); (ii) P16(INK4a) was an effective biomarker of prognosis in patients with osteosarcoma.The findings are valubale and encouraging. However, some flaws and imperfections rooted in this work.

Introduction: Osteosarcoma is the most common primary malignant bone tumor, and the grading of osteosarcoma cells relies on traditional histopathology and molecular biology methods, which require RNA extraction, protein isolation and immunohistological staining. All these methods require cell isolation, lysis or fixation, which is time-consuming and requires certain amount of tumor specimen. In this study, we report the use of Raman spectroscopy for grading of malignant osteosarcoma cells., Methods: We demonstrate that, based on the detection of differential production of mineral species, Raman spectroscopy can be used as a live cell analyzer to accurately assess the grades of osteosarcoma cells by evaluating their mineralization levels. Mineralization level was assessed by measuring amount of hydroxyapatite (HA), which is highly expressed in mature osteoblasts, but not in poorly differentiated osteosarcoma cell or mesenchymal stem cells, the putative cell-of-origin of osteosarcoma., Results: We found that under Raman spectroscopy, the level of HA production was high in MG-63 cells, which are low-grade. Moreover, hydroxyapatite production was low in high-grade osteosarcoma cells such as 143B and SaOS2 cells (p < 0.05). Matrix metalloproteinase MMP2, MMP9 were highly expressed in SaOS2, 143B and MSCs and decreased in human fetal osteoblast (FOB) and MG-63 cells as expected (p < 0.05). These results may highlight the inverse correlation between HA level and prognosis of osteosarcoma., Conclusions: The use of Raman spectroscopy for the measurement of HA production by the protocol reported in this study may serve as a useful tool to rapidly and accurately assess the degree of malignancy in osteosarcoma cells in a label-free manner. Such application may shorten the period of pathological diagnosis and may benefit patients who are inflicted with osteosarcoma.

Background/aim: It is known that the protein-unbound fraction (fp) of warfarin fluctuates in the plasma of cancer patients and the fluctuation of fp is correlated with albumin concentration. However, this mechanism remains unclear. The present study was performed with the objective of elucidating variations in the protein-binding rate of specific drugs in the presence of cancer, as well as the mechanisms involved., Materials and Methods: Experiments were performed using Fisher 344 model rats, that we have used in previous studies. A single i.v. injection of cefazolin (CEZ) 20 mg/kg or ibuprofen (IB) 10 mg/kg was administered to both tumor-bearing and control groups. We compared relevant pharmacokinetics. Purified albumin was checked for purity with SDS-PAGE and was used in experiments on protein binding of CEZ and IB., Results: The fp of CEZ in plasma from the tumor-bearing group increased approximately 2.9-fold and the fp of IB also increased about 2.7-fold. For that reason, we purified albumin from plasma and examined its spectroscopic signature. We showed that conformational changes (i.e., decreases in the hydrophobic region, increases in endogenous tryptophan fluorescence intensity and decrease in α-helical content) had occurred in albumin in the tumor-bearing group., Conclusion: In the present study, we confirmed that, in a state of cancer morbidity, fp was elevated for site I high-affinity CEZ and site II high-affinity IB. Moreover, our findings indicated that, as a cause of those elevations, a decrease in the albumin concentration and conformational changes due to an oxidation are involved., (Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Labeling internal structures within living cells with standard fluorescent probes is a challenging problem. Here, we introduce a novel intracellular staining method that enables us to carefully control the labeling process and provides instant access to the inner structures of living cells. Using a hollow glass capillary with a diameter of <100 nm, we deliver functionalized fluorescent probes directly into the cells by (di)electrophoretic forces. The label density can be adjusted and traced directly during the staining process by fluorescence microscopy. We demonstrate the potential of this technique by delivering and imaging a range of commercially available cell-permeable and nonpermeable fluorescent probes to cells.

Aim: We studied ERG expression in a large series of chondrogenic bone and soft tissue tumours to assess the value of ERG as a possible marker of chondroid/cartilaginous differentiation., Methods: Formalin-fixed, paraffin-embedded whole sections from 111 bone and soft tissue tumours with chondroid differentiation or a morphology that may mimic cartilaginous differentiation were retrieved. Immunohistochemistry was performed using anti-ERG monoclonal antibody directed against the N terminus. Nuclear staining was scored as negative (<5%), 1+ (5%-25%), 2+ (26%-50%), 3+ (>51%)., Results: Nuclear ERG expression was seen in all cases of soft tissue chondroma (8), chondromyxoid fibroma (7), chondroblastic osteosarcoma (6) and clear cell chondrosarcoma (1). 10/12 conventional chondrosarcomas were also positive for ERG. In cases of dedifferentiated chondrosarcoma, the well-differentiated component was positive in 7/9 cases, while all dedifferentiated foci were negative. In cases of mesenchymal chondrosarcoma, the hyaline cartilage component was positive in 2/4 cases, whereas the primitive component in all cases was negative. Variable positivity was identified in extraskeletal myxoid chondrosarcomas (4/9), chondroblastomas (3/8) and mixed tumours/myoepitheliomas (2/11). Only 1/12 chordoma was positive for ERG (1+). Interestingly, 15/17 enchondromas were negative for ERG., Conclusions: In this study, we further characterise the expression of ERG in mesenchymal tumours and found relatively constant nuclear ERG expression in selected chondrogenic tumours including conventional chondrosarcoma, chondromyxoid fibroma, chondroblastic osteosarcoma and clear cell chondrosarcoma. We also show that ERG may be a helpful ancillary tool in certain select diagnostic scenarios and that awareness of ERG expression in tumours with cartilaginous differentiation is important., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Introduction: This study aims to assess the degree of concordance of histological diagnosis of bone and soft tissue sarcomas between a Comprehensive Cancer Center (CCC) of Eastern Europe - not specialized in this area of pathology - and an important CCC of Western Europe, which is one of the coordinators of a clinical reference network in sarcoma pathology. The goal is to have an overview of the sarcomatous pathology in a region of Eastern Europe and to discover diagnostic discrepancies between the two centers, while determining their cause., Materials and Methods: The initial diagnosis was compared with the revised diagnosis on 110 specimens from 88 patients with bone or soft tissue sarcomas from East-European CCC, in a one-year period of time., Results: Complete diagnostic agreement was observed in 55 cases (62.5%), a partial agreement in 23 cases (26.1%) and a major disagreement in 10 cases (11.4%). Major discrepancies of the histological type was observed in only 3 cases (3.4%): one case of discordance benign/malignant and 2 cases of discordance mesenchymal/non mesenchymal. Minor histological discrepancies - not affecting the management of the patient - were observed in 18 cases (20.4%). A major discordance in grading - potentially changing the management of the patient - was noted in 7 cases (7.9%), and a minor discrepancy in 5 cases (5.7%)., Discussions: Some histological types were clearly overdiagnosed, like "adult fibrosarcomas" and "malignant peripheral nerve sheet tumors" (MPNST), mostly converted after the audit into "undifferentiated spindle cell sarcomas" or other types of sarcomas. Some "unclassified" sarcomas and "undifferentiated pleomorphic sarcomas" could be re-classified with the aid of an extensive panel of antibodies. Overall, immunohistochemistry was responsible, but not in exclusivity, for half of the minor discrepancies, and for 2 out of 3 cases of major histological discrepancies. Otherwise, the main cause of discrepancies was the difficulties in the interpretation of the morphology. Molecular biology was decisive in one case. Most grading discrepancies resulted from the appreciation of the mitotic index., Conclusions: The profile of the sarcomatous pathology in the northwest region of Romania does not appear to differ significantly from other parts of Europe or the world, but a prospective epidemiological study would be necessary to confirm this assessment. The expansion of immunohistochemical antibody panel, the over-specialization of pathologists and, in the future, the establishment of a national network of referral centers in sarcoma pathology, are required for a high level of histological diagnosis in Eastern Europe. A periodic external audit, continuing this trans-European collaboration between the two centers, would be beneficial for monitoring progress., (Copyright © 2014. Published by Elsevier Masson SAS.)

Background: The survival of patients who present with nonmetastatic extremity osteosarcoma has dramatically improved, but there are some patients who do not respond to chemotherapy. The ability to identify patients with a poorer prognosis might allow us to target different therapy for these patients. Glucose transporter protein-1 (Glut-1), one of the key factors in glucose metabolism, has been reported to be an independent prognostic factor in various tumors. However, little is known about the role of the Glut-1 pathway in osteosarcoma., Questions/purposes: We asked (1) if Glut-1 expression is a prognostic marker for survival in patients with osteosarcoma, and (2) if there is a relationship between Glut-1 expression and tumor angiogenesis., Patients and Methods: Thirty-seven patients with resectable high-grade osteosarcomas treated between 1982 and 2007 were reviewed retrospectively. Patients were excluded if representative biopsy material and followup data were not available. The expression of Glut-1 and the number of CD34-positive microvessels for angiogenic activity were measured immunohistochemically. The median followup was 6 years 6 months (range, 11-211 months). Survival analyses were evaluated using the Kaplan-Meier method and the Cox proportional hazards model. The association between Glut-1 expression and microvessel density was analyzed using Student's t-test and chi-square test. For 12 (32.4%) of 37 patients with osteosarcoma, the expression of Glut-1 was positive, with four patients (10.8%) showing strong expression of Glut-1 protein., Results: The expression of Glut-1 correlated with a shorter disease-free survival period (relative risk, 20.13; 95% CI, 1.77-229.3; p=0.0016). The microvessel density mean value of positive Glut-1 expression (mean±SD, 26.5±19.4) was lower than that of negative expression (mean±SD, 46.4±35.3; Student's t-test, p=0.038). When more than 50 was defined as a high microvessel density, positive expression of Glut-1 was significantly associated with low microvessel density (chi-square test, p=0.049)., Conclusions: These findings indicate that Glut-1 is a potential predictor of survival in patients with osteosarcoma and that glucose metabolism may be negatively associated with angiogenesis. If substantiated in larger numbers of patients, these findings might stimulate the development of novel treatments for patients with a poorer prognosis., Level of Evidence: Level III, prognostic study. See the Instructions for Authors for a complete description of levels of evidence.

Tumor suppressor gene p53 functions as the guardian of the human genome and mutations in p53 contribute to cancer development. However, studies that investigated the potential of p53 as a prognostic marker in osteosarcoma patients have yielded inconclusive results. Based on recommendation of the Cochrane Collaboration, this meta-analysis was conducted using data from the 17 published studies to evaluate the association of p53 alterations with clinical outcome of osteosarcoma patients. Different databases, including MEDLINE, PsycINFO, Scopus, EMBASE, and the Cochrane Central Register of Controlled Trials (CENTRAL) were searched. Prognostic value of p53 alterations was determined by risk ratio (RR). The data showed that p53-positive immunostaining tended to associate with decreased 2-year survival rates (RR, 1.94; 95% CI, 1.43 to 2.64; p < 0.0001, I(2) = 10%). However, the prediction value of RR was smaller with p53 expression than with p53 mutations. Moreover, patients who received neoadjuvant chemotherapy and surgery tended to have a stronger association between p53-positive staining and 2-year mortality compared to the patients treated with surgery only. However, p53-positive staining was not associated with 3-year (RR, 1.64; 95% CI, 0.84 to 3.20; P = 0.15; I(2) = 56%) and 5-year survival (RR, 1.25; 95% CI, 0.78 to 2.01; P = 0.36; I(2) = 70%). The data from the current study suggest that p53-positive osteosarcoma only predicted a decreased short-term survival rate, but not 3- or 5-year survival.

Osteosarcoma is one of the most common primary bone malignancies. Although there is a significant improvement of survival on osteosarcoma patients in the past decades, treatment of osteosarcoma is still unsatisfactory for the development of pulmonary metastasis. The potential prognostic value of p16(INK4a) in osteosarcoma has been investigated, however, the results from different studies were somewhat controversial. To elucidate whether p16(INK4a) is indeed a prognostic factor of osteosarcoma, we conducted a meta-analysis of the published literatures to provide a comprehensive evaluation of the significance of p16(INK4a) expression in patients with osteosarcoma. Eight studies with a total of 354 patients with osteosarcoma were examined. The pooled odds ratio (OR) with corresponding 95% confidence interval (95% CI) was calculated to evaluate the effect of p16(INK4a) expression on overall survival. Meta-analysis showed that patients with high p16(INK4a) expression were significantly associated with favourable overall survival when compared to their counterparts with low or undetectable p16(INK4a) expression (OR = 0.270, 95% CI 0.162-0.451, P < 0.001). Sensitivity analysis suggested the pooled OR was stable and not significantly changed when a single study was removed. In conclusion, the results from this meta-analysis highlight that p16(INK4a) is an effective biomarker of survival in patients with osteosarcoma.

Background: It is hardly possible to define osteosarcoma (OS) patients at greatest risk for non-response to chemotherapy, metastasis and short survival times. Our goal was the investigation of local expression of insulin-like growth factor (IGF-1) with regard to survival time of OS patients using a tissue microarray (TMA)., Materials and Methods: Tumor tissue specimens from surgical primary tumor resections were collected from patients with OS. A TMA was composed, sections were stained with rabbit anti-IGF-1 and grading was performed. Statistics involved Kaplan-Meier curves and the log-rank test., Results: We analyzed immunohistochemical expression of local IGF-1 on a TMA based on surgical primary tumor resections of 67 OS patients. The mean clinical follow-up time was 98 months. Twenty-two (33%) OS patients stained negatively and 44 (66%) OS patients stained positively for IGF-1. Significantly shorter survival was detected with expression of IGF-1 (p=0.007). The 5-year survival rate for patients expressing IGF-1 was 63% compared to 92% in patients without expression of IGF-1. Non-responders to chemotherapy and patients with metastasis, who also stained positively for IGF-1 manifested a significantly (p=0.002 and p<0.0001, respectively) shorter survival., Conclusion: Expression of local IGF-1 in primary tumor tissue appears to significantly affect the aggressiveness of OS, may predict survival time and, above all, may discriminate patients with non-response to chemotherapy and metastasis. This represents the basis for successful patient selection with regard to the decision process for or against chemotherapy and the choice of the most effective therapeutic drug. It may be a more important marker of tumor progression and indicator of prognosis than serum IGF-1. Novel tumor markers and therapeutic agents targeting the local IGF-1 pathway may increase the likelihood of therapeutic success., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Survival rates have plateaued at 70% for osteosarcoma. Proteins ectopically produced by malignant tumors may provide insight into new therapeutic targets. Osteosarcomas secreting human chorionic gonadotropin (hCG) have been suggested to have a worse prognosis. We examined the frequency of expression of β-subunit of hCG (β-hCG) in pretreatment osteosarcoma biopsies, and asked if it was associated with various clinical prognostic parameters, and the development of metastases. We subjected 51 pretreatment biopsies of high-grade osteosarcoma, from 51 patients, to β-hCG immunohistochemistry. In 19 of these patients, postchemotherapy metastatic biopsies also were examined for β-hCG expression. Clinical information (patient age, sex, survival status, and serum hCG in females only), and tumor characteristics (site, size, and presence of metastases) were recorded. The β-hCG positive and negative biopsies were separated and compared. Of 49 interpretable pretreatment biopsies, 28 (57%) showed positive cytoplasmic β-hCG expression: 27 with sparse positivity (1% of tumor cells) and 1 with frequent positivity (10% of tumor cells). The patient with frequent β-hCG positivity in her pretreatment biopsy had elevated serum hCG (88.2 mIU/mL) at diagnosis, decreasing to undetectable following chemotherapy and definitive resection. There was no difference in clinical parameters or rate of metastasis between β-hCG positive versus negative groups. Expression of β-hCG may be seen in high-grade osteosarcoma, but frequent β-hCG immunohistochemical expression by tumor cells, associated with clinically elevated serum β-hCG, is rare. Recognition that some nongerm cell tumors may produce β-hCG can prevent confusion with malignancies containing neoplastic syncytiotrophoblast cells, including germ cell and trophoblastic tumors.

Bone tumors are very rare. Diagnosis and treatment is an interdisciplinary task for experienced radiologists, pathologist, and surgeons that is ideally performed in specialized centers. For optimal processing of bone specimens, basic laboratory equipment and special techniques are required. The cornerstone of the histological diagnosis remains H&E staining, supplemented by special stains, immunohistochemistry, and molecular techniques. For an appropriate diagnosis, data on clinical history, age, location, topography within bone, and imaging are required. Major differences between histological and radiological diagnosis have to be clarified before starting treatment (e.g., by involving a reference registry).

The possibility to measure boron concentration with high precision in tissues that will be irradiated represents a fundamental step for a safe and effective BNCT treatment. In Pavia, two techniques have been used for this purpose, a quantitative method based on charged particles spectrometry and a boron biodistribution imaging based on neutron autoradiography. A quantitative method to determine boron concentration by neutron autoradiography has been recently set-up and calibrated for the measurement of biological samples, both solid and liquid, in the frame of the feasibility study of BNCT. This technique was calibrated and the obtained results were cross checked with those of α spectrometry, in order to validate them. The comparisons were performed using tissues taken form animals treated with different boron administration protocols. Subsequently the quantitative neutron autoradiography was employed to measure osteosarcoma cell samples treated with BPA and with new boronated formulations., (© 2013 Published by Elsevier Ltd.)

Several studies have focused on the relationships between the expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and the prognosis of patients with malignant tumors. However, few of these have investigated the expression of EMMPRIN in osteosarcoma. We examined expression levels of EMMPRIN immunohistochemically in 53 cases of high-grade osteosarcoma of the extremities and analyzed the correlation of its expression with patient prognosis. The correlation between matrix metalloproteinases (MMPs) and EMMPRIN expression and the prognostic value of co-expression were also analyzed. Staining positivity for EMMPRIN was negative in 7 cases, low in 17, moderate in 19, and strong in 10. The overall and disease-free survivals (OS and DFS) in patients with higher EMMPRIN expression (strong-moderate) were significantly lower than those in the lower (weak-negative) group (0.037 and 0.024, respectively). In multivariate analysis, age (P=0.004), location (P=0.046), and EMMPRIN expression (P=0.038) were significant prognostic factors for overall survival. EMMPRIN expression (P=0.024) was also a significant prognostic factor for disease-free survival. Co-expression analyses of EMMPRIN and MMPs revealed that strong co-expression of EMMPRIN and membrane-type 1 (MT1)-MMP had a poor prognostic value (P=0.056 for DFS, P=0.006 for OS). EMMPRIN expression and co-expression with MMPs well predict the prognosis of patients with extremity osteosarcoma, making EMMPRIN a possible therapeutic target in these patients.