Clinically relevant glioblastoma patient-derived xenograft models to guide drug development and identify molecular signatures.

Glioblastoma (GBM) heterogeneity, aggressiveness and infiltrative growth drastically limit success of current standard of care drugs and efficacy of various new therapeutic approaches. There is a need for new therapies and models reflecting the complex biology of these tumors to analyze the molecular mechanisms of tumor formation and resistance, as well as to identify new therapeutic targets. We established and screened a panel of 26 patient-derived subcutaneous (s.c.) xenograft (PDX) GBM models on immunodeficient mice, of which 15 were also established as orthotopic models. Sensitivity toward a drug panel, selected for their different modes of action, was determined. Best treatment responses were observed for standard of care temozolomide, irinotecan and bevacizumab. Matching orthotopic models frequently show reduced sensitivity, as the blood-brain barrier limits crossing of the drugs to the GBM. Molecular characterization of 23 PDX identified all of them as IDH-wt (R132) with frequent mutations in EGFR, TP53, FAT1, and within the PI3K/Akt/mTOR pathway. Their expression profiles resemble proposed molecular GBM subtypes mesenchymal, proneural and classical, with pronounced clustering for gene sets related to angiogenesis and MAPK signaling. Subsequent gene set enrichment analysis identified hallmark gene sets of hypoxia and mTORC1 signaling as enriched in temozolomide resistant PDX. In models sensitive for mTOR inhibitor everolimus, hypoxia-related gene sets reactive oxygen species pathway and angiogenesis were enriched. Our results highlight how our platform of s.c. GBM PDX can reflect the complex, heterogeneous biology of GBM. Combined with transcriptome analyses, it is a valuable tool in identification of molecular signatures correlating with monitored responses. Available matching orthotopic PDX models can be used to assess the impact of the tumor microenvironment and blood-brain barrier on efficacy. Our GBM PDX panel therefore represents a valuable platform for screening regarding molecular markers and pharmacologically active drugs, as well as optimizing delivery of active drugs to the tumor.

Immunohistochemical Evaluation of Hemostatic Changes in Glioblastoma Multiforme and Low-Grade Astrocytoma.

AIM: In glioblastoma multiforme, the balance between the procoagulant system, anticoagulant system and fibrinolytic system is impaired in favour of hypercoagulability. The aim of this study was to compare glioblastoma multiforme with astrocytoma grade II by subjectively evaluating the levels of prothrombin and biotinylation thrombin, and G protein serum protease activatin receptors, as tissue factors causing hypercoagulation and affecting coagulation. MATERIAL AND METHODS: Specimens from 35 cases with glioblastoma multiforme and 23 cases with astrocytoma grade II were evaluated immunohistochemically. The specimens were stained with hematoxylen-eosin and immunohistochemically for prothrombin, biotinylation thrombin and protease activating factor receptors to determine the correlation between the tumor malignancy and coagulation factor receptors. RESULTS: An increase in malignancy was seen to result in an increase in prothrombin, biotinylation thrombin, protein activator receptor 1, protein activator receptor 3, and protein activator receptor 4 levels, and a decrease in protein activator receptor 2 level. These data showedthat there was hypercoagulability in glioblastoma multiforme. Descriptive statistics and Mann-Whitney U analysis were used to evaluate the results. CONCLUSION: In glioblastoma multiforme, if there is no radiologicalevidence of hemorrhage, low molecular weight heparin should be administered peroperatively and continued for 3 months postoperatively to prevent the development of deep venous thrombosis. This will also be useful in the prevention of invasion, angiogenesis, metastasis and tumour progression.

[Low-grade gliomas in adults]. / Niedrig-gradige Gliome im Erwachsenenalter.

Low-grade gliomas (LGG) are brain tumors with a low or intermediate biological aggressiveness. According to histopathological features, they are further specified as grade I or II by WHO criteria. Diffuse astrocytomas, oligodendrogliomas, and mixed gliomas are the most common LGG. They mainly affect young patients in their 3rd to 5th decade and often manifest with epileptic seizures. A macroscopically complete or near-complete tumor resection that does not induce additional neurological deficits, is recommended as first line therapy in surgically accessible tumors, as a significant benefit for overall survival has been demonstrated. The indication for adjuvant chemo- or radiotherapy must be discussed interdisciplinary in each case. MGMT promotor methylation, LOH 1p/19q, as well as the status of somatic mutations within IDH1/2 gene constitute biomarkers that may predict response to adjuvant therapy and may correlate to overall survival. These and other biomarkers could be of benefit in future managing plans to offer patients with LGG an individually tailored, optimal treatment.

Les gliomes de bas grade (GBG) sont des lésions tumorales cérébrales primaires considérées à potentiel malin faible à modéré; l'Organisation Mondiale de la Santé les a classés de grades I et II. Les astrocytomes, les oligodendrogliomes et les gliomes mixtes constituent des sous-groupes. Les GBG sont principalement retrouvés chez les jeunes patients et se manifestent souvent par des crises épileptiques. La résection tumorale complète, dans la mesure où elle ne cause pas de dysfonction neurologique, est considérée comme le traitement de première ligne, car elle améliore significativement la survie globale. L'indication à une radiothérapie et/ou une chimiothérapie adjuvantes doit être discutée au cas par cas à la consultation multidisciplinaire. La connaissance de biomarqueurs tels que le MGMT (et de son éventuelle méthylation), et le status, muté ou non, de 1p/19q et IDH1/2- peuvent être utiles en raison de la répercussion sur le traitement et la survie globale. À l'avenir, ces marqueurs (et d'autres encore) pourraient être utilisés pour fournir un traitement personnalisé, par conséquent avec un résultat thérapeutique optimal pour les patients atteints de GBG.

Overexpression of protease-activated receptor type 1 (PAR-1) in glioblastoma multiforme WHO IV cells and blood vessels revealed by NCAM-assisted glioblastoma border labeling.

Glioblastomas are neuroepithelial tumors with lost cellular differentiation and tenfold increased growth rates compared to low-grade gliomas. Despite of very aggressive treatment options based on surgery, irradiation, and chemotherapy, the prognosis of affected patients has remained poor and showed only slight improvements during the last 30 years. Research on glioblastoma border zone was hindered by the tumor's intense invasion into the brain parenchyma and the lack of suitable tumor cell markers. Nevertheless, the compact tumor mass and tumor invasion zone are composed of distinct cell types that need to be distinguished from each other to be addressed selectively. As the isoform 140 of the neural cell adhesion molecule (NCAM-140) was recently demonstrated to be lost in human gliomas with rising WHO grade, human multiform glioblastomas were characterized as a NCAM-140 negative entity displaying three main distinct invasion patterns. Evaluation of putative therapy targets within the tumor tissue and tumor invasion zone has been made possible through NCAM-140 negativity. In the present study, brain tissue controls and human glioblastoma samples with compact tumor mass and invasion areas were analyzed for their vascularization at the tumor border and the expression of thrombin receptor protease-activated receptor type 1 (PAR-1) within tumor tissue and vascular vessel walls. Use of NCAM-140 enabled the identification of the tumor invasion zone and its experimental investigation. Tissue vascularization was found to be significantly increased in the compact tumor mass of glioblastomas compared to their invasion zone and tumor-free controls with a significantly high and specific overexpression of PAR-1 within tumor cells and within tumor blood vessels depending upon the tumor area. This suggests thereby a functional role of the thrombin receptor PAR-1 in glioma cell malignancy and glioblastoma neoangiogenesis.

Expression of coagulation factors and their receptors in tumor tissue and coagulation factor upregulation in peripheral blood of patients with cerebral carcinoma metastases.

BACKGROUND: Patients with malignancies often suffer from thrombembolic events that complicate the course of cancer disease and reduce the patients' quality of life or shorten the survival time in severe cases. This phenomenon is also known for patients with primary or secondary brain tumors; but the reasons are not identified. METHODS: We performed a prospective case-controlled study of patients with brain metastases but without any active peripheral tumor site. Blood of patients was collected perioperatively and investigated for coagulation factor activities. Moreover, we analyzed the expression of coagulation factors and their receptors within the tumor material of brain metastases from clear-cell renal cell carcinomas and small-cell carcinomas of the lung. RESULTS: Here, we show that even patients without an active peripheral tumor disease that means without any tumor masses outside the central nervous system after anticancer treatment by surgery, radiation therapy, or chemotherapy but with symptomatic brain metastasis develop an increased systemic activation of multiple coagulation factors. The pro-coagulatory state is expressed preoperatively, but also can be observed in the early postoperative period. Additionally to that, intracerebral metastases of clear-cell renal cell carcinomas and of small-cell carcinomas of the lung express prothrombin, thrombin, factor X, and the protease-activated receptors type 1, 2, 3, and 4. CONCLUSIONS: These observations support the hypothesis of a link between the hemostatic system in the periphery and the malignant tumor disease even when the tumor is an intracerebral metastasis and the affected patient currently is free of a systemically active tumor. The results of this study support the hypothesis that the concerted action of coagulation factors and their receptors within the metastasis tissue itself and the systemic coagulation system could control the malignant behavior of tumor disease and make larger prospective trials mandatory.

Neural cell adhesion molecule isoform 140 declines with rise of WHO grade in human gliomas and serves as indicator for the invasion zone of multiform glioblastomas and brain metastases.

PURPOSE: Gliomas are highly invasive neuroepithelial tumors with a propensity of malignant transformation and very restricted treatment options. The neural cell adhesion molecule (NCAM) modulates cellular migration, proliferation, and synaptic plasticity by homophilic and heterophilic interactions. Hereby, we investigated its relevance as a glioma tissue marker for the biological aggressiveness of these tumors and compared these features with the carcinoma brain metastasis invasion zone. MATERIALS AND METHODS: We analyzed 194 human brain samples. Human tumor-free brain specimens served as control for the white and gray matter. In addition to that, we used human glioblastomas from nude rats. All tissues were investigated immunohistochemically for the expression of the NCAM isoform 140. Additionally, the multiplanar MRI-CT fusion neuronavigation-guided serial stereotactic biopsy was performed and completed by histopathological workup. RESULTS: Human gliomas loose NCAM-140 with the rise of their WHO grade. Meningiomas are NCAM-140 negative. As the most striking feature, human brain metastases and the majority of human glioblastomas of our patients and of nude rats were totally NCAM-140 negative. This NCAM negativity led us to the conclusion of three different main glioblastoma invasion patterns. Surprisingly, the majority of brain metastasis samples that contained surrounding brain parenchyma demonstrated invasive tumor cell nests beyond the sharply demarcated metastasis border. We also found invasive metastatic cell nests outside the contrast enhancing tumor zone by means of the MRI-CT fusion neuronavigation-guided serial stereotactic biopsy. CONCLUSION: The expression of NCAM-140 inversely correlates with the WHO grade of human gliomas. The lost expression of NCAM-140 in human glioblastomas and in brain metastases enables the investigation of the brain-tumor interface and the definition of glioblastoma invasion patterns and shows that brain metastases are more invasive than ever thought.

Glioblastoma cells express functional cell membrane receptors activated by daily used medical drugs.

PURPOSE: Calcium ions are highly versatile spacial and temporal intracellular signals of non-excitable cells and have an important impact on nearly every aspect of cellular life controlling cell growth, metabolism, fluid secretion, information processing, transcription, apoptosis, and motility. Neurons and glia respond to stimuli, including neurotransmitters, neuromodulators, and hormones, which increase the intracellular calcium concentration. The function of intracellular calcium in gliomas is unknown. Lots of daily used drugs may act via receptors that can be linked to the intracellular calcium system and therefore could influence glioma biology. METHODS: Glioma cells were loaded with the calcium ion sensitive dye Fura 2-AM. Subsequently, cells were stimulated with 25 different medical drugs for 30 s. The increase of free intracellular calcium ions was measured and calculated by a microscope-camera-computer-unit. RESULTS: Except for the buffer solution HEPES that served as negative control and for the cortisol derivative dexamethasone, all other 24 tested drugs induced a rise of intracellular calcium ions. The cellular calcium responses were classified into seven functional groups. The tested substances activated several types of calcium channels and receptors. CONCLUSIONS: Our study impressively demonstrates that medical drugs are potent inducers of intracellular calcium signals. Totally unexpected, the results show a high amount of functional cellular receptors and channels on glioma cells, which could be responsible for certain biological effects like migration and cell growth. This calcium imaging study proves the usability of the calcium imaging as a screening system for functional receptors on human glioma cells.

Pulmonary meningioma and neurinoma associated with multiple CNS tumours in a patient with neurofibromatosis type 2.

OBJECTIVE: Neurofibromatosis type 2 (NF2) is a common neurocutaneous disorder that exhibits an autosomal dominant inheritance, with a mutation at chromosome 22q12.2. Two forms can be distinguished: the Wishart-phenotype with an early and aggressive course and the Feiling-Gardner-phenotype with a less dramatic presentation. In general, patients present bilateral vestibular schwannomas, meningiomas and neurinomas of the central and peripheral nervous system as well as neurofibromas and gliomas. There is no reported case of pulmonary meningiomas and neurinomas associated with NF2 until now. PATIENT AND METHODS: Here, we present a 16-year-old girl with NF-2 associated to CNS and pulmonary tumours and we discuss the case in the backlight of the literature. RESULTS: The reported patient presented a de novo NF2 germline mutation (R341X) and displayed the Wishart-type of NF-2 since she is 11 years old, with a huge anaplastic biparietal falx meningioma and a tentorium meningioma and a tumour-associated parietal mass as well as hypacusis starting at the infant age of 3 years. Multiple cranial and spinal tumours with extra- and intramedullary localization were also found. Moreover, recurrent pulmonary tumours developed and were classified as benign meningiomas and a single neurinoma. No direct evidence concerning a relationship between the pulmonary and cerebral tumours could be drawn. CONCLUSION: This rare case extends our knowledge of NF2 and also raises interesting questions about the pathogenesis of meningiomas outside the CNS.

Methylation-specific multiplex ligation-dependent probe amplification in meningiomas.

Genomic loss and promotor methylation contribute to inactivation of tumor suppressor genes (TSGs). Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is a relatively new method for simultaneous detection of both these alterations. Here, we apply MS-MLPA to a series of 15 meningiomas of different WHO grades. The two MS-MLPA probe sets used detect copy number changes in 55 unselected TSGs and promotor methylation in a subset of 36 of these genes. Our findings concerning genomic deletions are concordant with previously published studies using alternative techniques. The number of aberrations identified per tumor increased with histopathologically determined grading. The most frequent single event was deletion of the von Hippel-Lindau (VHL) gene in 12 of the 15 tumors. Moreover, VHL deletion status was associated with presence/absence of peritumoral edema. Methylation was rare, being observed in only four tumors and in each case restricted to a single gene. We conclude that a meningioma-specific MS-MLPA probe set would be a valuable tool for both research and diagnostic approaches in these tumors.

A paediatric supratentorial primitive neuroectodermal tumour associated with malignant astrocytic transformation and a clonal origin of both components.

The case of a 7-year-old boy suffering from a supratentorial primitive neuroectodermal tumour (sPNET) at the age of 5 is presented. The tumour has been characterized by astrocytic areas within the sPNET revealing malignant transformation up to a multiform glioblastoma during the course of the disease. The clonal origin of both tumour components was established by loss of heterozygosity (LOH) analysis. Clinically, the tumour showed an aggressive biological behaviour with two recurrences. We discuss this very rare case and the first description of the clonal origin of distinct and distinguishable tumour components taking into consideration published literature.

Brain slice invasion model reveals genes differentially regulated in glioma invasion.

Invasion of tumor cells into adjacent brain areas is one of the major problems in treatment of glioma patients. To identify genes that might contribute to invasion, fluorescent F98 glioma cells were allowed to invade an organotypic brain slice. Gene expression analysis revealed 5 up-regulated and 14 down-regulated genes in invasive glioma cells as compared to non-invasive glioma cells. Two gene products, ferritin and cyclin B1, were verified in human gliomas by immunohistochemistry. Ferritin exhibited high mRNA levels in migratory F98 cells and also showed higher protein expression in the infiltrating edge of human gliomas. Cyclin B1 with high mRNA expression levels in stationary F98 cells showed marked protein expression in the central portions of gliomas. These findings are compatible with the concept of tumor cells either proliferating or migrating. Our study is the first to apply brain slice cultures for the identification of differentially regulated genes in glioma invasion.

Microglia express GABA(B) receptors to modulate interleukin release.

gamma-Aminobutyric acid (GABA) can act as a neuroprotective agent besides its well-established role as the main inhibitory neurotransmitter in the CNS. Here we report that microglial cells express GABA(B) receptors indicating that these prominent immunocompetent cells in the brain are a target for GABA. Agonists of GABA(B) receptors triggered the induction of K(+) conductance in microglial cells from acute brain slices and in culture. Both subunits of GABA(B) receptors were identified in cultured microglia by Western blot analysis and immunocytochemistry, and were detected on a subpopulation of microglia in situ by immunohistochemistry. In response to facial nerve axotomy, we observed an increase in GABA(B) receptor expressing microglial cells in the facial nucleus. We activated microglial cells in culture with lipopolysaccharide (LPS) to induce the release of interleukin-6 and interleukin-12p40. This release activity was attenuated by simultaneous activation of the GABA(B) receptors indicating that GABA can modulate the microglial immune response.