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The 4(th) edition of the WHO Classification of Tumours of the Nervous System (WHO 2007) introduces changes that reflect both the recognition of new brain tumour types and a better understanding of neoplastic behavior. Three new tumours, angiocentric glioma (AG), pilomyxoid astrocytoma (PMA), and pituicytoma are added to the section on gliomas. AG is a slowly growing cerebral tumour that typically presents with seizures in children and young adults. It is characterized by monomorphous, bipolar tumour cells with a striking perivascular growth pattern. Although the 'cell of origin' of AG is not clear, ultrastructural evidence points to an ependymal derivation. Typically, AG can be cured by total resection, and is designated WHO grade I. PMA is a solid, circumscribed tumour occurring mainly in the hypothalamic region of young children. It is composed of a monomorphous population of bipolar tumour cells within a rich myxoid background, with a conspicuous anglocentric arrangement. While PMA is considered a more aggressive variant of pilocytic astrocytoma, this relationship awaits further clarification. The PMA has been designated WHO grade II. The pituicytoma, involves the posterior pituitary and/or its stalk and affects adults. It is solid in architecture, composed of spindle cells and presumably derived from pituicytes. Pituicytomas are indolent tumours, and are designated WHO grade I.

Objective: Astroblastomas are rare glial neoplasms that usually occur in young adults and have a predilection for the cerebral hemispheres. Patients typically present with signs of increased intracranial pressure and seizures. Imaging studies reveal circumscribed, contrast-enhancing tumors that contain both cystic and solid components with variable peritumoral edema. Hemorrhage, which suggested the presence of a vascular lesion in this patient, has not been previously described as a feature of this neoplasm., Clinical Presentation: The authors report the case of a 33-year-old woman who presented with spontaneous intraparenchymal hemorrhage. The collective radiographic data suggested the presence of a cavernous malformation., Intervention: A right frontotemporal craniotomy was performed under frameless stereotactic image guidance. An astroblastoma was diagnosed after resection and neuropathological examination., Conclusion: A rare radiological to pathological correlation of astroblastoma is presented in which the evolving hematoma, as observed on magnetic resonance imaging scans, complicated the radiographic diagnosis of this lesion. The clinical, radiographic, and pathological features of astroblastomas, as well as the natural history of these rare glial neoplasms, are reviewed. This case illustrates the capacity of astroblastomas to hemorrhage, disguising the classic radiographic findings typical of this glial neoplasm.

Grade 4 malignant glioma (GBM) is a fatal disease despite aggressive surgical and adjuvant therapies. The hallmark of GBM tumors is the presence of pseudopalisading necrosis and microvascular proliferation. These tumor cells are hypoxic and express hypoxia-inducible factor-1 (HIF-1), a prosurvival transcription factor that promotes formation of neovasculature through activation of target genes, such as vascular endothelial growth factor. Here, we evaluated whether 2-methoxyestradiol, a microtubule and HIF-1 inhibitor, would have therapeutic potential for this disease in a 9L rat orthotopic gliosarcoma model using a combination of noninvasive imaging methods: magnetic resonance imaging to measure the tumor volume and bioluminescence imaging for HIF-1 activity. After imaging, histologic data were subsequently evaluated to elucidate the drug action mechanism in vivo. Treatment with 2-methoxyestradiol (60-600 mg/kg/d) resulted in a dose-dependent inhibition of tumor growth. This effect was also associated with improved tumor oxygenation as assessed by pimonidazole staining, decreased HIF-1alpha protein levels, and microtubule destabilization as assessed by deacetylation. Our results indicate that 2-methoxyestradiol may be a promising chemotherapeutic agent for the treatment of malignant gliomas, with significant growth inhibition. Further studies are needed to assess the effect of low or intermediate doses of 2-methoxyestradiol in combination with chemotherapeutic agents in clinical studies focused on malignant gliomas. In addition to showing tumor growth inhibition, we identified three potential surrogate biomarkers to determine the efficacy of 2-methoxyestradiol therapy: decreased HIF-1alpha levels, alpha-tubulin acetylation, and degree of hypoxia as determined by pimonidazole staining.

CNS diseases are often accompanied by changes in the protein composition of cerebrospinal fluid (CSF). SELDI-TOF-MS provides an approach for identifying specific protein markers of disease in biological fluids. We compared the CSF proteomes from patients with neoplastic and reactive/inflammatory CNS diseases to identify potential biomarkers. SELDI-TOF-MS was performed on CSF derived from lumbar puncture of 32 patients, including 10 with CNS malignancies, 12 with inflammatory or reactive conditions, and 10 with unknown CNS disease. Using the SAX-2 (strong anionic exchange) chip, we uncovered three conserved protein peak ranges within each disease category. For neoplastic diseases, we identified conserved peaks at 7.5-8.0 kDa (9/10 samples), 15.1-15.9 kDa (8/10 samples), and 30.0-32.0 kDa (5/10 samples). In reactive/inflammatory diseases, conserved peaks were found at 6.7-7.1 kDa (10/12 samples), 11.5-11.9 kDa (12/12 samples), and 13.3-13.7 kDa (9/12 samples). A protein from the 30.0 to 32.0 kDa peak range found in neoplastic CSF was identified by MALDI analysis as carbonic anhydrase, a protein overexpressed in many malignancies including high-grade gliomas. Similarly, cystatin C was identified in the 13.3-13.7 kDa peak range in non-neoplastic CSF and was most prominent in inflammatory conditions. Our approach provides a rational basis for identifying biomarkers that could be used for detection, diagnosis, and monitoring of CNS diseases.

Purpose: There are a limited number of noninvasive methods available for the monitoring of neoplastic disease in the central nervous system. The goal of our study was to find reliable markers that could be used for disease monitoring as well as to identify new targets for the therapeutic intervention for malignant astrocytoma (WHO grades 3 and 4)., Experimental Design: We employed proteomic techniques to identify secreted proteins in the cerebrospinal fluid that were specific to patients with malignant astrocytoma., Results: Among 60 cerebrospinal fluid samples of patients with various central nervous system diseases, attractin was consistently found to be elevated in the samples of patients with malignant astrocytoma. To independently validate these results, we examined attractin expression in a new set of 108 normal and tumoral brain tissue specimens and found elevated expression in 97% of malignant astrocytomas, with the highest levels in grade 4 tumors. Using immunohistochemistry, we further showed that attractin is produced and secreted by the tumor cells. Finally, we showed that cerebrospinal fluid from brain tumor patients induces glioma cell migration and that attractin is largely responsible for this promigratory activity., Conclusions: Our results find attractin to be a reliable secreted marker for high-grade gliomas. Additionally, our migration studies suggest that it may be an important mediator of tumor invasiveness, and thus, a potential target in future therapies.

Hypoxia strongly up-regulates tissue factor and promotes plasma clotting by glioblastoma multiforme, but transcriptional mechanisms remain undefined. Here, we investigated the potential roles of early growth response gene-1 (Egr-1), Sp1, nuclear factor-kappaB (NF-kappaB), activator protein-1 (AP-1), and hypoxia-inducible factor-1 (HIF-1) in the hypoxic regulation of tissue factor by glioblastoma multiforme cells in vitro. Hypoxia (1% O2) strongly induced Egr-1 mRNA within 1 hour and led to nuclear localization of Egr-1 protein. Using luciferase reporter plasmids in glioma cells, we found that hypoxia dramatically increased luciferase activity in cells with constructs containing Egr-1-binding sites but not in cells with constructs containing AP-1- or NF-kappaB-binding sites. Electrophoretic mobility shift assays revealed hypoxia-induced Egr-1, but not Sp1, binding to oligonucleotides containing the Egr-1/Sp1 motif of tissue factor gene promoter. Using an expression vector containing the minimal tissue factor promoter (-111 to +14 bp) and small interfering RNA (siRNA) directed at Egr-1 and Sp1 mRNAs, we found that Egr-1 was required for maximal hypoxic induction of promoter activity. Forced overexpression of Egr-1 but not Sp1 by cDNA transfection caused up-regulation of tissue factor in glioma cells under normoxia (21% O2), whereas siRNA directed at Egr-1 strongly attenuated hypoxia-induced tissue factor expression. To examine the effects of HIF-1alpha on tissue factor expression, we used glioma cells stably transfected with a HIF-1alpha siRNA expression vector and found that HIF-1alpha mRNA silencing did not affect tissue factor expression under hypoxia. We conclude that hypoxic up-regulation of tissue factor in glioblastoma multiforme cells depends largely on Egr-1 and is independent of HIF-1.

Glioblastoma (GBM) is a highly malignant, rapidly progressive astrocytoma that is distinguished pathologically from lower grade tumors by necrosis and microvascular hyperplasia. Necrotic foci are typically surrounded by "pseudopalisading" cells-a configuration that is relatively unique to malignant gliomas and has long been recognized as an ominous prognostic feature. Precise mechanisms that relate morphology to biologic behavior have not been described. Recent investigations have demonstrated that pseudopalisades are severely hypoxic, overexpress hypoxia-inducible factor (HIF-1), and secrete proangiogenic factors such as VEGF and IL-8. Thus, the microvascular hyperplasia in GBM that provides a new vasculature and promotes peripheral tumor expansion is tightly linked with the emergence of pseudopalisades. Both pathologic observations and experimental evidence have indicated that the development of hypoxia and necrosis within astrocytomas could arise secondary to vaso-occlusion and intravascular thrombosis. This emerging model suggests that pseudopalisades represent a wave of tumor cells actively migrating away from central hypoxia that arises after a vascular insult. Experimental glioma models have shown that endothelial apoptosis, perhaps resulting from angiopoetin-2, initiates vascular pathology, whereas observations in human tumors have clearly demonstrated that intravascular thrombosis develops with high frequency in the transition to GBM. Tissue factor, the main cellular initiator of thrombosis, is dramatically upregulated in response to PTEN loss and hypoxia in human GBM and could promote a prothrombotic environment that precipitates these events. A prothrombotic environment also activates the family of protease activated receptors (PARs) on tumor cells, which are G-protein-coupled and enhance invasive and proangiogenic properties. Vaso-occlusive and prothrombotic mechanisms in GBM could readily explain the presence of pseudopalisading necrosis in tissue sections, the rapid peripheral expansion on neuroimaging, and the dramatic shift to an accelerated rate of clinical progression resulting from hypoxia-induced angiogenesis.

Expression of to nuclear antigen Ki-67 (MIB-1) has been linked to proliferative activity and prognosis in a variety of tumors. The authors assessed three techniques for quantitating MIB-1 (expression in oligodendrogliomas, correlating results with mitotic activity and prognosis. Formalin-fixed, paraffin-embedded sections of 38 oligodendrogliomas were immunostained using monoclonal MIB-l. Proliferation index (PI) was quantitated by visual estimation, CAS-200, and AC1S image analysis. MIB-1 expression and mitotic count were correlated with overall survival and recurrence (disease-free survival), defined clinically and radiographically as new tumor growth. Mean follow-up was 54 months (range 1-276). Mean PI quantitated by the three methods was statistically similar (Visual 10.5%, CAS-200, 12.2%, CAIS 11.2%). PI results by all three techniques correlated significantly with each other; visual and CAS-200 PI correlated with mitotic index. Overall and disease-free survivals were similar for patients with PIs above and below the mean by both image cytometric assays; visually estimated PIs below the mean, versus above the mean, correlated with improved disease-free survival. The authors show a significant correlation between MIB-1 PI using the visual method and recurrence in patients with oligodendrogliomas. The objectivity and speed of the image analysis systems make them an attractive alternative to visual estimation, and larger series should be analyzed for prognostic value.

Diffuse gliomas are the most common brain tumors and include astrocytomas, oligodendrogliomas, and oligoastrocytomas. Their correct pathologic diagnosis requires the ability to distinguish astrocytic from oligodendroglial differentiation in histologic sections, a challenging feat even for the most experienced neuropathologist. Interobserver variability in the diagnosis of diffuse gliomas has been high owing to subjective diagnostic criteria, overlapping morphologic features, and variations in training and practice among pathologists. A select, albeit imperfect, group of molecular and immunohistochemical tests are available to assist in diagnosis of these lesions. Combined loss of chromosomes 1p and 19q is a genetic signature of oligodendrogliomas, whereas gains of chromosome 7 in the setting of intact 1p/19q are more typical of astrocytomas. Detection of amplified epidermal growth factor receptor favors the diagnosis of high-grade astrocytomas over anaplastic oligodendroglioma, which is especially relevant for small cell astrocytomas. Strong nuclear staining for p53 often reflects TP53 mutation and is typical of low-grade astrocytomas. The Olig family of transcription factors has not demonstrated their diagnostic usefulness. Diffuse gliomas remain a diagnostic challenge, and new markers are needed for proper classification and directed therapies.

Background: Prolonged deep hypothermic circulatory arrest is known to cause neurological injury. Hypoxia inducible factor, a transcription factor that mediates adaptive changes during hypoxia, is neuroprotective in models of ischemic brain injury, in part by upregulating erythropoietin. This study tested the hypothesis that upregulation of hypoxia inducible factor and erythropoietin by preconditioning with hypoxia or the hypoxia-mimetic agents deferoxamine and cobalt chloride would be neuroprotective in a piglet model of deep hypothermic circulatory arrest., Methods: Anesthetized neonatal piglets were randomized to 4 preconditioning groups (15 per group): hypoxia, deferoxamine, cobalt chloride, or control (NaCl vehicle). Brain hypoxia inducible factor and erythropoietin contents were assessed by means of Western blotting at 3, 8, and 24 hours after treatment (n = 3 per time point). Twenty-four hours after treatment, 6 to 7 animals per group underwent cardiopulmonary bypass and 110 minutes of deep hypothermic circulatory arrest. After recovery, serial neurobehavioral examinations were conducted for 6 days, after which histopathologic brain injury and neuronal apoptosis (cleaved caspase 3) were assessed., Results: Erythropoietin expression was not significantly increased by any of the pretreatment strategies. In contrast, there was a significant upregulation of hypoxia inducible factor by pretreatment with deferoxamine and cobalt chloride (P = .002). Neurobehavioral measures revealed no significant differences in time to recovery or extent of injury. Examination of histopathologic brain injury in the hippocampus revealed that pretreatment with deferoxamine (0.4 +/- 0.3) and cobalt chloride (0.5 +/- 0.3) were associated with significantly less neuronal loss than pretreatment with hypoxia or control (2.8 +/- 0.5, P = .004). Finally, cleaved caspase 3 (a marker of apoptotic cell death) was also shown to be diminished in the cobalt and deferoxamine groups, but the difference was not significantly different from the value in the control group., Conclusions: In contrast to hypoxia, deferoxamine and cobalt chloride preconditioning upregulated hypoxia inducible factor and were associated with histopathologic neuroprotection after exposure to cardiopulmonary bypass and prolonged deep hypothermic circulatory arrest.

The neuronal PAS domain protein 3 (NPAS3) gene encoding a brain-enriched transcription factor was recently found to be disrupted in a family suffering from schizophrenia. Mice harboring compound disruptions in the NPAS3 and related NPAS1 genes manifest behavioral and neuroanatomical abnormalities reminiscent of schizophrenia. Herein we demonstrate that Npas3-/- mice are deficient in expression of hippocampal FGF receptor subtype 1 mRNA, most notably in the dentate gyrus. In vivo BrdUrd-labeling shows that basal neural precursor cell proliferation in the dentate gyrus of Npas3-/- mice is reduced by 84% relative to wild-type littermates. We propose that a deficiency in adult neurogenesis may cause the behavioral and neuroanatomical abnormalities seen in Npas3-/- mice, and we speculate that impaired neurogenesis may be involved in the pathophysiology of schizophrenia.

The authors report on 32-year-old woman with a history of a previously resected suprasellar clear cell meningioma (CCM), who returned to their institution after 3 years suffering from progressively worsening leg and back pain associated with leg weakness and bowel and bladder dysfunction. A magnetic resonance image of the thoracic and lumbar spine demonstrated a homogeneously enhancing intradural mass that filled and expanded the thecal sac. The patient underwent multiple-level laminectomies for resection of the lesion. Results of pathological studies confirmed distant recurrence of a CCM. Since its initial recognition as a rare but aggressive histological variant of meningothelial tumors, the body of literature on CCMs has grown to include more than 40 cases. Nevertheless, the natural history of this neoplastic entity remains ill defined, as are the recommendations for management. Of particular concern is the treatment of patients who have undergone subtotal resection or present with recurrence. To the authors' knowledge, the present case represents the sixth distant recurrence of CCM reported in the literature. The radiographic and histological studies are reviewed along with the current literature on this subtype of meningioma. Recommendations for surveillance and treatment are made.

Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with unknown function expressed primarily in normal but not tumoral brain. The finding of thrombospondin type 1 repeats in its extracellular domain suggested an antiangiogenic function, but the mechanisms by which a transmembrane receptor could inhibit angiogenesis remained unexplained. Here we demonstrate that BAI1 is proteolytically cleaved at a conserved G-protein-coupled receptor proteolytic cleavage site (GPS), releasing its 120 kDa extracellular domain. We named this secreted fragment Vasculostatin as it inhibited migration of endothelial cells in vitro and dramatically reduced in vivo angiogenesis. Both constitutive and doxycycline-induced expression of Vasculostatin elicited dose-dependent suppression of tumor growth and vascular density in mice, implicating Vasculostatin in the regulation of vascular homeostasis and tumor prevention. Generation of a soluble antiangiogenic factor by cleavage of a pre-existing transmembrane protein represents a novel mechanism for regulating vascular homeostasis and preventing tumorigenesis. Modulation of this cleavage or delivery of Vasculostatin may constitute novel treatment modalities for cancer and other diseases of aberrant angiogenesis, especially in the brain.

Interleukin-8 (IL-8, or CXCL8), which is a chemokine with a defining CXC amino acid motif that was initially characterized for its leukocyte chemotactic activity, is now known to possess tumorigenic and proangiogenic properties as well. In human gliomas, IL-8 is expressed and secreted at high levels both in vitro and in vivo, and recent experiments suggest it is critical to glial tumor neovascularity and progression. Levels of IL-8 correlate with histologic grade in glial neoplasms, and the most malignant form, glioblastoma, shows the highest expression in pseudopalisading cells around necrosis, suggesting that hypoxia/anoxia may stimulate expression. In addition to hypoxia/anoxia stimulation, increased IL-8 in gliomas occurs in response to Fas ligation, death receptor activation, cytosolic Ca(2+), TNF-alpha, IL-1, and other cytokines and various cellular stresses. The IL-8 promoter contains binding sites for the transcription factors NF-kappaB, AP-1, and C-EBP/NF-IL-6, among others. AP-1 has been shown to mediate IL-8 upregulation by anoxia in gliomas. The potential tumor suppressor ING4 was recently shown to be a critical regulator of NF-kappaB-mediated IL-8 transcription and subsequent angiogenesis in gliomas. The IL-8 receptors that could contribute to IL-8-mediated tumorigenic and angiogenic responses include CXCR1 and CXCR2, both of which are G-protein coupled, and the Duffy antigen receptor for cytokines, which has no defined intracellular signaling capabilities. The proangiogenic activity of IL-8 occurs predominantly following binding to CXCR2, but CXCR1 appears to contribute as well through independent, small-GTPase activity. A precise definition of the mechanisms by which IL-8 exerts its proangiogenic functions requires further study for the development of effective IL-8-targeted therapies.

Glioblastomas, like other solid tumors, have extensive areas of hypoxia and necrosis. The importance of hypoxia in driving tumor growth is receiving increased attention. Hypoxia-inducible factor 1 (HIF-1) is one of the master regulators that orchestrate the cellular responses to hypoxia. It is a heterodimeric transcription factor composed of alpha and beta subunits. The alpha subunit is stable in hypoxic conditions but is rapidly degraded in normoxia. The function of HIF-1 is also modulated by several molecular mechanisms that regulate its synthesis, degradation, and transcriptional activity. Upon stabilization or activation, HIF-1 translocates to the nucleus and induces transcription of its downstream target genes. Most important to gliomagenesis, HIF-1 is a potent activator of angiogenesis and invasion through its upregulation of target genes critical for these functions. Activation of the HIF-1 pathway is a common feature of gliomas and may explain the intense vascular hyperplasia often seen in glioblastoma multiforme. Activation of HIF results in the activation of vascular endothelial growth factors, vascular endothelial growth factor receptors, matrix metalloproteinases, plasminogen activator inhibitor, transforming growth factors alpha and beta, angiopoietin and Tie receptors, endothelin-1, inducible nitric oxide synthase, adrenomedullin, and erythropoietin, which all affect glioma angiogenesis. In conclusion, HIF is a critical regulatory factor in the tumor microenvironment because of its central role in promoting proangiogenic and invasive properties. While HIF activation strongly promotes angiogenesis, the emerging vasculature is often abnormal, leading to a vicious cycle that causes further hypoxia and HIF upregulation.

Tuberous sclerosis (TS) is a common autosomal dominant disorder caused by loss or malfunction of hamartin (tsc1) or tuberin (tsc2). Many lesions in TS do not demonstrate loss of heterozygosity for these genes, implying that dominant negative forms of these genes may account for some hamartomas and neoplasms in TS. To test this hypothesis, we expressed a dominant negative allele of tuberin (DeltaRG) behind the cytomegalovirus promoter in NIH3T3 cells and transgenic mice. This allele binds hamartin but has a deletion in the C terminus of tuberin, leading to constitutive activation of rap1 and rab5/rabaptin. Expression of DeltaRG in NIH3T3 cells led to a strong induction of reactive oxygen species, induction of vascular endothelial growth factor, and malignant transformation in vivo. Expression of DeltaRG driven by the constitutive cytomegalovirus promoter led to high level expression in all murine tissues examined, including skin, kidney, liver, and brain. Surprisingly, mice expressing the DeltaRG transgene developed a fibrovascular collagenoma in the dermis, which closely resembles the Shagreen patch observed in human patients with TS. In addition, numerous small subpial collections of external granule cells in the cerebellum were observed, which may be the murine equivalent of subependymal giant cell astrocytomas or tubers commonly seen in TS patients. Thus, expression of a dominant negative tuberin in multiple tissues can lead to a tissue-specific phenotype resembling some of the findings in human TS. Our data are the first to demonstrate that specific signaling abnormalities underlie specific hamartomas in a model of a human genetic disorder.

We have previously proposed that intravascular thrombosis and subsequent vasoocclusion contribute to the development of pseudopalisading necrosis, a pathologic hallmark that distinguishes glioblastoma (WHO grade 4) from lower grade astrocytomas. To better understand the potential prothrombotic mechanisms underlying the formation of these structures that drive tumor angiogenesis, we investigated tissue factor (TF), a potent procoagulant protein known to be overexpressed in astrocytomas. We hypothesized that PTEN loss and tumor hypoxia, which characterize glioblastoma but not lower grade astrocytomas, could up-regulate TF expression and cause intravascular thrombotic occlusion. We examined the effect of PTEN restoration and hypoxia on TF expression and plasma coagulation using a human glioma cell line containing an inducible wt-PTEN cDNA. Cell exposure to hypoxia (1% O(2)) markedly increased TF expression, whereas restoration of wt-PTEN caused decreased cellular TF. The latter effect was at least partially dependent on PTEN's protein phosphatase activity. Hypoxic cells accelerated plasma clotting in tilt tube assays and this effect was prevented by both inhibitory antibodies to TF and plasma lacking factor VII, implicating TF-dependent mechanisms. To further examine the genetic events leading to TF up-regulation during progression of astrocytomas, we investigated its expression in a series of human astrocytes sequentially infected with E6/E7/human telomerase, Ras, and Akt. Cells transformed with Akt showed the greatest incremental increase in hypoxia-induced TF expression and secretion. Together, our results show that PTEN loss and hypoxia up-regulate TF expression and promote plasma clotting by glioma cells, suggesting that these mechanisms may underlie intravascular thrombosis and pseudopalisading necrosis in glioblastoma.

Hypoxia plays a critical role in driving tumor malignancy and is associated with poor patient survival in many human cancers. Novel therapies targeting hypoxic tumor cells are urgently needed, because these cells hinder tumor eradication. Here we demonstrate than an anticancer strategy based on intratumoral delivery of a novel type of oncolytic adenovirus targeting tumor hypoxia is therapeutically efficient and can augment standard chemotherapy. We used a conditionally replicative adenovirus (HYPR-Ad) to specifically kill hypoxic tumor cells. Viral infection and conditional replication occurred efficiently in hypoxic/hypoxia-inducible factor-active cells in culture and in vivo, prevented tumor formation, and reduced the growth of established tumors. Combining HYPR-Ad with chemotherapy effective against normoxic cells resulted in strongly enhanced antitumor efficacy. These studies demonstrate that targeting the hypoxic microenvironment of tumors rather than an intrinsic gene expression defect is a viable and novel antitumor therapeutic strategy that can be used in combination with existing treatment regimens. The replication and oncolytic potential of this virus was made dependent on hypoxic/hypoxia-inducible factor, a transcription factor activated in the tumor hypoxic microenvironment, broadening its therapeutic use to solid tumors of any genetic make-up or tissue of origin.

The role of Notch signaling in tumorigenesis can vary; Notch1 acts as an oncogene in some neoplasms, and a tumor suppressor in others. Here, we show that different Notch receptors can have opposite effects in a single tumor type. Expression of truncated, constitutively active Notch1 or Notch2 in embryonal brain tumor cell lines caused antagonistic effects on tumor growth. Cell proliferation, soft agar colony formation, and xenograft growth were all promoted by Notch2 and inhibited by Notch1. We also found that Notch2 receptor transcripts are highly expressed in progenitor cell-derived brain tumors such as medulloblastomas, whereas Notch1 is scarce or undetectable. This parallels normal cerebellar development, during which Notch2 is predominantly expressed in proliferating progenitors and Notch1 in postmitotic differentiating cells. Given the oncogenic effects of Notch2, we analyzed its gene dosage in 40 embryonal brain tumors, detecting an increased copy number in 15% of cases. Notch2 gene amplification was confirmed by fluorescence in situ hybridization in one case with extremely high Notch2 mRNA levels. In addition, expression of the Notch pathway target gene Hes1 in medulloblastomas was associated with significantly shorter patient survival (P = 0.01). Finally, pharmacological inhibition of Notch signaling suppresses growth of medulloblastoma cells. Our data indicate that Notch1 and Notch2 can have opposite effects on the growth of a single tumor type, and show that Notch2 can be overexpressed after gene amplification in human tumors.

Anaplastic meningiomas (MIIIs) and meningeal hemangiopericytomas (HPCs) display significant morphologic and immunohistochemical overlap, including occasional cases of otherwise classic HPC with focal epithelial membrane antigen (EMA) positivity. The availability of several new biomarkers prompted us to examine the potential diagnostic roles of ancillary immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) studies. From the archival university neuropathology and consult files of 1 of the authors (A.P.), 19 meningeal HPCs and 19 MIIIs were retrieved for further study. IHC was performed by using EMA, CAM 5.2, CD99, Bcl-2, claudin-1 and Factor XIIIa (FXIIIa) antibodies. FISH was performed with NF2, 4.1B (DAL-1), chromosome 1p32, and 14q32 probes. HPCs showed strong CD99 (85% of cases), strong bcl-2 (86%), focal EMA (33%), focal claudin-1 (13%), and scattered individual cell FXIIIa (100%) positivity. MIIIs showed strong EMA (89%), strong claudin-1 (54%), weak or focal CD99 (15%), weak or focal bcl-2 (31%), and individual cell FXIIIa (84%) positivity. Focal CAM 5.2 expression was seen in 26% of HPCs and 15% of MIIIs. Deletions were extremely common in MIIIs: 1p (94%), 14q (67%), NF2 (100%), and 4.1B (67%). HPCs showed no 14q or 4.1B deletions, with 1 case each of 1p and NF2 deletions (6%). The sensitivities and specificities of the 3 most useful IHC markers (EMA, CD99, bcl-2) were 85%-89% and 67%-84%, respectively. The sensitivity and specificity of claudin-1 for MIII were 54% and 86%, respectively. The specificity and positive predictive value of combined CD99 and bcl-2 expression for the diagnosis of HPC was 95%. The sensitivities of individual genetic markers were 67%-100%, with specificities of 94%-100%. Our 3 conclusions were as follows: (1) EMA, CD99, bcl-2, and claudin-1 IHC and 1p, 14q, NF2, and 4.1B FISH are particularly useful for distinguishing anaplastic meningiomas from meningeal HPCs. (2) Focal EMA expression does not preclude a diagnosis of HPC. (3) The characteristic FXIIIa staining pattern reported for HPC also is encountered frequently in anaplastic meningiomas and therefore is nonspecific in this diagnostic setting.

Infiltrative astrocytic neoplasms are by far the most common malignant brain tumors in adults. Clinically, they are highly problematic due to their widely invasive nature which makes a complete resection almost impossible. Biologic progression of these tumors is inevitable and adjuvant therapies are only moderately effective in prolonging survival. Glioblastoma multiforme (GBM; WHO grade IV), the most malignant form of infiltrating astrocytoma, can evolve from a lower grade precursor tumor (secondary GBM) or can present as high grade lesion from the outset, so-called de novo GBM. Molecular genetic investigations suggest that GBMs are comprised of multiple molecular genetic subsets. Notwithstanding the diversity of genetic alterations leading to the GBM phenotype, the vascular changes that evolve in this disease, presumably favoring further growth, are remarkably similar. Underlying genetic alterations in GBM may tilt the balance in favor of an angiogenic phenotype by upregulation of pro-angiogenic factors and down-regulation of angiogenesis inhibitors. Increased vascularity and endothelial cell proliferation in GBMs are also driven by hypoxia-induced expression of pro-angiogenic cytokines, such vascular endothelial growth factor (VEGF). Understanding the contribution of genetic alterations and hypoxia in angiogenic dysregulation in astrocytic neoplasms will lead to the development of better anti-angiogenic therapies for this disease. This review will summarize the properties of angiogenic dysregulation that lead to the highly vascularized nature of these tumors.

TMS1/ASC is an intracellular signaling molecule with proposed roles in the regulation of apoptosis, nuclear factor-kappaB activation, and cytokine maturation. Previous studies have shown that TMS1/ASC is silenced by epigenetic means in human breast tumors. In this study, we examined methylation and expression of TMS1/ASC in glioblastoma multiforme (GBM). Whereas normal brain tissue was unmethylated at the TMS1 locus and expressed TMS1 message, 11 of 23 human GBM cell lines exhibited reduced or absent expression of TMS1 that was associated with aberrant methylation of a CpG island in the promoter of the TMS1 gene. Quantitative analysis showed that there was an inverse correlation between the degree of methylation and level of TMS1 expression. Treatment of GBM cell lines lacking TMS1 expression with the methyltransferase inhibitor 5-aza-2'deoxycytidine resulted in partial demethylation and re-expression of TMS1. Analysis of primary tissues indicated that the TMS1 gene is unmethylated and expressed in normal brain, where its expression is restricted to astrocytes. In contrast, TMS1 was aberrantly methylated in 43% (10 of 23) primary GBM specimens. Tumors that exhibited aberrant methylation of TMS1 generally expressed reduced or absent expression of TMS1 as compared to unmethylated cases. Methylation of TMS1 was not associated with patient age, gender, or treatment status. Although the relationship did not reach statistical significance, there was a trend toward increased overall survival for patients with unmethylated tumors. For one patient, disease progression from astrocytic astrocytoma (World Health Organization grade III) to GBM (World Health Organization grade IV) was associated with selective expansion of TMS1-negative cells. The data suggest a role for the epigenetic silencing of TMS1 in the pathogenesis of human GBM. Methylation of TMS1 may prove to be a useful prognostic marker and/or predictor of patient survival and tumor malignancy.

Protease-activated receptor-1 (PAR1) is a G-protein coupled receptor that is proteolytically activated by blood-derived serine proteases. Although PAR1 is best known for its role in coagulation and hemostasis, recent findings demonstrate that PAR1 activation has actions in the central nervous system (CNS) apart from its role in the vasculature. Rodent studies have demonstrated that PAR1 is expressed throughout the brain on neurons and astrocytes. PAR1 activation in vitro and in vivo appears to influence neurodegeneration and neuroprotection in animal models of stroke and brain injury. Because of increasing evidence that PAR1 has important and diverse roles in the CNS, we explored the protein localization and function of PAR1 in human brain. PAR1 is most intensely expressed in astrocytes of white and gray matter and moderately expressed in neurons. PAR1 and GFAP co-localization demonstrates that PAR1 is expressed on the cell body and on astrocytic endfeet that invest capillaries. PAR1 activation in the U178MG human glioblastoma cell line increased PI hydrolysis and intracellular Ca(2+), indicating that PAR1 is functional in human glial-derived tumor cells. Primary cultures of human astrocytes and human glioblastoma cells respond to PAR1 activation by increasing intracellular Ca(2+). Together, these results demonstrate that PAR1 is expressed in human brain and functional in glial tumors and cultures derived from it. Because serine proteases may enter brain tissue and activate PAR1 when the blood brain barrier (BBB) breaks down, pharmacological manipulation of PAR1 signaling may provide a potential therapeutic target for neuroprotection in human neurological disorders.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor of adults and is in great need of novel diagnostic and therapeutic approaches. Diagnosis is beginning to consider a tumor's genetic status and in the future may incorporate gene expression or proteomic profiles. Genetic alterations in gliomas that are being used in classification include TP53 and retinoblastoma pathway disruption, PTEN mutations, epidermal growth factor receptor amplification and 1p/19q losses. Molecular mechanisms are being exploited to treat glioblastoma multiforme. Tyrosine kinase inhibitors directed at epidermal growth factor receptor (ZD1839, OSI-774) are being explored. Farnesyltransferase inhibitors (R115777) block activation of the ras pathway and may be effective. Antagonists of the endothelin receptor (e.g., atrasentan) expressed on blood vessels may block the high degree of angiogenesis in gliomas. Tumors lacking methylthioadenosine phosphorylase are sensitive to inhibitors of de novo adenosine synthesis (SDX-102) since they lack a salvage pathway. Future goals are to tailor therapies to a tumor's molecular, proteomic or genomic status ,and manage glioblastoma multiformes as in chronic diseases in a multidisciplinary clinical setting.

The invasion of neoplastic cells into healthy brain tissue is a pathologic hallmark of gliomas and contributes to the failure of current therapeutic modalities (surgery, radiation and chemotherapy). Transformed glial cells share the common attributes of the invasion process, including cell adhesion to extracellular matrix (ECM) components, cell locomotion, and the ability to remodel extracellular space. However, glioma cells have the ability to invade as single cells through the unique environment of the normal central nervous system (CNS). The brain parenchyma has a unique composition, mainly hyaluronan and is devoid of rigid protein barriers composed of collagen, fibronectin and laminin. The integrins and the hyaluronan receptor CD44 are specific adhesion receptors active in glioma-ECM adhesion. These adhesion molecules play a major role in glioma cell-matrix interactions because the neoplastic cells use these receptors to adhere to and migrate along the components of the brain ECM. They also interact with the proteases secreted during glioma progression that degrade ECM allowing tumor cells to spread and diffusely infiltrate the brain parenchyma. The plasminogen activators (PAs), matrix metalloproteinases (MMPs) and lysosomal cysteine peptidases called cathepsins are also induced during the invasive process. Understanding the mechanisms of tumor cell invasion is critical as it plays a central role in glioma progression and failure of current treatment due to tumor recurrence from micro-disseminated disease. This review will focus on the impact of microregional heterogeneity of the ECM on glioma invasion in the normal adult brain and its modifications in tumoral brain.

Glioblastoma (GBM) has explosive biologic properties with rapid clinical progression leading to death. Its distinguishing pathologic features, necrosis with surrounding pseudopalisades and microvascular hyperplasia, are believed to be instrumental to its accelerated growth. Microvascular hyperplasia arises in response to the secretion of proangiogenic factors by hypoxic pseudopalisades and allows for peripheral neoplastic expansion. Mechanisms underlying necrosis and hypoxia remain obscure, but vaso-occlusive and prothrombotic contributions could be substantial. Recent investigations on the origin of pseudopalisades suggest that this morphologic phenomenon is created by a tumor cell population actively migrating away from a central hypoxic region and that, in at least a significant subset, hypoxia-induced migration appears due to vaso-occlusion caused by intravascular thrombosis. Both vascular endothelial growth factor induced vascular permeability to plasma coagulation factors and the increased neoplastic expression of tissue factor likely contribute to a prothrombotic state favoring intravascular thrombosis. In addition to prothrombotic mechanisms, vaso-occlusion could also result from angiopoietin-2-mediated endothelial cell apoptosis and vascular regression, which follows neoplastic co-option of native vessels in animal models of gliomas. Vaso-occlusive and prothrombotic mechanisms in GBM could readily explain the presence of pseudopalisades and coagulative necrosis in tissue sections, the emergence of central contrast enhancement and its rapid peripheral expansion on neuroimaging, and the dramatic shift to an accelerated rate of clinical progression. Since the hypoxic induction of angiogenesis appears to support further neoplastic growth, therapeutic targeting of the underlying vascular pathology and thrombosis could provide a new means to prolong time to progression.

Survival periods vary considerably for patients with high-grade astrocytomas, and reliable prognostic markers are not currently available. We therefore investigated whether genetic losses from chromosomes 1p, 19q, 9p, or 10q were associated with survival in 89 high-grade astrocytomas using tissue microarrays (TMAs) derived from Radiation Therapy Oncology Group clinical trials. Cases included 15 anaplastic astrocytomas (AAs) and 74 glioblastomas (GBMs) selected on the basis of survival times significantly shorter or longer than the expected median. Genetic analysis was performed by TMA-fluorescence in situ hybridization (FISH) on array sections using 8 DNA probes, including those directed at 1p32, 19q13.4, 9p21 (p16/CDKN2A), and 10q (PTEN and DMBT1). Genetic status for each locus was correlated with patient survival group, and data were analyzed by using Fisher's exact test of association (adjusted P = 0.025). Losses of chromosome 1p, either alone or in combination with 19q, were encountered in only 2 cases, both AAs. This contrasts with oligodendrogliomas, in which combined 1p and 19q losses are frequent and predictive of prolonged survival. Solitary 19q loss was noted in 3/15 AAs and in 7/70 GBMs and was more frequent in the long-term survival group (P = 0.041, AA and GBM combined). Chromosome 9p loss was seen in 5/8 AAs and 39/57 GBMs, whereas chromosome 10q loss was detected in 4/15 AAs and 48/68 GBMs. The 9p and 10q deletions were slightly more frequent in short-term survivors, though none of the comparisons achieved statistical significance. Long-term and short-term survival groups of high-grade astrocytomas appear to have dissimilar frequencies of 19q, 9p, and 10q deletions. TMA-FISH is a rapid and efficient way of evaluating genetic alterations in such tumors.

Objective and Importance: The pituicytoma is a rare primary tumor of the neurohypophysis. Although histologically benign, the location and vascular nature of these tumors can make surgical resection difficult. We present a report of two patients with pituicytomas and review the literature regarding treatment and prognosis for this unusual lesion. Possible histogenetic relationships of this tumor with other pituitary neoplasms are presented., Clinical Presentation: Patient 1 was a 45-year-old man who presented with a 5-year history of decreased libido. He was found to have a 2-cm suprasellar mass on a magnetic resonance imaging scan. Patient 2 was a 48-year-old man who presented with multiple endocrine complaints. He was found to have an intrasellar mass on magnetic resonance imaging., Intervention: Patient 1 underwent a right frontal craniotomy, with a subtotal resection of the suprasellar mass through the lamina terminalis. The residual tumor was treated with fractionated stereotactic radiotherapy. The intrasellar mass in Patient 2 was resected via a transsphenoidal approach. On surveillance magnetic resonance imaging, the tumor was found to have recurred and expanded into the suprasellar space. The patient underwent a right frontal craniotomy for decompression and a subtotal resection of the tumor. The patient experienced a second recurrence 7 years after the initial procedure and was subsequently treated with fractionated stereotactic radiotherapy., Conclusion: Pituicytomas are a distinct form of pituitary gland neoplasia that may recur if subtotally resected. These neurohypophysial tumors may contain a small subpopulation of previously unrecognized bcl-2-immunoreactive cells, whose role in the histogenesis of pituicytoma deserves further study.

Posttransplant lymphoproliferative disorders (PTLDs) represent a spectrum ranging from Epstein-Barr virus (EBV)-driven polyclonal lymphoid proliferations to EBV+ or EBV- malignant lymphomas. Central nervous system (CNS) PTLDs have not been characterized fully. We reviewed the clinical, radiologic, and pathologic features of 12 primary CNS PTLDs to define them more precisely. Patients included 10 males and 2 females (median age, 43.4 years) who were recipients of kidney (n = 5), liver (n = 2), heart (n = 2), peripheral blood stem cells (n = 2), or bone marrow (n = 1). All received immunosuppressive therapy. CNS symptoms developed 3 to 131 months (mean, 31 months) after transplantation. By neuroimaging, most showed multiple (3 to 9) intra-axial, contrast-enhancing lesions. Histologic sections showed marked expansion of perivascular spaces by large, cytologically malignant lymphoid cells that were CD45+, CD20+, EBV+ and showed light chain restriction or immunoglobulin gene rearrangement. In distinction to PTLDs in other organ systems, CNS PTLDs were uniformly high-grade lymphomas that fulfilled the World Health Organization criteria for monomorphic PTLDs. Extremely short survival periods were noted for each CNS PTLD that followed peripheral blood stem cell transplantation. Survival of others with CNS PTLD varied; some lived more than 2 years.

Necrosis and vascular proliferation are the pathologic features that distinguish the most malignant infiltrative astrocytoma, glioblastoma (GBM), from those of lower grades. In GBM, hypercellular zones called pseudopalisades typically surround necrotic foci. Although these cells are known to secrete high levels of proangiogenic factors that promote tumor growth, their origins are ill defined. We propose that pseudopalisades represent differing stages and histologic samplings of astrocytoma cells migrating away from a hypoxic/anoxic focus, often triggered by a central vaso-occlusive event. This proposition is based on our findings that pseudopalisading cells are 5-50% less proliferative and 6-20 times more apoptotic than adjacent astrocytoma, indicating that cell accumulation does not result from increased proliferation or resistance to apoptosis. Coexisting inflammatory cells account for <2% of pseudopalisading cells and cannot account for hypercellularity. Pseudopalisading cells show nuclear expression of hypoxia-inducible factor 1 alpha, consistent with their hypoxic nature, and hypoxia induces a 20-60% increase in glioma cell migration in vitro. Hypoxic cells in vitro and pseudopalisades in GBM specimens show enhanced gelatinase activity, typical of an invasive phenotype. These results suggest that pseudopalisading cells are migrating at the periphery of a hypoxic center. To uncover a potential source of hypoxia and sequence of structural events leading to pseudopalisade formation, we performed a morphometric analysis of 234 pseudopalisades from 85 pretreatment GBMs. We found distorted, degenerating, or thrombosed blood vessels within the center of more than half the pseudopalisades, suggesting that at least a subset of pseudopalisades are two-dimensional histologic representations of tumor cells migrating away from a vaso-occlusive event.

West Nile virus (WNV), a mosquito-transmitted single-stranded RNA flavivirus, causes human disease of variable severity. We report clinical and pathologic findings of fatal encephalitis from the transmission of WNV from an organ donor to a kidney transplant recipient. The patient developed a febrile illness 18 days after transplantation, which progressed to encephalitis. Postmortem examination demonstrated extensive viral encephalopathic changes. Immunohistochemical studies highlighted WNV antigens within neurons, especially in the cerebellum and brainstem. Flavivirus virions were detected ultrastructurally within the cerebellum, and WNV was isolated from the brain and the brainstem. Thus, this case demonstrates the first death in the first solid organ transplant-associated transmission of WNV. Immunosuppression of the transplant recipient might have been responsible for the fulminant viral effects. The pathologic diagnosis helped guide subsequent epidemiologic and laboratory studies.

Ependymomas are glial neoplasms whose clinical behavior is difficult to predict based on histology alone. Recently, a comparative genomic hybridization study identified frequent chromosome 9p and 13q losses in anaplastic ependymomas, suggesting that p16 and RB alterations may be involved in tumor progression. In order to test this hypothesis further, 101 myxopapillary, conventional, and anaplastic ependymomas (51 spinal and 50 intracranial tumors) were tested for RB and p16 deletions using fluorescence in situ hybridization. Clinical follow-up, ranging from 2 to 198 months (median 46 months), was obtained in 90 cases (91%). RB and p16 deletions were seen in 22 of 92 (24%) and 22 of 89 (25%) informative cases, respectively. Polysomies were more frequent in the grade I and II spinal tumors, consistent with prior reports of increased aneuploidy in such cases. No significant genetic associations were seen with tumor grade, recurrence, or death, suggesting that 9p and 13q deletions do not play a prominent role in the malignant progression of ependymomas, as has been implicated in other glioma subtypes.

The serine proteases tissue plasminogen activator, plasmin, and thrombin and their receptors have previously been suggested to contribute to neuronal damage in certain pathological situations. Here we demonstrate that mice lacking protease-activated receptor 1 (PAR1) have a 3.1-fold reduction in infarct volume after transient focal cerebral ischemia. Intracerebroventricular injection of PAR1 antagonist BMS-200261 reduced infarct volume 2.7-fold. There are no detectable differences between PAR1-/- and WT mice in cerebrovascular anatomy, capillary density, or capillary diameter, demonstrating that the neuroprotective phenotype is not likely related to congenital abnormalities in vascular development. We also show that the exogenously applied serine proteases thrombin, plasmin, and tissue plasminogen activator can activate PAR1 signaling in brain tissue. These data together suggest that if blood-derived serine proteases that enter brain tissue in ischemic situations can activate PAR1, this sequence of events may contribute to the harmful effects observed. Furthermore, PAR1 immunoreactivity is present in human brain, suggesting that inhibition of PAR1 may provide a novel potential therapeutic strategy for decreasing neuronal damage associated with ischemia and blood-brain barrier breakdown.

Recent advances in our understanding of the molecular genetic mechanisms underlying diffusely infiltrating brain neoplasms have important implications for the classification and therapy of these tumors. Traditionally, primary brain tumors have been classified histologically; however, it is now clear that tumors within a single histologically defined category are heterogeneous from a molecular genetic perspective. Furthermore, many new experimental therapeutic strategies directed against these almost invariably fatal tumors are aimed at molecular rather than histologic abnormalities. Consequently, the classification of these tumors is in the process of being re-evaluated as underlying molecular genetic mechanisms continue to be elucidated. This review covers traditional histologic based classification of infiltrating glial neoplasms together with molecular abnormalities of these tumors involving p53, epidermal growth factor receptor, the retinoblastoma pathway, platelet derived growth factor receptor, genetic losses on chromosome 10, and loss of heterozygosity on chromosomes 1p and 19q. The contribution of these molecular genetic abnormalities to the classification and therapy of these tumors is discussed. Although at the present no molecular system for the classification of brain tumors has been generally accepted, microarray technologies offer the exciting prospect of practical molecular classification in the future.

The May 2003 COM. A 57-year-old woman presented with slurring of her speech and right arm weakness. Her past medical history included idiopathic hypertrophic subendocardial stenosis (IHSS), arthritis, asthma, congestive heart failure, hypertension and NIDDM. Neurological examination showed persistent word finding difficulty but her motor and sensory function had essentially returned to normal. Extensive laboratory studies were unrevealing. Imaging studies showed a meningeal lesion over the left posterior parietal lobe and the findings suggested an infectious or inflammatory process. A biopsy of the involved dura and meninges was performed and revealed leptomeningeal Rosai-Dorfman disease. Emperipolesis was noted. The finding of emperipolesis is characteristic of Rosai-Dorfman disease of the leptomeninges, but in 30% of cases, this feature will not be identified. Large pale histiocytes of Rosai-Dorfman disease are immunoreactive for S-100 protein and KP1, but negative for CD1a. The differential diagnosis of a chronic inflammatory infiltrate containing numerous, large histiocytes includes granulomatous diseases such as Wegener graulomatosis and sarcoid, Hodgkin disease, and Langerhans histiocytosis. CNS Rosai-Dorfman most commonly involves patients between 20- and 40-years-old, with a slight male predominance. Approximately 75% of cases are intracranial, whereas 20% involve the spine. Over 90% of CNS Rosai-Dorfman cases involve the leptomeninges and are seen by neuroimaging as a dural-based, contrast-enhancing masses that often elicit vasogenic edema in the underlying brain. Thus, clinically and radiologically, the disease is thought to represent meningioma. Leptomeningeal Rosai-Dorfman disease is considered a benign condition and in most cases surgical resection is the treatment of choice. Although the number of cases in the literature is small, disease progression following surgical resection is uncommon. Little is known regarding the pathogenesis of Rosai-Dorfman disease. Most have suggested that it represents either an autoimmune disease or a reaction to an infectious agent that has yet to be discovered. Currently it is best considered a benign, idiopathic histiocytosis.

Pituitary adenomas cause significant morbidity caused by compression of regional structures or the inappropriate expression of pituitary hormones. However, little is known about the molecular changes that contribute to the development of these tumors. To investigate these changes, we recently used cDNA microarray analysis to identify several genes with altered expression patterns in pituitary adenomas. The folate receptor (FRalpha) was significantly overexpressed in clinically nonfunctional (NF) adenomas but not in functional adenomas (adrenocorticorticotropic hormone, growth hormone, and prolactin). FRalpha is a high affinity folate transporter that is overexpressed by other tumors and could provide a growth advantage to cells that express it. Analysis of FRalpha expression by Western blotting confirmed that FRalpha protein was specifically overexpressed in NF tumors. The FRalpha was capable of binding folates from measurements of [(3)H] folic acid binding, indicating that the overexpressed receptor was properly folded and may mediate vitamin uptake. Comparison of protein and specific [(3)H] folic acid binding levels in subtypes of NF adenomas suggested that the immunohistochemically negative adenomas produced more properly folded FRalpha than adenomas that stained positively for anterior pituitary hormones. Finally, immunohistochemistry demonstrated that FRalpha was specifically expressed in NF adenoma cells. These results demonstrate that overexpression of FRalpha mRNA by NF pituitary adenomas results in production of properly folded FRalpha protein, may mediate vitamin transport, and could potentially facilitate the growth of these tumors.

Glioblastoma multiforme (GBM) is the highest-grade infiltrative astrocytoma and also the most common. It is generally associated with a dismal prognosis (mean survival 11 months), yet individual patient survivals vary. Histologic parameters have had limited value in predicting survival among patients with GBM. The current view of GBM as a histopathologic entity consisting of several genetic subtypes raises the possibility that molecular alterations could be predictive of survival. Common genetic alterations in GBM include gene amplification of epidermal growth factor receptor (EGFR), mutations in the tumor suppressors TP53 and PTEN, and genetic losses on chromosome 10. Less common in GBMs is the combined loss of chromosomes 1p and 19q-a combination that has proven prognostically favorable in oligodendrogliomas. A recent article on prognostic factors in a series of 97 GBMs by Schmidt et al. finds that both TP53 mutations and young patient age at presentation are independent factors associated with a long survival. Loss of heterozygosity (LOH) of chromosome 10q was predictive of a poor outcome. Perhaps most intriguing, the finding of combined LOH of 1p and 19q, which was noted in only five GBMs, was associated with a significantly longer survival. Thus, combined losses of 1p and 19 may be associated with a favorable prognosis in a wider range of infiltrative gliomas that includes GBM. While these findings will be debated and need to be confirmed, it is clear that genotyping of infiltrative gliomas will be an important component of neuro-oncology in the future. Not only will genetic alterations offer prognostication, but they will also serve as targets for directed therapies. Treatments directed against tumors with EGFR amplification, TP53, mutations and PTEN mutations are being developed and tested in clinical trials. It remains to be seen if GBMs with 1p and 19q losses are chemosensitive in the same manner as oligodendrogliomas.

High-level gains at 5p15, a chromosomal region including the human telomerase catalytic protein subunit (hTERT) gene, have been documented in several medulloblastomas. We therefore analyzed hTERT gene dosage in a group of medulloblastomas and other embryonal brain tumors using differential PCR. Amplification of the hTERT locus was detected in 15 of 36 (42%) tumors examined. To correlate gene amplification with message level, we used real-time quantitative PCR to measure hTERT mRNA in 50 embryonal brain tumors. hTERT mRNA was detected in all but one of these cases, and mRNA level correlated significantly with gene dosage (r = 0.82). Log-rank analysis of survival data revealed a trend toward poor clinical outcomes in patients with medulloblastomas containing high hTERT mRNA levels, but clinical follow-up was relatively short and the association was not statistically significant (P = 0.078). Comparative genomic hybridization was used to further analyze the tumor with the greatest hTERT gene dosage and mRNA level, a recurrent medulloepithelioma. hTERT was amplified in the recurrent tumor but not in the primary lesion, suggesting this locus can be involved in tumor progression. Our data indicate that hTERT gene amplification is relatively common in embryonal brain tumors, and that increased expression of hTERT mRNA may be associated with biologically aggressive tumor behavior.

Granular cell astrocytomas (GCA) are an uncommon morphologic variant of infiltrative glioma that contains a prominent population of atypical granular cells. As a rule, they are biologically aggressive compared to similar tumors without granular features. We sought to determine whether GCAs possess distinct genotypic alterations that might reflect their unique morphology or clinical behavior. Eleven GCAs occurring in 7 men and 4 women ranging in age from 46 to 75 years were investigated for genetic alterations of known significance in glial tumorigenesis, including LOH at 1p, 9p, 10q, 17p, and 19q, point mutations of TP53, deletions of p16(CDKN2A) and p14ARF, as well as EGFR amplifications. Tumors included had an infiltrative growth pattern and consisted of large, round cells packed with eosinophilic, PAS-positive granules that varied in quantity, ranging from 30 to 100% of tumor cells. Three tumors were of WHO grade II, one was grade III, and 7 were grade IV lesions. Overall, the tumors showed higher frequencies of LOH at 1p, 9p, 10q, 17p, and 19q than typical infiltrating astrocytomas of similar grades. Losses on 9p and 10q occurred in nearly all cases, including low grade lesions. TP53 mutations were identified in 2 grade IV GCAs, while combined p14ARF and p16(CDKN2A) homozygous deletions were noted in only one grade IV lesion. None showed EGFR amplification. We found no genetic alterations specific for GCA. Instead, it appears that granular cell change occurs across genetic subsets. The high frequency of allelic loss, especially on 9p and 10q, may confer aggressive growth potential and be related to their rapid clinical progression.

Angiogenesis is required for the development and biologic progression of infiltrative astrocytomas and takes the form of "microvascular hyperplasia" in glioblastoma multiforme, the most malignant astrocytoma. This pathologic term refers to an abnormal vascular proliferation that is often associated with necrosis and likely originates in hypoxic zones. Both the physiologic response to hypoxia and genetic alterations contribute to this process. The presence of hypoxic regions within an expanding tumor mass leads to upregulation of pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), through increased activity of the transcriptional complex HIF-1 (hypoxia-inducible factor-1). HIF-1 mediated gene expression may be directly or indirectly modulated by alterations in oncogenes/tumor suppressor genes that occur during astrocytoma development, including PTEN, TP53, p16(CDKN2A), p14ARF, EGFR, and PDGFR. Genetic alterations are also believed to influence the HIF-independent expression of pro- and anti- angiogenic factors, such as basic fibroblast growth factor (bFGF) and thrombospondin-1 (TSP-1), respectively. Thus, genetic events that occur during the progression of infiltrating astrocytomas promote angiogenesis, both by modulating hypoxia induced gene expression and by regulating of pro- and anti- angiogenic factors.

Deletions of chromosomes 1p and 19q are associated with chemosensitivity and enhanced survival in oligodendrogliomas. Therefore, we have utilized FISH analysis as an ancillary tool for diffuse gliomas with suspected oligodendroglial features. To date, 246 gliomas have been analyzed in 131 male and 93 female patients, including 109 oligodendrogliomas (O), 109 mixed oligoastrocytomas/equivocal gliomas (MOA), and 28 astrocytomas (A). To address specificity, we also analyzed 41 oligodendroglioma mimics, including 12 central and 12 extraventricular neurocytomas (EVN), 12 dysembryoplastic neuroepithelial tumors, and 5 clear cell ependymomas. Aside from 2 EVNs, no mimics demonstrated codeletion. Three patterns were associated with glioma cell type (O vs. MOA/A): -1p/19q, -19q alone, and polysomies. Long-term survivals of >5-years (N=47) and >10-years (N=16) were associated with 1p/19q codeletion in 60% and 75% respectively, whereas solitary 19q deletion accounted for 11% and 6% respectively. Survivals<2-years (N=10) were associated with lack of deletions in 70%. A few older patients with high-grade, "genetically favorable" tumors did poorly, whereas prolonged survival was observed in several low-grade glioma patients despite a lack of the "genetically favorable" pattern. Our data suggests that: 1) FISH-detectable 1p/19q codeletion is relatively specific for oligodendrogliomas with long survival, 2) solitary 19q deletion may also portend a favorable prognosis in a smaller subset, and 3) combined clinicopathologic and genetic assessment likely provides a more accurate means of patient stratification than either one alone.

Brain angiogenesis inhibitors (BAI) are putative transmembrane proteins containing an extracellular domain with thrombospondin type-1 repeats which can exhibit anti-angiogenic activity. BAI1 mRNA is expressed mainly in the brain, while BAI2 and BAI3 mRNAs are more widely expressed. We hypothesized that the BAI family might have anti-tumoral properties and studied the expression of BAI1 protein in normal human brain and in glioblastoma multiforme. We generated an anti-BAI1 antibody and showed that BAI1 was widely expressed in normal brain but was absent in 28 glioma cell lines and in the majority of human glioblastoma investigated. BAI1 expression did not correlate with TP53 status and we did not confirm previous findings that p53 regulates BAI1 mRNA expression in glioma cells. The finding that expression of BAI proteins may be lost during tumor formation is of special interest as restoration of their function in tumors may be of therapeutic benefit.

Granular cell astrocytomas (GCAs) are rare, incompletely characterized infiltrative gliomas that contain a prominent component of granular cells. Such tumors can readily be mistaken for reactive conditions. We studied 22 cases to explore their morphologic spectrum, establish features useful in distinguishing GCA from nonneoplastic diseases, and to determine which parameters correlate with biologic behavior. Tumors occurred in 17 men and five women, ranging in age from 29 to 75 years, who presented mainly with seizures, headache, aphasia, or hemiparesis. Radiologically, high-grade GCAs were contrast-enhancing, cerebral hemispheric masses with prominent peritumoral edema. All contained sheets or interspersed large, round cells packed with eosinophilic, PAS-positive granules. Lymphocytic infiltrates, either perivascular or admixed with neoplastic cells, were present in 14 tumors. Transition to typical infiltrating astrocytoma was noted in 16 cases; of these, granular cells comprised 30-95% of cells. Six tumors consisted almost entirely of atypical granular cells. By WHO criteria, four GCA were grade 2, seven were grade 3, and 11 were grade 4. Glial fibrillary acidic protein staining was seen in all but one tumor, and the majority were immunoreactive for S-100 protein, KP-1, ubiquitin, and epithelial membrane antigen. Although MIB-1 proliferation indices increased with tumor grade, granular cells accounted for only a minority of immunoreactive cells. Among 18 cases with follow-up, 15 recurred after surgery and resulted in death (mean survival, 7.6 months). Two patients died postoperatively, and one was alive at 51 months. Granular cell astrocytoma is an uncommon morphologic variant that appears to be rapidly progressive and usually fatal.

Fibrillary astrocytoma, the most common primary central nervous system neoplasm, is infiltrating, rapidly proliferating, and almost invariably fatal. This contrasts with the biologically distinct pilocytic astrocytoma, which is circumscribed, often cystic, slowly proliferating, and associated with a favorable long-term outcome. Diagnostic markers for distinguishing pilocytic astrocytomas from infiltrating anaplastic astrocytomas are currently not available. To identify genes that might either serve as markers or explain these distinct biologic behaviors, cDNA microarray analysis was used to compare the expression of 7,073 genes (nearly one quarter of the human genome) between these 2 types of astrocytoma. Messenger RNAs pooled from 3 pilocytic astrocytomas and from 4 infiltrating anaplastic astrocytomas were compared. Apolipoprotein D (apoD), which expressed 8.5-fold higher in pilocytic astrocytomas, showed the greatest level of differential expression and emerged as a potential marker for pilocytic tumors. By immunohistochemistry, 10 of 13 pilocytic astrocytomas stained positively for apoD, while none of 21 infiltrating astrocytomas showed similar staining. ApoD immunostaining was also seen in 9 of 14 of gangliogliomas, 4 of 5 subependymal giant cell astrocytomas (SEGAs), and a single pleomorphic xanthoastrocytomas (PXAs). By in situ hybridization, pilocytic astrocytomas, in contrast with infiltrating astrocytomas, showed widespread increased apoD expression. SAGE analysis using the NCBI database showed a higher level of expression of apoD RNA in pilocytic astrocytoma than in any of the other 94 neoplastic and non-neoplastic tissues in the database. ApoD is associated with decreased proliferation in some cell lines, and is the protein found in highest concentration in cyst fluid from benign cystic disease of the breast. ApoD might play a role in either decreased proliferation or cyst formation in pilocytic astrocytomas, gangliogliomas, SEGAs, and PXAs.

Summary: Astroblastoma is a rare primary glial tumor with a characteristic appearance on neuroradiologic images. Typically, astroblastomas are large, lobulated, peripheral, supratentorial, solid, and cystic masses with relatively little associated vasogenic edema and tumor infiltration for their large size. The solid component of the mass has a bubbly appearance and a T2 signal that is isointense to gray matter. Punctate calcifications are often present. Neuroradiologists should be familiar with the characteristic appearance of this tumor.

Infiltrative astrocytic neoplasms are the most common malignancies of the central nervous system. They remain clinically problematic because of their involvement of brain structures critical to proper cognitive, behavioral, and motor function; their widely invasive properties, which make them difficult to resect totally; and their nearly inevitable biologic progression in spite of adjuvant therapy. Glioblastoma multiforme (GBM, World Health Organization grade IV), the most malignant form of infiltrating astrocytoma, can present as a high-grade lesion from the outset (so-called de novo GBM) or can evolve from a lower grade precursor lesion (secondary GBM). Molecular genetic investigations suggest that GBM is best regarded as a clinicopathologic entity composed of multiple molecular genetic subsets. Molecular alterations associated with progression to GBM and that define genetic subsets include epidermal growth factor receptor amplifications, p53 mutations, retinoblastoma pathway alterations [most commonly, p16(CDKN2A) losses], and chromosome 10 alterations, including PTEN mutations. Despite the wide range of genetic events that ultimately lead to GBM, the vascular changes that evolve are remarkably similar. Microvascular hyperplasia is spatially and temporally associated with pseudopalisading necrosis in GBM and is believed to be driven by hypoxia-induced expression of proangiogenic cytokines such vascular endothelial growth factor. In addition, genetic alterations in GBM are thought to contribute directly or indirectly to angiogenic dysregulation. Both p53 mutations and genetic losses on chromosome 10 may tip the balance toward an angiogenic phenotype through upregulation of proangiogenic factors and/or downregulation of angiogenesis inhibitors. Understanding genetic events and their relation to angiogenic regulation in astrocytic neoplasms may eventually lead to therapies that are specifically directed at molecularly defined subsets of these diseases.

Chordoid gliomas are uncommon primary brain tumors that arise in the region of the third ventricle. Reports of this entity to date have been limited to adults. We present a case of a chordoid glioma arising in the hypothalamic/third ventricle region of a 12-year-old male who presented with visual symptoms. The neoplasm consisted of cords and clusters of well-differentiated, spindled-to-rounded cells containing abundant eosinophilic cytoplasm within a prominent mucinous matrix. Unlike other chordoid gliomas, this lesion contained islands and sheets showing cartilaginous differentiation intermixed with the glial component. A graded transition between neoplastic glial and chondroid regions was evident, and cells in both regions were strongly immunoreactive for GFAP and S-100. Cartilaginous metaplasia is infrequent in gliomas, but occurs most often in pediatric neoplasms of the midline such as this chordoid glioma. Thus, chondroid metaplasia represents an unusual histopathologic feature of chordoid glioma-in this case, presenting in a child.