[Show abstract][Hide abstract] ABSTRACT: Anti-angiogenic therapy in glioblastoma (GBM) has unfortunately not led to the anticipated improvement in patient prognosis. We here describe how human GBM adapts to bevacizumab treatment at the metabolic level. By performing (13)C6-glucose metabolic flux analysis, we show for the first time that the tumors undergo metabolic re-programming toward anaerobic metabolism, thereby uncoupling glycolysis from oxidative phosphorylation. Following treatment, an increased influx of (13)C6-glucose was observed into the tumors, concomitant to increased lactate levels and a reduction of metabolites associated with the tricarboxylic acid cycle. This was confirmed by increased expression of glycolytic enzymes including pyruvate dehydrogenase kinase in the treated tumors. Interestingly, L-glutamine levels were also reduced. These results were further confirmed by the assessment of in vivo metabolic data obtained by magnetic resonance spectroscopy and positron emission tomography. Moreover, bevacizumab led to a depletion in glutathione levels indicating that the treatment caused oxidative stress in the tumors. Confirming the metabolic flux results, immunohistochemical analysis showed an up-regulation of lactate dehydrogenase in the bevacizumab-treated tumor core as well as in single tumor cells infiltrating the brain, which may explain the increased invasion observed after bevacizumab treatment. These observations were further validated in a panel of eight human GBM patients in which paired biopsy samples were obtained before and after bevacizumab treatment. Importantly, we show that the GBM adaptation to bevacizumab therapy is not mediated by clonal selection mechanisms, but represents an adaptive response to therapy.
[Show abstract][Hide abstract] ABSTRACT: AimsThe paired box gene 8 (PAX8) plays crucial roles in organ patterning and cellular differentiation during development and tumorigenesis. While its function is partly understood in vertebrate development, there is poor data concerning human CNS development and brain tumors.Methods
We investigated developing human (n=19) and mouse (n=3) brains as well as medulloblastomas (n=113) for PAX8 expression by immunohistochemistry. Human medulloblastoma cell lines were assessed for PAX8 expression using PCR and immunoblotting and analysed for growth and migration following PAX8 knockdown by siRNA.ResultsPAX8 protein expression was associated with germinal layers in human and murine forebrain and hindbrain development. PAX8 expression significantly decreased over time in the external granule cell layer, but increased in the internal granule cell layer. In medulloblastoma (MB) subtypes we observed an association of PAX8 expression with SHH (sonic hedgehog) and WNT subtypes but not with group 3 and 4 MBs. Beyond that, we detected high PAX8 levels in desmoplastic MB subtypes. Univariate analyses revealed high PAX8 levels as a prognostic factor associated with a significantly better patient prognosis in human MB (overall survival: Log-Rank p=0.0404, Wilcoxon p=0.0280; progression-free survival: Log-Rank p=0.0225; Wilcoxon p=0.0136). In vitro assays revealed increased proliferation and migration of medulloblastoma cell lines after PAX8 siRNA knockdown.Conclusion
In summary, high PAX8 expression is linked to better prognosis in medulloblastomas potentially by suppressing both proliferative and migratory properties of MB cells. The distinct spatio-temporal expression pattern of PAX8 during brain development might contribute to the understanding of distinct MB subtype histogenesis.
Neuropathology and Applied Neurobiology 10/2014; · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pre-B-cell leukemia homeobox (Pbx)-regulating protein-1 (Prep1) is a ubiquitous homeoprotein involved in early development, genomic stability, insulin sensitivity, and hematopoiesis. Previously we have shown that Prep1 is a haploinsufficient tumor suppressor that inhibits neoplastic transformation by competing with myeloid ecotropic integration site 1 for binding to the common heterodimeric partner Pbx1. Epithelial-mesenchymal transition (EMT) is controlled by complex networks of proinvasive transcription factors responsive to paracrine factors such as TGF-β. Here we show that, in addition to inhibiting primary tumor growth, PREP1 is a novel EMT inducer and prometastatic transcription factor. In human non-small cell lung cancer (NSCLC) cells, PREP1 overexpression is sufficient to trigger EMT, whereas PREP1 down-regulation inhibits the induction of EMT in response to TGF-β. PREP1 modulates the cellular sensitivity to TGF-β by inducing the small mothers against decapentaplegic homolog 3 (SMAD3) nuclear translocation through mechanisms dependent, at least in part, on PREP1-mediated transactivation of a regulatory element in the SMAD3 first intron. Along with the stabilization and accumulation of PBX1, PREP1 induces the expression of multiple activator protein 1 components including the proinvasive Fos-related antigen 1 (FRA-1) oncoprotein. Both FRA-1 and PBX1 are required for the mesenchymal changes triggered by PREP1 in lung tumor cells. Finally, we show that the PREP1-induced mesenchymal transformation correlates with significantly increased lung colonization by cells overexpressing PREP1. Accordingly, we have detected PREP1 accumulation in a large number of human brain metastases of various solid tumors, including NSCLC. These findings point to a novel role of the PREP1 homeoprotein in the control of the TGF-β pathway, EMT, and metastasis in NSCLC.
Proceedings of the National Academy of Sciences 08/2014; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:We investigated the prognostic role of tumour-associated macrophages (TAMs) in patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT).Methods:The expression of CD68+, CD163+ and CD11b+ cells was assessed using immunohistochemistry in n=106 pre-treatment tumour biopsy samples and was correlated with clinicopathological characteristics, including T-stage, N-stage, grading, tumour localisation, age and sex as well as local failure-free survival (LFFS), distant metastases-free survival (DMFS), progression-free (PFS), and overall survival (OS). Finally, TAMs expression and vessel density (CD31) were examined in n=12 available early local recurrence samples and compared with their matched primary tumours . The diagnostic images and radiotherapy plans of these 12 patients were also analysed. All local recurrences occurred in the high radiation dose region (⩾70 Gy).Results:With a median follow-up of 40 months, OS at 2 years was 60.5%. High CD163 expression in primary tumours was associated with decreased OS (P=0.010), PFS (P=0.033), LFFS (P=0.036) and DMFS (P=0.038) in multivariate analysis. CD163 demonstrated a strong prognostic value only in human papillomavirus (p16(INK4))-negative patients. Early local recurrence specimens demonstrated a significantly increased infiltration of CD11b+ myeloid cells (P=0.0097) but decreased CD31-positive vessel density (P=0.0004) compared with their matched primary samples.Conclusions:Altogether, baseline CD163 expression predicts for an unfavourable clinical outcome in HNSCC after definitive CRT. Early local recurrences showed increased infiltration by CD11b+ cells. These data provide important insight on the role of TAMs in mediating response to CRT in patients with HNSCC.British Journal of Cancer advance online publication, 5 August 2014; doi:10.1038/bjc.2014.446 www.bjcancer.com.
[Show abstract][Hide abstract] ABSTRACT: The macrophage migration inhibitory factor (MIF) receptor CD74 is overexpressed in various neoplasms, mainly in hematological tumors, and currently investigated in clinical studies. CD74 is quickly internalized and recycles after antibody binding therefore it constitutes an attractive target for antibody-based treatment strategies. CD74 has been further described as one of the most upregulated molecules in human glioblastomas. To assess the potential relevance for anti-CD74 treatment we determined the cellular source and clinico-pathological relevance of CD74 expression in human gliomas by immunohistochemistry, immunofluorescence, immunoblotting, cell sorting analysis and qPCR. Furthermore, we fractionated glioblastoma cells and glioma-associated microglia/macrophages (GAMs) from primary tumors and compared CD74 expression in cellular fractions with whole tumor lysates. Our results show that CD74 is restricted to GAMs in vivo, while being absent on tumor cells, the latter strongly expressing its ligand MIF. Most interestingly, a higher amount of CD74-positive GAMs was associated with beneficial patient survival constituting an independent prognostic parameter and with an anti-tumoral M1-polarization. In summary, CD74 expression in human gliomas is restricted to GAMs and positively associated with patient survival. In conclusion, CD74 represents a positive prognostic marker most probably due to its association with a M1-polarized immune milieu in high-grade gliomas.
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis inhibitors have evolved in the past decade as one of the most promising biology based therapeutic strategies. Promising pre-clinical studies (published in 1993 and 1994) provided proof of principle that blocking of VEGF dramatically inhibits tumor growth. These observations led to the successful development of inhibitors for VEGF and VEGF receptors by various pharmaceutical companies. Bevacizumab, a monoclonal antibody neutralizing VEGF, was the first-in class drug to achieve FDA approval for the treatment of colorectal carcinoma in 2004 and later on in other cancer types, including glioblastoma. Anti-VEGF therapy led to an increase in progression free survival in recurrent and primary GBM, but its role in first-line treatment is less clear. Importantly, several preclinical studies suggested that resistance to anti-angiogenic therapy might evolve in the course of the treatment due to an infiltration of specially polarized myeloid cells.
[Show abstract][Hide abstract] ABSTRACT: To investigate the relevance of bevacizumab (BEV)-induced diffusion-restricted lesions and T1-hyperintense lesions in patients with recurrent glioblastoma.METHODS: We prospectively screened 74 BEV-treated patients with recurrent glioblastoma for (1) diffusion-restricted lesions and/or, (2) lesions with a hyperintense signal on precontrast T1-weighted images. We further evaluated overall survival (OS), histopathology of the lesions, and patterns of progression.RESULTS: Twenty-five of 74 patients (34%) developed T1-hyperintense lesions, whereas diffusion-restricted lesions could be detected in 35 of 74 patients (47%). In 21 of 74 patients (28%), the lesions displayed both features ("double-positive"). OS for patients with double-positive lesions was 13.0 months; patients with neither of these lesions had an OS of 6.6 months (p < 0.005). Histologic evaluation of double-positive lesions revealed extensive calcified necrosis in 4 of 4 patients. Notably, these double-positive lesions were rarely involved in further tumor progression. However, they were associated with an increase in distant recurrences at BEV failure.CONCLUSIONS: BEV-induced double-positive MRI lesions are a predictive imaging marker associated with a substantial survival benefit and with improved local control in patients with recurrent glioblastoma. Our data suggest that these lesions are the result of a sustained focal antitumor activity of BEV.
[Show abstract][Hide abstract] ABSTRACT: The transcription factor OCT4 is an established diagnostic marker for central nervous system (CNS) germinoma. However, no data are available to date concerning the expression of its downstream target undifferentiated embryonic cell transcription factor 1 (UTF1) in CNS germ cell tumours.We examined 21 CNS germinomas and two mixed CNS germ cell tumours for UTF1 and the post-transcriptional regulator LIN28 immunohistochemical expression. We compared the profile to established diagnostic germinoma markers and to the expression in six testicular and four metastatic germ cell tumours as well as 150 CNS tumours of various backgrounds.We found UTF1 expression in 23 of 23 and LIN28 in 20 of 23 CNS germ cell tumours. The established germinoma markers cKIT (23/23), OCT4 (21/23) and placental alkaline phosphatase (PLAP) (19/21) were also frequently expressed in our cohort. In terms of signal intensity and frequency, UTF1 showed similar results as cKIT but staining was superior to OCT4, PLAP and LIN28. OCT4 was absent in all CNS metastases and haemangioblastomas, while UTF1 was weakly observed in two metastases.With a sensitivity of 100% and a specificity of 97% in the detection of CNS germinomas, UTF1 serves as a new reliable alternative in the diagnostic setting of CNS germ cell tumours.
[Show abstract][Hide abstract] ABSTRACT: AimsThe prognosis of patients with malignant gliomas is still dismal despite maximum treatment. Novel therapeutic alternatives targeting tumorigenic pathways are, therefore, demanded. In murine glioma models, targeting of tumor necrosis factor receptor superfamily (TNFRSF) 9 led to complete tumor eradication. Thus, TNFRSF9 might also constitute a promising target in human diffuse gliomas. Since there is a lack of data, we aimed to define the expression pattern and cellular source of TNFRSF9 in human gliomas. Methods
We investigated TNFRSF9 expression in normal human CNS tissue and glioma specimens using immunohistochemistry, immunofluorescence and western blotting techniques. ResultsOur results show that TNFRSF9 is considerably upregulated in human gliomas when compared to normal brain tissue. In addition, our data provides evidence for an immune cell-independent de novo expression pattern of TNFRSF9 in mainly non-neoplastic reactive astrocytes and excludes classic immunological cell types, namely lymphocytes and microglia as the source of TNFRSF9. Moreover, TNFRSF9 is predominantly expressed in a perivascular and peri-tumoral distribution with significantly higher expression in IDH1 mutant gliomas. Conclusions
Our findings provide a novel, TNFRSF9-positive, reactive astrocytic phenotype and challenge the therapeutic suitability of TNFRSF9 as a promising target for human gliomas.
Neuropathology and Applied Neurobiology 03/2014; · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the treatment of glioblastoma (GBM) the impact of radical tumor resection as first line therapy is beyond controversy. The significance of a second resection in case of tumor-recurrence remains unclear and is an issue of debate. Since GBMs always recur, it is important to determine whether or not patients will benefit from repeat surgery. We performed a retrospective analysis of our prospectively collected database and evaluated all re-resected patients with primary GBM who underwent second surgery during a 3 years period. All patients underwent early postoperative magnetic resonance imaging. We determined survival after re-resection with regard to possible prognostic factors using Kaplan-Meier estimates and Cox regression analyses. Forty patients were included in this study. Median age was 58 years and median KPS score was 80. Average tumor volume was 5.5 cm(3). A radiologically confirmed complete resection was achieved in 29 patients (72.5 %). Median follow-up was 18.8 months, and median survival after re-resection was 13.5 months. Only complete removal of contrast enhancing tumor was significantly correlated with survival after re-resection according to multivariate analysis. There was a statistical trend for KPS score influencing survival. In contrast, time between first diagnosis and tumor-recurrence, tumor volume at recurrence, MGMT status and MSM score were not significantly correlated with survival after second surgery. In the event of tumor recurrence, patients in good clinical condition with recurrent GBM amenable to complete resection should thus not be withheld second surgery as a treatment option.
Journal of Neuro-Oncology 02/2014; · 3.12 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: B10 is a glycosylated derivative of betulinic acid with promising activity against glioma cells. Lysosomal cell death pathways appear to be essential for its cytotoxicity. We investigated the influence of hypoxia, nutrient deprivation and current standard therapies on B10 cytotoxicity. The human glioma cell lines LN-308 and LNT-229 were exposed to B10 alone or together with irradiation, temozolomide, nutrient deprivation or hypoxia. Cell growth and viability were evaluated by crystal violet staining, clonogenicity assays, propidium iodide uptake and LDH release assays. Cell death was examined using an inhibitor of lysosomal acidification (bafilomycin A1), a cathepsin inhibitor (CA074-Me) and a short-hairpin RNA targeting cathepsin B. Hypoxia substantially enhanced B10-induced cell death. This effect was sensitive to bafilomycin A1 and thus dependent on hypoxia-induced lysosomal acidification. Cathepsin B appeared to mediate cell death because either the inhibitor CA074-Me or cathepsin B gene silencing rescued glioma cells from B10 toxicity under hypoxia. B10 is a novel antitumor agent with substantially enhanced cytotoxicity under hypoxia conferred by increased lysosomal cell death pathway activation. Given the importance of hypoxia for therapy resistance, malignant progression, and as a result of antiangiogenic therapies, B10 might be a promising strategy for hypoxic tumors like malignant glioma.
PLoS ONE 01/2014; 9(4):e94921. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The formation of new blood vessels is a major hallmark in the process of malignant transformation in human glioblastomas. In diffusely infiltrating gliomas, enhanced angiogenesis is associated with decreased patient survival rates and therefore serves as a central diagnostic criterion according to the WHO (World Health Organization) classification of tumors of the central nervous system (CNS). However, the assessment of what a newly built blood vessel really is and how the extent of glioma-associated angiogenesis can be estimated in vivo is often a highly subjective procedure with imprecise criteria depending on the experience of the neuropathologist. The increased interest in translational medicine and anti-angiogenic treatment strategies implies that basic researchers in glioma angiogenesis are frequently asked to validate their findings in patient material to provide evidence for potential clinical relevance of their results. Therefore, more precise methods and measurement techniques are needed to objectively measure the extent of angiogenesis in human glioblastoma samples. The present synopsis provides an overview about morphological methods to assess the formation of new blood vessels by quantitative imaging using histological and immunohistochemical marker profiles.
[Show abstract][Hide abstract] ABSTRACT: The multifunctional molecule netrin-1 is upregulated in various malignancies and has recently been presented as a major general player in tumorigenesis leading to tumor progression and maintenance in various animal models. However, there is still a lack of clinico-epidemiological data related to netrin-1 expression. Therefore, the aim of our study was to elucidate the association of netrin-1 expression and patient survival in brain metastases since those constitute one of the most limiting factors for patient prognosis. We investigated 104 brain metastases cases for netrin-1 expression using in-situ hybridization and immunohistochemistry with regard to clinical parameters such as patient survival and MRI data. Our data show that netrin-1 is strongly upregulated in most cancer subtypes. Univariate analyses revealed netrin-1 expression as a significant factor associated with poor patient survival in the total cohort of brain metastasis patients and in sub-entities such as non-small cell lung carcinomas. Interestingly, many cancer samples showed a strong nuclear netrin-1 signal which was recently linked to a truncated netrin-1 variant that enhances tumor growth. Nuclear netrin-1 expression was associated with poor patient survival in univariate as well as in multivariate analyses. Our data indicate both total and nuclear netrin-1 expression as prognostic factors in brain metastases patients in contrast to other prognostic markers in oncology such as patient age, number of brain metastases or Ki67 proliferation index. Therefore, nuclear netrin-1 expression constitutes one of the first reported molecular biomarkers for patient survival in brain metastases. Furthermore, netrin-1 may constitute a promising target for future anti-cancer treatment approaches in brain metastases.
PLoS ONE 01/2014; 9(3):e92311. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Our goal was to develop strategies to quantify the accumulation of model therapeutics in small brain metastases using multimodal imaging, in order to enhance the potential for successful treatment. Human melanoma cells were injected into the left cardiac ventricle of immunodeficient mice. Bioluminescent, MR and PET imaging were applied to evaluate the limits of detection and potential for contrast agent extravasation in small brain metastases. A pharmacokinetic model was applied to estimate vascular permeability. Bioluminescent imaging after injecting D-Luciferin (molecular weight (MW) 320D) suggested tumor cell extravasation had already occurred at week 1, which was confirmed by histology. 7T T1w MRI at week 4 was able to detect non-leaky 100 μm sized lesions and leaky tumors with diameters down to 200μm after contrast injection at week 5. PET imaging showed that (18)F-FLT (MW 244D) accumulated in the brain at week 4. Gadolinium-based MRI tracers (MW 559D and 2.066kD) extravasated after 5weeks (tumor diameter 600 μm), and the lower MW agent cleared more rapidly from the tumor (mean apparent permeabilities 2.27x10(-5)cm/s versus 1.12x10(-5)cm/s). PET imaging further demonstrated tumor permeability to (64)Cu-BSA (MW 65.55kD) at week 6 (tumor diameter 700 μm). In conclusion, high field T1w MRI without contrast may improve the detection limit of small brain metastases, allowing for earlier diagnosis of patients, although the smallest lesions detected with T1w MRI were permeable only to D-Luciferin and the amphipathic small molecule (18)F-FLT. Different-sized MR and PET contrast agents demonstrated the gradual increase in leakiness of the blood tumor barrier during metastatic progression, which could guide clinicians in choosing tailored treatment strategies.
Journal of Controlled Release 10/2013; · 7.63 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Far Upstream Element [FUSE] Binding Protein 1 (FUBP1) regulates target genes, such as the cell cycle regulators MYC and p21. FUBP1 is up-regulated in many tumours and acts as an oncoprotein by stimulating proliferation and inhibiting apoptosis. Recently, FUBP1 mutations were identified in approximately 15% of oligodendrogliomas. To date, all reported FUBP1 mutations have been predicted to inactivate FUBP1, which suggests that in contrast to most other tumours FUBP1 may act as a tumour suppressor in oligodendrogliomas.
As no data are currently available concerning FUBP1 protein levels in gliomas, we examined the FUBP1 expression profiles of human glial tumours by immunohistochemistry and immunofluorescence. We analysed FUBP1 expression related to morphological differentiation, IDH1 and FUBP1 mutation status, 1p/19q loss of heterozygosity (LOH) as well as proliferation rate.
Our findings demonstrate that FUBP1 expression levels are increased in all glioma subtypes as compared to normal central nervous system (CNS) control tissue and are associated with increased proliferation. In contrast, FUBP1 immunonegativity predicted FUBP1 mutation with a sensitivity of 100% and a specificity of 90% in our cohort and was associated with oligodendroglial differentiation, IDH1 mutation and 1p/19q loss of heterozygosity (LOH). Using this approach, we detected a to-date undescribed FUBP1 mutation in an oligodendroglioma.
In summary, our data indicate an association between of FUBP1 expression and proliferation in gliomas. Furthermore, our findings present FUBP1 immunohistochemical analysis as a helpful additional tool for neuropathological glioma diagnostics predicting FUBP1 mutation.
Neuropathology and Applied Neurobiology 10/2013; · 4.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The scaffold protein A-kinase anchor protein 12 (AKAP12) exerts tumor suppressor activity and is downregulated in several tumor entities. We characterized AKAP12 expression and regulation in astrocytomas, including pilocytic and diffusely infiltrating astrocytomas. We examined 194 human gliomas and 23 normal brain white matter samples by immunohistochemistry or immunoblotting for AKAP12 expression. We further performed quantitative methylation analysis of the AKAP12 promoter by MassARRAY® of normal brain, World Health Organization (WHO) grade I to IV astrocytomas, and glioma cell lines. Our results show that AKAP12 is expressed in a perivascular distribution in normal CNS, strongly upregulated in tumor cells in pilocytic astrocytomas, and weakly expressed in diffuse astrocytomas of WHO grade II to IV. Methylation analyses revealed specific hypermethylation of AKAP12α promoter in WHO grade II to IV astrocytomas. Restoration experiments using 5-aza-2'-deoxycytidine in primary glioblastoma cells decreased AKAP12α promoter methylation and markedly increased AKAP12α mRNA levels. In summary, we demonstrate that AKAP12 is differentially expressed in human astrocytomas showing high expression in pilocytic but low expression in diffuse astrocytomas of all WHO-grades. Our results further indicate that epigenetic mechanisms are involved in silencing AKAP12 in diffuse astrocytomas; however, a tumor suppressive role of AKAP12 in distinct astrocytoma subtypes remains to be determined.
Journal of Neuropathology and Experimental Neurology 09/2013; 72(10):933. · 4.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diffuse human gliomas constitute a group of most treatment-refractory tumors even if maximum treatment strategies including neurosurgical resection followed by combined radio-/chemotherapy are applied. In contrast to most other neoplasms, diffusely infiltrating gliomas invade the brain along pre-existing structures such as axonal tracts and perivascular spaces. Even in cases of early diagnosis single or small clusters of glioma cells are already encountered far away from the main tumor bulk. Complex interactions between glioma cells and the surrounding extracellular matrix and considerable changes in the cytoskeletal apparatus are prerequisites for the cellular movement of glioma cells through the brain thereby escaping from most current treatments. This review provides an overview about classical and current concepts of glioma cell migration/invasion and promising preclinical treatment approaches.