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Background Deregulation of microRNA (miRNA) transcript levels has been observed in many types of tumors including osteosarcoma. Molecular pathways regulated by differentially expressed miRNAs may contribute to the heterogeneous tumor behaviors observed in naturally occurring cancers. Thus, tumor-associated miRNA expression may provide informative biomarkers for disease outcome and metastatic potential in osteosarcoma patients. We showed previously that clusters of miRNAs at the 14q32 locus are downregulated in human osteosarcoma. Methods Human and canine osteosarcoma patient’s samples with clinical follow-up data were used in this study. We used bioinformatics and comparative genomics approaches to identify miRNA based prognostic biomarkers in osteosarcoma. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance. Results Here we show that an inverse correlation exists between aggressive tumor behavior (increased metastatic potential and accelerated time to death) and the residual expression of 14q32 miRNAs (using miR-382 as a representative of 14q32 miRNAs) in a series of clinically annotated samples from human osteosarcoma patients. We also show a comparable decrease in expression of orthologous 14q32 miRNAs in canine osteosarcoma samples, with conservation of the inverse correlation between aggressive behavior and expression of orthologous miRNA miR-134 and miR-544. Conclusions We conclude that downregulation of 14q32 miRNA expression is an evolutionarily conserved mechanism that contributes to the biological behavior of osteosarcoma, and that quantification of representative transcripts from this family, such as miR-382, miR-134, and miR-544, provide prognostic and predictive markers that can assist in the management of patients with this disease.

Rhabdomyosarcomas with TFCP2 fusions represent an emerging subtype of tumors, initially discovered by RNA-sequencing. We report herein the clinicopathological, transcriptional, and genomic features of a series of 14 cases. Cases were retrospectively and prospectively recruited and studied by immunohistochemistry (MYF4, MYOD1, S100, AE1/E3, ALK), fluorescence in situ hybridization with TFCP2 break-apart probe ( n  = 10/14), array-comparative genomic hybridization (Agilent), whole RNA-sequencing (Truseq Exome, Illumina), or anchored multiplex PCR-based targeted next-generation sequencing (Archer® FusionPlex® Sarcoma kit). Patient’s age ranged between 11 and 86 years, including 5 pediatric cases. Tumors were located in the bone ( n  = 12/14) and soft tissue ( n  = 2/14). Most bone tumors invaded surrounding soft tissue. Craniofacial bones were over-represented ( n  = 8/12). Median survival was 8 months and five patients are currently alive with a median follow-up of 20 months. Most tumors displayed a mixed spindle cell and epithelioid pattern with frequent vesicular nuclei. All tumors expressed keratins and showed a rhabdomyogenic phenotype (defined as expression of MYF4 and/or MYOD1). ALK was overexpressed in all but three cases without underlying ALK fusion on break-apart FISH ( n  = 5) nor next-generation sequencing ( n  = 14). ALK upregulation was frequently associated with an internal deletion at genomic level. TFCP2 was fused in 5′ either to EWSR1 ( n  = 6) or FUS ( n  = 8). EWSR1 was involved in both soft tissue cases. FISH with TFCP2 break-apart probe was positive in all tested cases ( n  = 8), including one case with unbalanced signal. On array-CGH, all tested tumors displayed complex genetic profiles with genomic indexes ranging from 13 to 107.55 and recurrent CDKN2A deletions. FET-TFCP2 rhabdomyosarcomas clustered together and distinctly from other rhabdomyosarcomas subgroups. Altogether, our data confirm and expand the spectrum of the new family of FET-TFCP2 rhabdomyosarcomas, which are associated with a predilection for the craniofacial bones, an aggressive course, and recurrent pathological features. Their association with ALK overexpression might represent a therapeutic vulnerability.

Osteosarcomas are the most common primary malignant tumors of bone and show multiple and complex genomic aberrations. miRNAs are non-coding RNAs capable of regulating gene expression at the post transcriptional level, and miRNAs and their target genes may represent novel therapeutic targets or biomarkers for osteosarcoma. In order to investigate the involvement of miRNAs in osteosarcoma development, global microarray analyses of a panel of 19 human osteosarcoma cell lines was performed. We identified 177 miRNAs that were differentially expressed in osteosarcoma cell lines relative to normal bone. Among these, miR-126/miR-126*, miR-142-3p, miR-150, miR-223, miR-486-5p and members of the miR-1/miR-133a, miR-144/miR-451, miR-195/miR-497 and miR-206/miR-133b clusters were found to be downregulated in osteosarcoma cell lines. All miRNAs in the paralogous clusters miR-17-92, miR-106b-25 and miR-106a-92 were overexpressed. Furthermore, the upregulated miRNAs included miR-9/miR-9*, miR-21*, miR-31/miR-31*, miR-196a/miR-196b, miR-374a and members of the miR-29 and miR-130/301 families. The most interesting inversely correlated miRNA/mRNA pairs in osteosarcoma cell lines included miR-9/TGFBR2 and miR-29/p85α regulatory subunit of PI3K. PTEN mRNA correlated inversely with miR-92a and members of the miR-17 and miR-130/301 families. Expression profiles of selected miRNAs were confirmed in clinical samples. A set of miRNAs, miR-1, miR-18a, miR-18b, miR-19b, miR-31, miR-126, miR-142-3p, miR-133b, miR-144, miR-195, miR-223, miR-451 and miR-497 was identified with an intermediate expression level in osteosarcoma clinical samples compared to osteoblasts and bone, which may reflect the differentiation level of osteosarcoma relative to the undifferentiated osteoblast and fully differentiated normal bone. This study provides an integrated analysis of miRNA and mRNA in osteosarcoma, and gives new insight into the complex genetic mechanisms of osteosarcoma development and progression.

Bone tumours are difficult to diagnose and treat, as they are rare and over 60 different subtypes are recognised. The emergence of next-generation sequencing has partly elucidated the molecular mechanisms behind these tumours, including the group of bone forming tumours (osteoma, osteoid osteoma, osteoblastoma and osteosarcoma). Increased knowledge on the molecular mechanism could help to identify novel diagnostic markers and/or treatment options. Osteoid osteoma and osteoblastoma are bone forming tumours without malignant potential that have overlapping morphology. They were recently shown to carry FOS and—to a lesser extent—FOSB rearrangements suggesting that these tumours are closely related. The presence of these rearrangements could help discriminate these entities from other lesions with woven bone deposition. Osteosarcoma is a malignant bone forming tumour for which different histological subtypes are recognised. High-grade osteosarcoma is the prototype of a complex karyotype tumour, and extensive research exploring its molecular background has identified phenomena like chromothripsis and kataegis and some recurrent alterations. Due to lack of specificity, this has not led to a valuable novel diagnostic marker so far. Nevertheless, these studies have also pointed towards potential targetable drivers of which the therapeutic merit remains to be further explored.

Cancer cell lines represent in vitro models for studying malignancies, general cell biology, drug discovery and more. Whether they can be considered as exact representative models of the parental tumors remains uncertain given the acquisition of additional ex vivo changes of the cells and the lack of tissue architecture and stroma. Previously, within the EuroBoNeT consortium, we characterized a collection of bone sarcoma cell lines on genomic and proteomic level. Here, we address the phenotypical and functional characterization of the unique set of osteosarcoma cell lines (n=19) in vitro and in vivo. For functional analysis of differentiation capacity, cells were stimulated towards osteoblasts, adipocytes and chondrocytes. Furthermore, all cell lines were injected subcutaneously and intramuscularly into nude mice to assay their in vivo tumor formation capacity as well as for phenotypical analysis of the tumors. All formed tumors were further characterized histologically and immunohistochemically. Out of 19 cell lines, 17 (89%) showed adipogenic differentiation, 13/19 (68%) could differentiate towards osteoblasts and in 6/19 (32%) cell lines chondrogenic differentiation was evident. About half of the cell lines (8/19, 42%) produced tumors in vivo after subcutaneous and intramuscular injections. Several cell lines showed invasion into adjacent tissues and one tumor developed several lung metastases. The use of cell lines, especially in cancer research, is of paramount importance. Here, we identify comprehensively characterized osteosarcoma cell lines, which robustly represent clinical osteosarcoma providing researchers useful in vitro and in vivo models to study the genetics and functional characteristics of this highly malignant neoplasm.

Background As major regulators of normal chondrogenesis, the bone morphogenic protein (BMP) and transforming growth factor β (TGFB) signaling pathways may be involved in the development and progression of central chondrosarcoma. In order to uncover their possible implication, the aim of this study was to perform a systematic quantitative study of the expression of BMPs, TGFBs and their receptors and to assess activity of the corresponding pathways in central chondrosarcoma. Methods Gene expression analysis was performed by quantitative RT-PCR in 26 central chondrosarcoma and 6 healthy articular cartilage samples. Expression of endoglin and nuclear localization of phosphorylated Smad1/5/8 and Smad2 was assessed by immunohistochemical analysis. Results The expression of TGFB3 and of the activin receptor-like kinase ALK2 was found to be significantly higher in grade III compared to grade I chondrosarcoma. Nuclear phosphorylated Smad1/5/8 and Smad2 were found in all tumors analyzed and the activity of both signaling pathways was confirmed by functional reporter assays in 2 chondrosarcoma cell lines. Immunohistochemical analysis furthermore revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other. Conclusions The BMP and TGFβ signaling pathways were found to be active in central chondrosarcoma cells. The correlation of Smad1/5/8 activity to endoglin expression suggests that, as described in other cell types, endoglin could enhance Smad1/5/8 signaling in high-grade chondrosarcoma cells. Endoglin expression coupled to Smad1/5/8 activation could thus represent a functionally important signaling axis for the progression of chondrosarcoma and a regulator of the undifferentiated phenotype of high-grade tumor cells.

Osteosarcoma is a high-grade bone-forming neoplasm, with a complex genome. Tumours frequently show chromothripsis, many deletions, translocations and copy number alterations. Alterations in the p53 or Rb pathway are the most common genetic alterations identified in osteosarcoma. Using spontaneously transformed murine mesenchymal stem cells (MSCs) which formed sarcoma after subcutaneous injection into mice, it was previously demonstrated that p53 is most often involved in the transformation towards sarcomas with complex genomics, including osteosarcoma. In the current study, not only loss of p53 but also loss of p16Ink4a is shown to be a driver of osteosarcomagenesis: murine MSCs with deficient p15Ink4b, p16Ink4a, or p19Arf transform earlier compared to wild-type murine MSCs. Furthermore, in a panel of nine spontaneously transformed murine MSCs, alterations in p15Ink4b, p16Ink4a, or p19Arf were observed in eight out of nine cases. Alterations in the Rb/p16 pathway could indicate that osteosarcoma cells are vulnerable to CDK4/CDK6 inhibitor treatment. Indeed, using two-dimensional (n = 7) and three-dimensional (n = 3) cultures of human osteosarcoma cell lines, it was shown that osteosarcoma cells with defective p16INK4A are sensitive to the CDK4/CDK6 inhibitor palbociclib after 72-hour treatment. A tissue microarray analysis of 109 primary tumour biopsies revealed a subset of patients (20–23%) with intact Rb, but defective p16 or overexpression of CDK4 and/or CDK6. These patients might benefit from CDK4/CDK6 inhibition, therefore our results are promising and might be translated to the clinic. Osteosarcoma is a tumour with a highly complex genome, which hampers the identification of driver genes. Using a model of murine mesenchymal stem cells (MSCs) with deficient p15Ink4b, p16Ink4a, or p19Arf that transform earlier compared to wild-type MSCs, the authors demonstrated that loss of p16Ink4a is a driver of osteosarcomagenesis. This can be exploited with a CDK4/CDK6 inhibitor, as osteosarcoma cells showed sensitivity to palbociclib which might be used as a novel therapeutic option.

Sarcomas are a rare group of tumors of mesenchymal origin. Metastatic sarcomas are often difficult to treat and unresponsive to standard radio- and chemotherapy, resulting in a poor survival rate for patients. Novel treatments with immune checkpoint inhibitors have been proven to prolong survival of patients with a variety of cancers, including metastatic melanoma, lung, and renal cell carcinoma. Since immune checkpoint inhibitors could provide a novel treatment option for patients with sarcomas, clinical trials investigating their efficacy in these group of tumors are ongoing. However, the discrimination of patients that are the most likely to respond to these treatments is still an obstacle in the design of clinical trials. In this review, we provide a brief overview of the mechanisms of action of immune checkpoint inhibitors and discuss the proposed biomarkers of therapy response, such as lymphocytic infiltration, intratumoral PD-L1 expression, and mutational load in sarcomas.

IntroductionChondrosarcoma is a malignant cartilage-forming bone tumour in which mutations in IDH1 and IDH2 frequently occur. Previous studies suggest an increased dependency on glutaminolysis in IDH1/2 mutant cells, which resulted in clinical trials with the drugs CB-839, metformin and chloroquine. In this study, the preclinical rationale for using these drugs as a treatment for chondrosarcoma was evaluated.MethodsExpression of glutaminase was determined in 120 cartilage tumours by immunohistochemistry. Ten chondrosarcoma cell lines were treated with the metabolic compounds CB-849, metformin, phenformin (lipophilic analogue of metformin) and chloroquine.ResultsA difference in glutaminase expression levels between the different tumour grades (p = 0.001, one-way ANOVA) was identified, with the highest expression observed in high-grade chondrosarcomas. Treatment with CB-839, metformin, phenformin or chloroquine revealed that chondrosarcoma cell lines are sensitive to glutaminolysis inhibition. Metformin and phenformin decreased mTOR activity in chondrosarcoma cells, and metformin decreased LC3B-II levels, which is counteracted by chloroquine.ConclusionTargeting glutaminolysis with CB-839, metformin, phenformin or chloroquine is a potential therapeutic strategy for a subset of high-grade chondrosarcomas, irrespective of the presence or absence of an IDH1/2 mutation.

Annexin A1 (AnxA1) is a candidate regulator of the epithelial- to mesenchymal (EMT)-like phenotypic switch, a pivotal event in breast cancer progression. We show here that AnxA1 expression is associated with a highly invasive basal-like breast cancer subtype both in a panel of human breast cancer cell lines as in breast cancer patients and that AnxA1 is functionally related to breast cancer progression. AnxA1 knockdown in invasive basal-like breast cancer cells reduced the number of spontaneous lung metastasis, whereas additional expression of AnxA1 enhanced metastatic spread. AnxA1 promotes metastasis formation by enhancing TGFβ/Smad signaling and actin reorganization, which facilitates an EMT-like switch, thereby allowing efficient cell migration and invasion of metastatic breast cancer cells.