A potential diagnostic marker for osteosarcoma: hsa_circ_0005721.

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.

Anti-neoplastic characteristics and potential targets of calycosin against bisphenol A-related osteosarcoma: bioinformatics analysis.

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].

Usefulness of ß-catenin expression in the differential diagnosis of osteosarcoma, osteoblastoma, and chondroblastoma.

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.

Ethanolic extract of gabirobeira (Campomanesia adamantium) leaves reduces proliferation of osteosarcoma cells in vitro

Osteosarcoma is the most commonly diagnosed malignant bone tumor in humans, with a higher incidence in children and young people. It is highly aggressive and has a high metastatic potential. Its treatment is based on both chemotherapy and surgical intervention. However, currently used chemotherapeutic agents, such as doxorubicin, have several adverse effects on the patient. Therefore, there is a growing demand for new chemotherapeutic agents that stimulate new researches, such as those involving compounds extracted from plants, such as the gabirobeira. In this study, we aimed to evaluate the cytotoxic effects of ethanolic extract, both crude and ethyl acetate, of gabirobeira leaves on osteosarcoma cells in vitro. Cytotoxicity was evaluated using the Trypan blue exclusion method and the IC50 values were calculated using the tetrazolium reduction method. The ethanolic extract of gabirobeira leaves showed a cytotoxic effect on osteosarcoma cells in vitro. The group treated with the crude extract at 1. 0& 956;L mL-1 concentration for 48 hours showed higher cytotoxicity and the lowest IC50 value for this extract was found in the 24 to 48 hours interval. The ethanolic extract of gabirobeira leaves is cytotoxic for osteosarcoma cells.

Clinical significance and in vitro cellular regulation of hypoxia mimicry on HIF-1α and downstream genes in canine appendicular osteosarcoma.

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 (CoCl2). 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.

Quantitative Lipidomic Analysis of Osteosarcoma Cell-Derived Products by UHPLC-MS/MS.

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 Spectral Imaging Study of DAPT, a γ-secretase Inhibitor, Induced Physiological and Biochemical Reponses in Osteosarcoma Cells.

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.

Proteomic profiling and identification of significant markers from high-grade osteosarcoma after cryotherapy and irradiation.

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.

Parathyroid hormone receptor 1 (PTHR1) is a prognostic indicator in canine osteosarcoma.

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.

PD-1 and PD-L1 Expression in Osteosarcoma: Which Specimen to Evaluate?

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.

Clinicopathological and prognostic values of fibronectin and integrin αvß3 expression in primary osteosarcoma.

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 αvß3 in soft-tissue sarcoma promotes cell migration, invasion, and lung metastasis. This study aimed to investigate the prognostic significance of FN and αvß3 in osteosarcoma. METHODS: Immunohistochemistry and western blotting were used to detect the expression of FN and αvß3 in 60 osteosarcoma specimens and in 30 osteochondroma specimens. Furthermore, correlations of FN and αvß3 with the clinicopathological features of osteosarcoma patients were analyzed using the χ2 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 αvß3 (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 αvß3 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 αvß3 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 αvß3 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.

Label-Free Volumetric Quantitative Imaging of the Human Somatic Cell Division by Hyperspectral Coherent Anti-Stokes Raman Scattering.

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.

Untangling the response of bone tumor cells and bone forming cells to matrix stiffness and adhesion ligand density by means of hydrogels.

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.

Raman spectroscopy of osteosarcoma cells.

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.

Elevated Expression of Transforming Acidic Coiled-Coil Containing Protein 3 (TACC3) Is Associated With a Poor Prognosis in Osteosarcoma.

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.

Fluorescent In Situ Hybridization for TP53 in the Diagnosis of Pediatric Osteogenic Sarcoma.

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.

[Detection and clinical significance of circulating tumor cells in osteosarcoma using immunofluorescence combined with in situ hybridization].

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.

Assessment of programmed death-ligand 1 expression and tumor-associated immune cells in pediatric cancer tissues.

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.

Interobserver Reproducibility Among Gynecologic Pathologists in Diagnosing Heterologous Osteosarcomatous Component in Gynecologic Tract Carcinosarcomas.

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.