SUMOylation is associated with aggressive behavior in chondrosarcoma of bone

Simple Summary SUMO is a ubiquitin-like post-translational modification important for many cellular processes and is suggested to play a role in cancer cell cycle progression. The aim of our study is to understand the role of SUMOylation in tumor progression and aggressiveness. Chondrosarcoma of bone was employed as a model to investigate if SUMOylation contributes to its aggressiveness. We confirmed that SUMO expression levels correlate with aggressiveness of chondrosarcoma and disease outcome. Inhibition of SUMOylation showed promising effects on reduction of chondrosarcoma growth in vitro. Our study implies that SUMO expression could be used as a potential biomarker for disease outcome in chondrosarcoma. Multiple components of the SUMOylation machinery are deregulated in various cancers and could represent potential therapeutic targets. Understanding the role of SUMOylation in tumor progression and aggressiveness would increase our insight in the role of SUMO in cancer and clarify its potential as a therapeutic target. Here we investigate SUMO in relation to conventional chondrosarcomas, which are malignant cartilage forming tumors of the bone. Aggressiveness of chondrosarcoma increases with increasing histological grade, and a multistep progression model is assumed. High-grade chondrosarcomas have acquired an increased number of genetic alterations. Using immunohistochemistry on tissue microarrays (TMA) containing 137 chondrosarcomas, we showed that higher expression of SUMO1 and SUMO2/3 correlates with increased histological grade. In addition, high SUMO2/3 expression was associated with decreased overall survival chances (p = 0. 0312) in chondrosarcoma patients as determined by log-rank analysis and Cox regression. Various chondrosarcoma cell lines (n = 7), especially those derived from dedifferentiated chondrosarcoma, were sensitive to SUMO inhibition in vitro. Mechanistically, we found that SUMO E1 inhibition interferes with cell division and as a consequence DNA bridges are frequently formed between daughter cells. In conclusion, SUMO expression could potentially serve as a prognostic biomarker.MTG6Molecular tumour pathology - and tumour genetic

Inhibition of PARP Sensitizes Chondrosarcoma Cell Lines to Chemo- and Radiotherapy Irrespective of the IDH1 or IDH2 Mutation Status

Chondrosarcomas are chemo- and radiotherapy resistant and frequently harbor mutationsin isocitrate dehydrogenase (IDH1 or IDH2), causing increased levels of D-2-hydroxyglutarate(D-2-HG). DNA repair defects and synthetic lethality with poly(ADP-ribose) polymerase (PARP)inhibition occur in IDH mutant glioma and leukemia models. Here we evaluated DNA repairand PARP inhibition, alone or combined with chemo- or radiotherapy, in chondrosarcoma celllines with or without endogenous IDH mutations. Chondrosarcoma cell lines treated with thePARP inhibitor talazoparib were examined for dose–response relationships, as well as underlyingcell death mechanisms and DNA repair functionality. Talazoparib was combined with chemo- orradiotherapy to evaluate potential synergy. Cell lines treated long termwith an inhibitor normalizingD-2-HG levels were investigated for synthetic lethality with talazoparib. We report that talazoparibsensitivity was variable and irrespective of IDH mutation status. All cell lines expressed AtaxiaTelangiectasia Mutated (ATM), but a subset was impaired in poly(ADP-ribosyl)ation (PARylation)capacity, homologous recombination, andO-6-methylguanine-DNAmethyltransferase (MGMT) expression.Talazoparib synergized with temozolomide or radiation, independent of IDH1 mutant inhibition.This study suggests that talazoparib combined with temozolomide or radiation are promisingtherapeutic strategies for chondrosarcoma, irrespective of IDH mutation status. A subset ofchondrosarcomas may be deficient in nonclassical DNA repair pathways, suggesting that PARPinhibitor sensitivity is multifactorial in chondrosarcoma.Toxicolog

FOS rearrangement and expression in cementoblastoma

Cementoblastomas are rare odontogenic tumors developing in close proximity to the roots of teeth. Due to their striking morphologic resemblance to osteoblastomas of the peripheral skeleton, we set out to determine whether cementoblastomas harbor the same FOS rearrangements with overexpression of c-FOS as has recently been described for osteoblastomas. In total, 16 cementoblastomas were analyzed for FOS expression by immunohistochemistry and for FOS rearrangements by fluorescence in situ hybridization (FISH). We observed strong and diffuse staining of c-FOS in 71% of cementoblastomas and identified a FOS rearrangement in all cases (n=3) applicable for FISH. In the remaining cases, FISH failed due to decalcification. Cementoblastomas harbor similar FOS rearrangements and show overexpression of c-FOS like osteoblastomas, suggesting that both entities might represent parts of the spectrum of the same disease.Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Hypoxia-related microRNA-210 is a diagnostic marker for discriminating osteoblastoma and osteosarcoma

MTG6Molecular tumour pathology - and tumour genetic

Oestrogen receptor expression distinguishes non-ossifying fibroma from other giant cell containing bone tumours

Non-ossifying fibroma (NOF) and central giant cell granuloma (CGCG) are both benign tumours of bone with overlapping morphology and similar mutations in the RAS/MAPK pathway. However, NOF is located in the long bones with regression after puberty in contrast to CGCG which is located in the jaw bones and does not regress spontaneously. We hypothesised that endocrine regulation by oestrogen plays a role in the spontaneous regression in NOF. Therefore, we examined the expression of ER alpha in a series of NOF and CGCG. ER alpha expression (EP1) was determined using immunohistochemistry on 16 NOFs (whole slides), and 47 CGCGs (tissue microarrays (TMA's n = 41 and whole slide n = 6)). As comparison, we included TMAs of other giant cell containing bone lesions: giant cell tumour of bone (n = 75), chondroblastoma (n = 12), chondromyxoid fibroma (n = 12), aneurysmal bone cyst (n = 6) and telangiectatic osteosarcoma (n = 6). All 16 NOF samples demonstrated ER alpha protein expression, while all 47 CGCG and all other giant cell containing bone tumours were negative. Most NOF samples had moderate staining intensity and between 24 and 49% of the spindle cells were ER alpha-positive. Our findings further support the role of endocrine regulation via oestrogen in the spontaneous regression in NOF. Whether oestrogen signalling at puberty is involved in the induction of senescence in the neoplastic cells of NOF harbouring RAS/MAPK pathway mutations needs further research. Since ER alpha expression was not observed in other giant cell containing bone lesions with overlapping morphological features, positive ER alpha expression may favour the diagnosis of NOF in challenging diagnostic cases.Molecular tumour pathology - and tumour geneticsMTG

A translocation t(6;14) in two cases of leiomyosarcoma: Molecular cytogenetic and array-based comparative genomic hybridization characterization

Molecular tumour pathology - and tumour genetic

M-CSF and IL-34 expression as indicators for growth in sporadic vestibular schwannoma

Macrophage colony stimulating factor and IL-34 are associated with clinical vestibular schwannoma progression. Investigating the biology behind vestibular schwannoma progression helps understanding tumor growth. Inflammation is important in the microenvironment of neoplasms. Macrophages are major players in the intratumoral infiltrate. These tumor-associated macrophages are known to stimulate angiogenesis and cell growth. M-CSF and IL-34 are cytokines that can regulate tumor-infiltrating macrophages. They are expressed by tumors and form potential targets for therapy. The goal of this study was to investigate these cytokines in vestibular schwannomas and to see if their expression is related to angiogenesis, macrophage numbers, cystic degeneration, and volumetric tumor progression. Immunohistochemical expression of M-CSF and IL-34 was analyzed in ten fast-growing vestibular schwannomas and in ten slow-growing vestibular schwannomas. Expression M-CSF and IL-34 were compared between fast- versus slow-growing and cystic versus non-cystic tumors. Data on macrophage numbers and microvessel density, known from earlier research, was also included. All tumors expressed M-CSF and its expression was higher in fast-growing tumors (p=0.003) and in cystic tumors (p=0.035). CD163 expression was higher in tumors with strong M-CSF expression (p=0.003). All tumors expressed IL-34 as well, but no significant differences were found in relation to clinicopathological characteristics. This study demonstrated the expression of M-CSF and IL-34 in vestibular schwannomas. The results suggest that M-CSF is related to macrophage activity and tumor progression, making it a potential target for therapy. If a similar assumption can be made for IL-34 remains unclear.Otorhinolaryngolog

Utility of FOS as diagnostic marker for osteoid osteoma and osteoblastoma

Osteoid osteoma and osteoblastoma are bone-forming tumors shown to harbor FOS (87%) and FOSB (3%) rearrangements. The aim was to evaluate the immunohistochemical expression of FOS and FOSB in these tumors in comparison to other bone tumors, to evaluate the influence of decalcification, and to correlate immunohistochemical findings with the underlying genetic alteration using fluorescence in situ hybridization (FISH). Immunohistochemistry using whole sections was performed on osteoid osteoma (n=23), osteoblastoma (n=22), osteoblastoma-like osteosarcoma (n=3), reactive (n=3), and proliferative (n=11) bone lesions. Immunoreactivity in giant cell tumor of bone (n=74), aneurysmal bone cyst (n=6), chondromyxoid fibroma (n=20), osteosarcoma (n=85), chondroblastoma (n=17), and clear cell chondrosarcoma (n=20) was assessed using tissue micro arrays. Strong nuclear expression of FOS in > 50% of the tumor cells was observed in all osteoid osteomas (22/22), in 57% of osteoblastomas (12/21) and in 3/197 control cases. FOS immunoreactivity disappeared after > 3 days decalcification. FOS rearrangements were present in 94% of osteoid osteomas and osteoblastomas, with a concordance of 86% between FISH and immunohistochemistry. Two osteoblastomas (5%) were positive for FOSB, as opposed to 8/177 control cases. Additional FISH revealed no FOSB rearrangements in these cases. To conclude, in short decalcified biopsies, FOS immunohistochemistry can be used to diagnose osteoid osteoma and osteoblastoma, as overexpression is seen in the majority, being rare in their mimics. FOS immunohistochemistry should not be used after long decalcification. Moreover, low level of focal expression found in other lesions and tissues might cause diagnostic problems, in which case FISH could be employed

Selection of Effective Therapies Using Three-Dimensional in vitro Modeling of Chondrosarcoma

Purpose: Chondrosarcomas are a group of cartilaginous malignant neoplasms characterized by the deposition of chondrogenic extracellular matrix. Surgical resection is currently the only curative treatment option, due to their high resistance to conventional chemotherapy and radiotherapy. Novel therapeutic treatment options may improve outcome. Predominantly used cell line monolayer in vitro models lack in vivo complexity, such as the presence of extracellular matrix, and differing oxygen access. Hence, we aimed to improve pre-clinical chondrosarcoma research by developing an alginate-based 3D cell culture model.Method: An alginate scaffold was applied to generate spheroids of three chondrosarcoma cell lines (CH2879, JJ012, SW1353). Morphological, histological and immunohistochemical assessment of the spheroids were used to characterize the chondrosarcoma model. Presto blue assay, morphological and immunohistochemical assessment were applied to assess spheroid response to a panel of chemotherapeutics and targeted therapies, which was compared to conventional 2D monolayer models. Synergistic effect of doxorubicin and ABT-737 (Bcl-2 inhibitor) was compared between monolayer and spheroid models using excess over Bliss. A 3D colony formation assay was developed for assessment of radiotherapy response.Results: Chondrosarcoma spheroids produced chondrogenic matrix and remained proliferative after 2 weeks of culture. When treated with chemotherapeutics, the spheroids were more resistant than their monolayer counterparts, in line with animal models and clinical data. Moreover, for sapanisertib (mTOR inhibitor) treatment, a recovery in chondrosarcoma growth, previously observed in mice models, was also observed using long-term treatment. Morphological assessment was useful in the case of YM-155 (survivin inhibitor) treatment where a fraction of the spheroids underwent cell death, however a large fraction remained proliferative and unaffected. Synergy was less pronounced in 3D compared to 2D. A 3D clonogenic assay confirmed increased resistance to radiotherapy in 3D chondrosarcoma spheroids.Conclusion: We demonstrate that the chondrosarcoma alginate spheroid model is more representative of chondrosarcoma in vivo and should be used instead of the monolayer model for therapy testing. Improved selection at in vitro stage of therapeutic testing will increase the amount of information available for experimental design of in vivo animal testing and later, clinical stages. This can potentially lead to increased likelihood of approval and success at clinical trials.Molecular tumour pathology - and tumour geneticsMTG