Angiogenesis in gliomas

Brain gliomas are characterized by invasive growth and neovascularisation potential. Angiogenesis plays a major role in the progression of gliomas and its determination has a great prognostic value. The aim of the study was to assess the vascularisation of chosen brain gliomas and to estimate how it is correlated with tumour histological type, malignancy grade, location and size, and with age and sex of patients. Tumour vascularisation analysis was based on the determination of microvascular proliferation (MVP) and microvessel density (MVD). Microvascular proliferation was measured with immunohistochemical methods using mouse monoclonal antibodies to detect cell proliferation antigens. The following antibodies were used Ki-67 and PCNA (DAKO). Identification of vessels was performed by CD31 antibody and anti-human von Willebrand factor (DAKO). The highest microvascular proliferation and microvascular density were observed in multiform glioblastomas and the lowest in oligodendrogliomas. Significant correlation was observed between the vascularisation and malignancy grade.     

MicroRNA and target protein patterns reveal physiopathological features of glioma subtypes

Gliomas such as oligodendrogliomas (ODG) and glioblastomas (GBM) are brain tumours with different clinical outcomes. Histology-based classification of these tumour types is often difficult. Therefore the first aim of this study was to gain microRNA data that can be used as reliable signatures of oligodendrogliomas and glioblastomas. We investigated the levels of 282 microRNAs using membrane-array hybridisation and real-time PCR in ODG, GBM and control brain tissues. In comparison to these control tissues, 26 deregulated microRNAs were identified in tumours and the tissue levels of seven microRNAs (miR-21, miR-128, miR-132, miR-134, miR-155, miR-210 and miR-409-5p) appropriately discriminated oligodendrogliomas from glioblastomas. Genomic, epigenomic and host gene expression studies were conducted to investigate the mechanisms involved in these deregulations. Another aim of this study was to better understand glioma physiopathology looking for targets of deregulated microRNAs. We discovered that some targets of these microRNAs such as STAT3, PTBP1 or SIRT1 are differentially expressed in gliomas consistent with deregulation of microRNA expression. Moreover, MDH1, the target of several deregulated microRNAs, is repressed in glioblastomas, making an intramitochondrial-NAD reduction mediated by the mitochondrial aspartate-malate shuttle unlikely. Understanding the connections between microRNAs and bioenergetic pathways in gliomas may lead to identification of novel therapeutic targets.     

Lectins as differentiation markers of human gliomas.

The lectins Concanavalin A (Con A), Ricinus communis agglutinin (RCA-I), Peanut agglutinin (PNA) and Wheat germ agglutinin (WGA) as well as the immunomarkers for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were used in a series of 21 glial tumors (4 pylocytic astrocytomas, 5 grade II astrocytomas, 3 anaplastic astrocytomas, 4 glioblastomas and 5 oligodendrogliomas). ConA binds to all tumoral astrocytes in low grade astrocytomas, as well as to well differentiated tumoral astrocytes in anaplastic astrocytomas and glioblastomas. RCA-I has a similar behaviour. PNA, and to a lesser degree WGA, binds selectively to the oligodendroglial plasma membrane in well differentiated oligodendrogliomas. The results suggest that these lectins are markers of differentiation in gliomas rather than of malignancy.     

Cyclooxygenase-2 expression in astrocytes and microglia in human oligodendroglioma and astrocytoma

Cyclooxygenases (cox) are potent mediators of inflamation and two cox-izoenzymes, cox-1, cox-2, are described to date. Cox-2 is cytokine-inducible in inflammatory cells and enhanced cox-2 expression has been attributed a key role in the development of edema and immunomodulation in pathologically altered brain tissues. In normal cerebral cortex cox-2 is present only in neurons, but not in the glial or vascular endothelial cells. The function of microglia in glioma biology is unclear. Microglia have both neurotrophic and neurotoxic functions and have been shown to release a variety of cytokines. Our preliminary results showed that the expression pattern of cox-2 is predominantly neuronal although glial expression was observed with the correlation of high malignancy. In this study we aimed to assess the phenotypes (astrocyte, microglia) of the cox-2-expressing glial cells in various types of human gliomas and to compare their expression patterns. For this purpose we employed dual immunohistochemistry for cox-2 and GFAP (astrocyte) or LCA-MAC (microglia-macrophage) in archival formalin-fixed, paraffin embedded human tissue diagnosed as oligodendroglioma and/or astrocytoma. The results showed that cox-2 immunoreactivity is up-regulated in the neurons according to the tumor grade. Most of the cox-2 immunoreactive glia were GFAP-positive in anaplastic oligodendrogliomas and at lesser extend in glioblastomas. Cox-2 and LCA co-localization was detected in more glial cells in glioblastomas. It may be speculated that the induction of cox-2 in microglia may contribute to the deleterious effects of prostanoids in cerebral edema formation during the progression of oligodendrogliomas. The detection of cox-2 in astrocytes surrounding the necrotic areas might be important to develop new strategies, such as the usage of cox-2 inhibitors combine with chemotherapy and radiotherapy in the treatment of glioma patients.     

Nondestructive Detection of Glutamate by 1H Nuclear Magnetic Resonance Spectroscopy in Cortical Brain Slices from the Guinea Pig: Evidence for Changes in Detectability During Severe Anoxic Insults

31P and 1H nuclear magnetic resonance spectroscopy (NMR) was used to study the metabolism of intact superfused cortical brain slices during normoxia and anoxia. Attention was focused on quantification of 1H NMR-detected glutamate by a water-suppressed spin-echo method, using N-acetyl aspartate as an internal concentration reference. To quantify the 1H NMR signals, the spin-spin relaxation times and saturation effects were estimated for given metabolites. In addition, absolute concentrations of metabolites were determined by biochemical methods from acid extracts of the preparations after NMR experiments. Under aerobic conditions, 1H NMR detected 79% of the glutamate determined biochemically from the brain slice extracts. During anoxia in the absence of glucose when a severe energetic failure was evident, both 1H NMR and biochemical assays gave closely matching levels for glutamate. We conclude that in the brain cortex 21% of glutamate is located in an intracellular compartment in which this amino acid does not contribute to the 1H NMR signal. However, during severe anoxia an intracellular reorganisation occurs increasing the detectability of this amino acid neurotransmitter by NMR.     

Genetic analysis to complement histopathological diagnosis of brain tumors

Gliomas, the most frequent tumors originating in the human nervous system, are divided into various subtypes. Currently, microscopic examination alone is insufficient for classification and grading so that genetic profiles are increasingly being emphasized in recognition of the emerging role of molecular diagnostic approaches to glioma classification. Glioblastomas (WHO grade IV) may develop de novo (primary glioblastomas) or through progression from lower-grade astrocytomas (secondary glioblastomas), while both glioblastomas show similar histological features. In contrast, they do constitute distinct disease entities that evolve through different genetic pathways, and are likely to differ in prognosis and response to therapy. Oligodendrogliomas (WHO grade II) account for 2.7% of brain tumors and 5-18% of all gliomas. Since this tumor is recognized as a particular subtype of glioma that shows remarkable responses to chemotherapy, a correct diagnosis is of prime importance. The difficulty is that histological differentiation of oligodendrogliomas from diffuse astrocytomas is highly subjective in cases without typical morphological features and there is a lack of reliable immunohistochemical markers. While histological distinction of low-grade gliomas from reactive astrocytes is also often difficult, reactive astrocytes usually lack genetic alterations. More biological and molecular approaches to glioma classification thus appear warranted to provide improved means to achieve correct diagnoses.     

Combined use of 1H-NMR and GC-MS for metabolite monitoring and in vivo 1H-NMR assignments

Thirty-three metabolites were observed in perchloric acid extracts of four different tissues by in vitro 1H-NMR, GC-MS and alcohol dehydrogenase assay, and the information was used to interpret an in vivo two-dimensional nuclear Overhauser effect 1H-NMR spectrum. The metabolite profiles of the different tissues indicate a number of potential tissue-specific markers: N-acetylaspartate and gamma-aminobutyric acid for rat brain, glutamine/glutamic acid ratio for dog heart, arginine and sucrose for carrot, and t-aconitate, sucrose, asparagine/aspartic acid concentration ratios for corn roots. gamma-Aminobutyric acid and malate can be regarded as metabolic indicators for stressed corn roots. Concentrations of threonine and valine in corn roots were constant under hypoxic and salt stress, and can serve as internal standards for both in vivo and in vitro NMR studies. The in vitro information was further used to identify 12 compounds from the in vivo 1H-NMR spectra (including the two-dimensional nuclear Overhauser effect spectrum) of a carrot cylinder by correlating the chemical shift and nuclear Overhauser effect information. Thus, our choice of methods with a capability for structural determination allows the characterization of complex tissue extracts with minimum sample preparation, and supports, as well as complements, in vivo 1H-NMR investigations of metabolism.     

Aurora A is differentially expressed in gliomas,is associated with patient survival in glioblastoma and is a potential chemotherapeutic target in gliomas

Aurora A is critical for mitosis and is overexpressed in several neoplasms. Its overexpression transforms cultured cells, and both its overexpression and knockdown cause genomic instability. In transgenic mice, Aurora A haploinsufficiency, not overexpression, leads to increased malignant tumor formation. Aurora A thus appears to have both tumor-promoting and tumor-suppressor functions. Here, we report that Aurora A protein, measured by quantitative protein gel blotting, is differentially expressed in major glioma types in lineage-specific patterns. Aurora A protein levels in WHO grade II oligodendrogliomas (n = 16) and grade III anaplastic oligodendrogliomas (n = 16) are generally low, similar to control epilepsy cerebral tissue (n = 11). In contrast, pilocytic astrocytomas (n = 6) and ependymomas (n = 12) express high Aurora A levels. Among grade II to grade III astrocytomas (n = 7, n = 14, respectively) and grade IV glioblastomas (n = 31), Aurora A protein increases with increasing tumor grade. We also found that Aurora A expression is induced by hypoxia in cultured glioblastoma cells and is overexpressed in hypoxic regions of glioblastoma tumors. Retrospective Kaplan-Meier analysis revealed that both lower Aurora A protein measured by quantitative protein gel blot (n = 31) and Aurora A mRNA levels measured by real-time quantitative RT-PCR (n = 58) are significantly associated with poorer patient survival in glioblastoma. Furthermore, we report that the selective Aurora A inhibitor MLN8237 is potently cytotoxic to glioblastoma cells, and that MLN8237 cytotoxicty is potentiated by ionizing radiation. MLN8237 also appeared to induce senescence and differentiation of glioblastoma cells. Thus, in addition to being significantly associated with survival in glioblastoma, Aurora A is a potential new drug target for the treatment of glioblastoma and possibly other glial neoplasms.     

Choline-Containing Compounds in Human Astrocytomas Studied by 1H NMR Spectroscopy In Vivo and In Vitro

Abstract: We have studied 14 patients with different grades of astrocytomas using ¹H NMR spectroscopy in vivo. Typically, astrocytomas exhibited a low N -acetyl-aspartate peak, a prominent signal from choline group-containing compounds, and lactate in the ¹H NMR spectra in vivo. The uncorrected choline/creatine + phosphocreatine peak area ratios were higher in tumors than in normal brain tissue. Absolute concentration of choline-containing compounds (1.74 ± 0.09 mmol/L) in the normal brain tissue was not different in any grade of astrocytoma, but total creatine concentration in healthy brain (7.49 ± 0.30 mmol/L) was higher than that in grade IV astrocytomas (4.84 ± 0.89 mmol/L). Relaxation constants of choline-containing compounds did not differ in tumors from those determined in normal brain. Perchloric acid extracts of biopsy samples from 35 astrocytomas and 13 samples of normal temporal white matter were analyzed with ¹H NMR. Total concentration of choline-containing compounds did not differ between controls and any grade of astrocytoma when the quantification was done in vitro. It is interesting that phosphorylcholine concentration was about twofold greater in grade IV astrocytomas than in controls or other grades of astrocytomas. We conclude that high phosphorylcholine in grade IV astrocytomas may be an indicator of degree of malignancy. The proportional changes within the group of choline-containing compounds observed in vitro were not reflected in the NMR properties of choline signal in vivo.     

Determination of metabolite and nucleotide concentrations in proliferating lymphocytes by 1H-NMR of acid extracts

Nuclear magnetic resonance (NMR) studies of extracts have proven to be a powerful window onto the intracellular machinery of cells and tissues. The major advantages of in vitro 1H-NMR, namely chemical preservation, simultaneous detection, identification, and quantitation of compounds, and sensitivity to a large variety of classes of compounds, are employed in this study to characterize the metabolic course of mitogen-stimulated proliferation of human peripheral lymphocytes. A reliable method to quantitate amino acids, metabolic intermediates, soluble membrane lipid precursors, and purine, pyridine and pyrimidine nucleotides is presented, using samples as small as 30 mg wet weight. A total of 53 substances were detected in lymphocytes and other blood cells. During the course of lymphocyte culture, changes in intracellular concentrations of lactate, taurine, inositol and nucleotides, including NAD, IMP and high-energy phosphates, were especially marked. 1H-NMR compares favorably to 31P-NMR and to HPLC, and is especially attractive in light of expectations for future in vivo application.     

SMOC1 is a tenascin-C interacting protein over-expressed in brain tumors

Tenascin-C is an extracellular matrix protein over-expressed in a large variety of cancers. In the present study, we aimed at identifying new interactors of tenascin-C by purifying secreted proteins on a tenascin-C affinity column. Analysis of eluates by mass spectrometry revealed phosphoglycerate kinase 1, clusterin, fibronectin, SPARC-related modular calcium-binding protein 1 (SMOC1) and nidogen-2 as potential interactors of tenascin-C. The interaction between tenascin-C and SMOC1 was confirmed by co-immunoprecipitation and further analyzed by Surface Plasmon Resonance Spectroscopy, which revealed an apparent dissociation constant (K_D) value of 2.59 ∗ 10⁻⁹M. Further analyses showed that this binding is reduced in the presence of EDTA. To investigate whether SMOC1 itself could be over-expressed in the context of tumorigenesis, we analyzed data of two independent RNA profiling studies and found that mRNA levels of SMOC1 are significantly increased in oligodendrogliomas compared to control brain samples. In support of these data, western blot analysis of protein extracts from 12 oligodendrogliomas, 4 astrocytomas and 13 glioblastomas revealed elevated levels compared to healthy brain extract. Interestingly, cell migration experiments revealed that SMOC1 can counteract the chemo-attractive effect of tenascin-C on U87 glioma cells. The present study thus identified SMOC1 as a new cancer-associated protein capable of interacting with tenascin-C in vitro.     

Effect of lipid peroxidation on molecular arrangement of phospholipids in liposomes prepared from egg yolk phosphatidylcholine or total rat brain lipids. A 31P NMR study.

Changes in molecular arrangement of membrane phospholipids in the course of lipid autoxidation were studied by means of broad-band 31-P NMR spectroscopy. Multilamellar liposomes prepared from egg yolk phosphatidylcholine (PC) or total lipid extracts from rat brains (TL) were used as models. The initial lamellar arrangement of phospholipids of both types changed as lipid peroxidation proceeded and a narrow isotropic signal appeared in the spectra at 0 ppm, this phenomenon being more prominent for TL than for PC. Probably the isotropic signal represents some nonlamellar structures within the membranes of peroxidized lipids.     

Assessment of in vivo oxidative lipid metabolism following acute microcystin-LR-induced hepatotoxicity in rats

Oxidative lipid metabolism as a result of acute cyanobacterial toxin-induced hepatotoxicity was monitored in male Sprague-Dawley rats using electron spin resonance (ESR) spectroscopy and image-guided proton nuclear magnetic resonance (1H-NMR) spectroscopy. ESR spectroscopy, coupled with spin trapping, was used to trap and detect lipid-derived radicals, formed in rat livers after acute in vivo exposure (LD50) to the cyanobacterial toxin, microcystin-LR (MCLR). A statistically significant increase in the levels (spectral peak integrals) of lipid radicals was detected in MCLR-treated livers (p < 0.05) (n = 8), in comparison to control livers (n = 6). In order to monitor lipid metabolism, before and for a period of 3 h, following toxin exposure, in vivo proton image-guided NMR spectroscopy was used. A statistically significant decrease in the levels of lipid methylene hydrogen resonances (spectral peak integrals) was observed from MCLR-treated livers (n = 6) 2 and 3 h post-exposure (p < 0.05), in comparison to controls (n = 6). Image-guided NMR spectroscopy was also used to detect significant decreasing levels of in vivo glutamine/glutamate, following exposure to MCLR. Biochemical assessment of perchloric extracts of liver glutamine and glutamate levels correlated with NMR spectroscopy results. Lactate levels measured as perchloric acid extracts, were also found to significantly decrease. In addition, assessment of serum enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were used to confirm hepatotoxicity (n = 20). This study strongly suggests that oxidative stress related processes are involved in in vivo microcystin-induced hepatotoxicity in mammals, and may play an integral role in MCLR-induced toxicity.     

Protein Tyrosine Kinases in Human Brain and Gliomas

Tyrosine kinase activity was determined in neonatal and adult human brain, oligodendrogliomas, and astrocytomas. The astrocytomas were divided into low- (grade I and grade II) and high-grade (grade III and grade IV) tumors. We measured the tyrosine kinase activity in the cytosolic and membrane fraction using poly(glutamic acid:tyrosine, 4:1) as an artificial substrate. The cytosolic activity in oligodendrogliomas (n = 7), low-grade astrocytomas (n = 7), and neonatal brain (n = 1) was increased, on average, two- to fourfold compared with that in normal adult brain (n = 14). The cytosolic activities of high-grade astrocytomas (n = 11) were in approximately the same range as found in normal adult brain. The absence of an increase in cytosolic activity in high-grade astrocytomas compared with adult brain is likely due to the occurrence of necrosis in these tumors. In contrast to the cytosolic activity, no differences were found in the membrane-bound activity. By fast protein liquid chromatography, at least three forms of cytosolic protein tyrosine kinase could be separated, which eluted at 0, 115, and 210 mM NaCl. In most cases the highest amount of activity eluted at 210 mM NaCl. However, in oligodendrogliomas, high-grade astrocytomas, and neonatal brain, more activity eluted at 115 mM NaCl than in normal adult brain (p = 0.043). Nevertheless, protein tyrosine kinases from all three peaks contributed to the elevated levels of total cytosolic activity of oligodendrogliomas and low-grade astrocytomas.     

Glioma-associated antigen expression in oligodendroglial neoplasms. Tenascin and epidermal growth factor receptor.

Epidermal growth factor receptor (EGFR), its variant, EGFRvIII, and tenascin are glioma-associated antigens that are hyperexpressed by neoplastic glial cells relative to normal brain, making them attractive antigenic targets for immunotherapy. Preliminary surveys indicate that oligodendroglial tumors also produce these proteins, although the exact patterns and degrees of reactivity are not known. In this study we examined the immunoreactivity of tenascin among 50 oligodendroglial tumors, including 25 well-differentiated oligodendrogliomas (WDOs) and 12 glioblastomas (GBMs) exhibiting high proportions of oligodendroglia-like cells. We used well-characterized immunoreagents with defined specificities against the target antigens on formalin-fixed, paraffin-embedded archival tissue. The tumors were graded according to WHO guidelines. Immunoreactivity was reported on a 1-3 scale according to staining intensity multiplied by a 1-3 distribution scale distribution within tumor as focal (1), multifocal (2), and diffuse (3) for both the parenchymal and the perivascular components. Although there is considerable overlap in antigen production among the grades of tumor, this study establishes the production of tenascin and wild-type EGFR (but not EGFR vIII) in oligodendroglial neoplasms and supports the concept that antigen production increases with tumor grade.     

The lipid composition of isolated cytoplasmic lipid droplets from a human cancer cell line, BE(2)M17

(1)H nuclear magnetic resonance spectroscopy (NMR) resonances from lipids in tumours are associated with tumour grade and treatment response. The origin of these NMR signals is mainly considered to be cytoplasmic lipid droplets (LDs). Techniques exist for isolating LDs but little is known about their composition and its relationship to NMR signals. In this work, density-gradient ultracentrifugation was performed on homogenised human cancer cells to isolate LDs. (1)H NMR was performed on whole cells, isolated LDs and their extracts. Heteronuclear single quantum coherence spectroscopy (HSQC) and liquid chromatography mass spectroscopy (LC-MS) were performed on lipid extracts of LDs. Staining and microscopy were used to characterize isolated LDs. An excellent agreement in chemical shift and relative signal intensity was observed between lipid resonances in cells and isolated LD spectra supporting that NMR-visible lipids originate primarily from LDs. Isolated LDs showed high concentrations of unsaturated lipids, a oleic-to-linoleic acid ratio greater than two and a cholesteryl ester (ChE)-to-cholesterol (Ch) ratio close to unity. These ratios were several-fold greater than respective ratios in whole cells, demonstrating isolation is important to characterize LD composition. LDs contain a specific group of lipid species that are likely to contribute to the (1)H NMR spectrum of cells.     

Grade classification of neuroepithelial tumors using high-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy and pattern recognition

Clinical data have shown that survival rates vary considerably among brain tumor patients, according to the type and grade of the tumor. Metabolite profiles of intact tumor tissues measured with high-resolution magic-angle spinning proton nuclear magnetic resonance spectroscopy (HRMAS (1)H NMRS) can provide important information on tumor biology and metabolism. These metabolic fingerprints can then be used for tumor classification and grading, with great potential value for tumor diagnosis. We studied the metabolic characteristics of 30 neuroepithelial tumor biopsies, including two astrocytomas (grade I), 12 astrocytomas (grade II), eight anaplastic astrocytomas (grade III), three glioblastomas (grade IV) and five medulloblastomas (grade IV) from 30 patients using HRMAS (1)H NMRS. The results were correlated with pathological features using multivariate data analysis, including principal component analysis (PCA). There were significant differences in the levels of N-acetyl-aspartate (NAA), creatine, myo-inositol, glycine and lactate between tumors of different grades (P<0.05). There were also significant differences in the ratios of NAA/creatine, lactate/creatine, myo-inositol/creatine, glycine/creatine, scyllo-inositol/creatine and alanine/creatine (P<0.05). A soft independent modeling of class analogy model produced a predictive accuracy of 87% for high-grade (grade III-IV) brain tumors with a sensitivity of 87% and a specificity of 93%. HRMAS (1)H NMR spectroscopy in conjunction with pattern recognition thus provides a potentially useful tool for the rapid and accurate classification of human brain tumor grades.     

Metabolite changes in BT4C rat gliomas undergoing ganciclovir-thymidine kinase gene therapy-induced programmed cell death as studied by 1H NMR spectroscopy in vivo,ex vivo,and in vitro

Programmed cell death was induced by HSV-tk gene therapy in rat BT4C glioma cells, and metabolite changes associated with cell damage were monitored in vivo by 1H NMR spectroscopy and ex vivo by high resolution magic angle spinning (HRMAS) 1H NMR, and in vitro in perchloric acid extracts of tumors. Metabolite concentrations, as quantified in vivo using water as an internal reference and in vitro in extracts, were correlated with cell density. The results showed that both in vivo and in vitro glycine and creatine concentrations followed volume-averaged cell density, whereas that of total choline-containing compounds was unaffected by a cell loss approaching 60%. Meanwhile, both saturated and unsaturated 1H NMR visible lipids increased. HRMAS 1H NMR spectroscopy of the tumor samples at 14.1 tesla demonstrated the presence of nucleotide peaks from adenosine and uridine nucleotides in glioma samples ex vivo. The assignment of a doublet at 7.95 ppm to UDP was confirmed by spiking experiments of tumor extracts in conjunction with 1H and 31P NMR spectroscopy. HRMAS also resolved the choline-containing peak at 3.2 ppm in vivo into resonances from choline (3.20 ppm), phosphocholine (3.22 ppm), glycerophosphocholine (3.24 ppm), and taurine (3.26 ppm). These resonances were uncorrelated with temporal progression through programmed cell death. Our results show that 1H NMR-detected lipids and some of the small molecular weight metabolites respond to gene therapy. However, the choline-containing compounds are unaffected by severe decline in cell density. The latter observation supports the idea that triacylglycerols, rather than membrane phospholipids, are the key components of 1H NMR visible lipids, and it also casts doubt on the validity of resonance of choline-containing compounds as a diagnostic marker of programmed cell death in vivo.     

Epithelial differentiation in gliomas,meningiomas and choroid plexus papillomas

The immunohistological findings using antibodies to different intermediate filaments (glial fibrillary acidic protein, vimentin and two types of cytokeratin) and epithelial membrane antigen are described in 89 gliomas, 19 meningiomas and 8 choroid plexus papillomas (CPPs) from adult patients. All the patients had total or subtotal surgical excision of their tumours with clinical follow up for between 3 and 7 years. The immunohistological results were correlated with the histological features and patient survival. Tumours other than low grade astrocytomas, oligodendrogliomas and anaplastic ependymomas expressed one or more epithelial markers. This immunohistological evidence of epithelial differentiation in the absence of histological epithelial features in gliomas confirms that the two are not necessarily correlated. It is concluded that the expression of epithelial markers in some intradural tumours may reflect aberrant differentiation related to the degree of anaplasia in poorly differentiated astrocytomas and glioblastomas. All the patients with anaplastic epithelial marker-positive gliomas died within 1 year, whereas only 68% of patients with marker-negative tumours died within the follow-up period. In ependymomas and meningiomas, the expression of epithelial markers may reflect their histogenesis, while in malignant CPPs such expression could denote either their aberrant differentiation or histogenetic derivation.