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In recent years there has been considerable progress in brain tumor neuropathology. Several new diagnostic entities have been recognized, subclassification schemes have been modified, and new concepts on the histogenesis and cell biology of brain tumors have emerged. In 1993, a revised WHO classification of brain tumors was published by an international committee. This article summarizes the pertinent new aspects. As novel tumor entities, the central neurocytoma, the dysembryoplastic neuroepithelial tumor (DNT), desmoplastic infantile ganglioglioma (DIG) and pleomorphic xanthoastrocytoma (PXA) have been included. Several histopathological variants of meningiomas have been added of which only the papillary meningioma and the atypical meningioma are characterized by an increased rate of recurrence. Meningeal hemangiopericytomas and hemangioblastomas are classified as tumors of non-meningothelial origin. The glioblastoma multiforme, which had previously been listed as an embryonal tumor, is now recognized as an astrocytic glioma. Immunohistochemistry has greatly advanced the practical diagnosis and classification of brain tumors. There are specific markers for all normal and neoplastic cell types except for oligodendroglioma cells. The prognosis of and therapeutic approaches to brain tumors greatly depend on histopathological grading. The WHO proposes four tumor grades, i.e., I, II, III, and IV. As a rule, grades I and II tumors are viewed as benign or semi-benign neoplasms and grades III and IV tumors as malignant. There are attempts to use new biological parameters for the grading of brain tumors. Antibodies to proliferation-associated proteins reflect tumor growth. Molecular genetic approaches to tumor-associated genes and gene loci are particularly promising new tools for the future.

Using a combination of transplacental carcinogen exposure and retrovirus-mediated oncogene transfer into fetal brain transplants, we have studied complementary transformation by N-ethyl-N-nitrosourea (NEU) and the v-myc oncogene in the nervous system. Previous experiments had demonstrated that both agents will not induce tumors independently whereas simultaneous expression of v-H-ras and v-gag/myc exerted a powerful transforming potential in neural grafts. In order to identify other genetic alterations that co-operate with an activated myc gene, the neurotropic carcinogen NEU was used to generate mutations of cellular genes. On embryonic day 14 (ED14), pregnant donor animals (F344 rats) received a single i.v. dose of NEU (50 mg/kg). Twenty-four hours later (ED15), the fetal brains were removed, triturated and incubated with a retroviral vector carrying the v-gag/myc oncogene. Subsequently, these primary cell suspensions were transplanted stereotactically into the caudate-putamen of syngenic adult recipients. After latency periods of 3-6 months, 5 of 10 recipients harboring ED15 fetal brain transplants developed malignant, poorly differentiated neuroectodermal tumors in the grafts. No tumor development was observed in seven recipients harboring ED16 neural grafts. Cell lines were established from three tumors and the 110 kd gag/myc fusion protein encoded by the retroviral construct was identified in the tumors by Western blotting. Several candidate genes for mutational activation by NEU including the H-ras, K-ras and neu oncogenes were analyzed for specific point mutations by polymerase chain reaction (PCR) and direct DNA sequencing of the PCR products. However, no mutations were found in any of these genes. These findings lend further support to the multistep hypothesis of neoplastic transformation in the brain. The tumors induced in this model provide an interesting tool for the identification of genes that co-operate with an activated myc gene in neurocarcinogenesis.

Object Accumulating data suggest more aggressive surgery in patients with malignant glioma to improve outcome. However, extended surgery may increase morbidity. The randomized Phase III 5-aminolevulinic acid (ALA) study investigated 5-ALA–induced fluorescence as a tool for improving resections. An interim analysis demonstrated more frequent complete resections with longer progression-free survival (PFS). However, marginal differences were found regarding neurological deterioration and the frequency of additional therapies. Presently, the authors focus on the latter aspects in the final study population, and attempt to determine how safety might be affected by cytoreductive surgery. Methods Patients with malignant gliomas were randomized for fluorescence-guided (ALA group) or conventional white light (WL) (WL group) microsurgery. The final intent-to-treat population consisted of 176 patients in the ALA and 173 in the WL group. Primary efficacy variables were contrast-enhancing tumor on early MR imaging and 6-month PFS. Among secondary outcome measures, the National Institutes of Health Stroke Scale (NIH-SS) score and the Karnofsky Performance Scale (KPS) score were used for assessing neurological function. Results More frequent complete resections and improved PFS were confirmed, with higher median residual tumor volumes in the WL group (0.5 vs 0 cm3, p = 0.001). Patients in the ALA group had more frequent deterioration on the NIH-SS at 48 hours. Patients at risk were those with deficits unresponsive to steroids. No differences were found in the KPS score. Regarding outcome, a combined end point of risks and neurological deficits was attempted, which demonstrated results in patients in the ALA group to be superior to those in participants in the WL group. Interestingly, the cumulative incidence of repeat surgery was significantly reduced in ALA patients. When stratified by completeness of resection, patients with incomplete resections were quicker to deteriorate neurologically (p = 0.0036). Conclusions Extended resections performed using a tool such as 5-ALA–derived tumor fluorescence, carries the risk of temporary impairment of neurological function. However, risks are higher in patients with deficits unresponsive to steroids.

Frequency, pathogenesis and morphological features of toxoplasmosis were assessed in a consecutive autopsy study. Among 204 patients who died from AIDS in Zurich during 1981–1990, 46 (23%) showed morphological evidence of cerebral toxoplasmosis. In 38 out of 46 cases (83%), toxoplasmosis was restricted to the central nervous system (CNS) and, therefore, pathogenetically classified as reactivation of a latent infection. Acute, systemic toxoplasmosis most frequently involved heart and lungs in addition to the CNS and was observed in 7 cases (15%). These patients probably acquired the infection during HIV-induced immunosuppression. Latent infection with intracerebral tissue cysts but no inflammatory response was present in only one case. Diffuse, necrotizing toxoplasma encephalitis with widespread, confluent areas of necrosis was mainly observed during the early period of the AIDS epidemic and restricted to 6 patients (13%) who did not receive chemotherapy. The majority of patients (83%) had multiple, macroscopically well-circumscribed abscesses with preferential location in the cerebral hemispheres. Of all CNS regions, the rostral basal ganglia were most frequently affected (78% of cases). Since 1989, chronic, burnt-out lesions were observed. These were mainly composed of lipid-laden macrophages and immunocyto-chemistry for Toxoplasma gondii usually failed to detect the parasite. This changing pattern of CNS lesions probably reflects improved clinical management of patients with AIDS.

This report describes an unusual medullo-myoblastoma which developed in the cerebellar vermis of a 6-year-old girl. Histological investigation showed a highly cellular and predominantly undifferentiated tumor. Myogenic differentiation was prominent in clusters of large tumor cells with eosinophilic cytoplasm and immunoreactivity for desmin and myoglobin. Electron microscopy revealed the presence of immature Z-bands. Immunohistochemically, numerous cells showed incipient expression of myoblastic marker antigens, supporting the view that medulloblastomas and related primitive neuroectodermal tumors possess the potential for non-neural differentiation. In addition, there was evidence of advanced neuronal differentiation, with expression of neuron-specific enolase, synaptophysin, retinal S-antigen, and the formation of ganglioid tumor cells. Occassional neoplastic cells expressed glial fibrillary acidic protein without morphologically detectable astrocytic differentiation. Associated with the neoplasm was brain tissue containing clusters of neuronal cells and focal accumulations of immature oligodendroglia-like cells which expressed neuronal marker antigens. This unusual component resembled a hamartomatous lesion and would support the hypothesis that the cerebellar medullomyoblastoma originated from a teratomatous or malformative lesion. Alternatively, this component may constitute the end stage of advanced neuronal differentiation of a primitive neuroectodermal tumor.

Induction of apoptosis by the death ligand TRAIL might be a promising therapeutic approach in colorectal cancer therapy. However, some colon cancer cells are resistant to TRAIL because of the expression of anti-apoptotic proteins, such as FLIP. We studied the role of FLIP for apoptosis resistance in colon cancer and developed an approach to overcome the resistance to TRAIL.The mechanisms of TRAIL-induced cell death in colon cancer cells were studied by Western blot analysis, apoptosis assays, transient and stable transfections, siRNA-mediated knockdown, and FACS analysis.The anti-apoptotic protein FLIP is expressed in the majority of colon carcinoma. Stable over-expression of FLIP renders colon carcinoma cells resistant to the death ligand, TRAIL. siRNA-mediated down-regulation of FLIP sensitizes the cells to TRAIL-induced apoptosis. FLIP-expressing colon cancer cells can be sensitized to TRAIL-induced apoptosis by the anti-diabetic drug troglitazone. Troglitazone induces a pronounced reduction in protein expression levels of FLIP. The troglitazone-dependent down-regulation of FLIP occurs on a post-translational level and involves the accelerated FLIP degradation by the proteasome. Moreover, troglitazone suppresses the expression of the anti-apoptotic protein, survivin, and induces the cell surface expression of the TRAIL receptor 2.The anti-apoptotic FLIP protein plays an important role in apoptosis resistance of colon carcinoma cells. Troglitazone down-regulates FLIP and sensitizes the cells to TRAIL-induced apoptosis. A combined treatment with troglitazone and TRAIL might be a promising experimental therapy for some forms of colorectal cancer because troglitazone sensitizes tumor cells to TRAIL-induced apoptosis via various mechanisms, thereby minimizing the risk of acquired tumor cell resistance.

PEA-15 (Phosphoprotein enriched in astrocytes 15 kD) is a death effector domain-containing protein, which is involved in the regulation of apoptotic cell death. Since PEA-15 is highly expressed in cells of glial origin, we studied the role of PEA-15 in human malignant brain tumors. Immunohistochemical analysis of PEA-15 expression shows strong immunoreactivity in astrocytomas and glioblastomas. Phosphorylation of PEA-15 at Ser116 is found in vivo in perinecrotic areas in glioblastomas and in vitro after glucose deprivation of glioblastoma cells. Overexpression of PEA-15 induces a marked resistance against glucose deprivation-induced apoptosis, whereas siRNA-mediated down-regulation of endogenous PEA-15 results in the sensitization to glucose withdrawal-mediated cell death. This anti-apoptotic activity of PEA-15 under low glucose conditions depends on its phosphorylation at Ser116 Moreover, siRNA-mediated knockdown of PEA-15 abolishes the tumorigenicity of U87MG glioblastoma cells in vivo. PEA-15 regulates the level of phosphorylated ERK1/2 in glioblastoma cells and the PEA-15-dependent protection from glucose deprivation-induced cell death requires ERK1/2 signaling. PEA-15 transcriptionally up-regulates the glucose transporter 3, which is abrogated by the inhibition of ERK1/2 phosphorylation. Taken together, our findings suggest that Ser116-phosphorylated PEA-15 renders glioma cells resistant to glucose deprivation-mediated cell death as encountered in poor microenvironments, e.g. in perinecrotic areas of glioblastomas.

✓ Surgical specimens of 104 craniopharyngiomas from 93 patients were reviewed and characterized histopathologically. They were found to have either a classic adamantinous or a squamous papillary structure. The clinical features of each group were then assessed. The frequently solid (50%), always uncalcified squamous papillary tumor type was found in one-third of the adult patients (≥ 20 years) but did not occur in children. It was associated with a good functional postoperative outcome (84.6%). There have been no cases of tumor recurrence in the squamous papillary group. However, in the group with the adamantinous type of craniopharyngioma, the recurrence rate was 13% in adult patients and 9% in children. When compared to the adult adamantinous cases, the incidence of visual deficits was lower in the squamous papillary group (75% vs. 84%) but the incidence of endocrine abnormalities was higher (75% vs. 52%). Thus, the preoperative, operative, and postoperative features of the two types of craniopharyngioma were found to be distinctly different in adults and children.

Central neurocytoma has been characterised by its intraventricular localisation, predominant occurrence in young adults, oligodendroglioma-like histology, benign course and ultrastructural evidence for neuronal differentiation. Eleven intraventricular central neurocytomas were studied histopathologically, employing cell type-specific immunocytochemical markers and electron microscopic analysis. In the past, these lesions have caused diagnostic problems since central neurocytomas share basic histopathological features with other periventricular neoplasms. Accordingly, several tumours of this series had previously been classified as ependymomas of the foramen of Monro or oligodendrogliomas. Although generally regarded as benign lesions, two central neurocytomas of this series showed histopathological evidence of anaplasia, with focal necrosis, mitotic activity and vascular proliferation. All central neurocytomas exhibited immunoreactivity for neuronspecific enolase and synaptophysin, indicating consistent neuronal differentiation. Three tumours were studied by electron microscopy and contained synaptic vesicles, neuritic processes and neurosecretory granules. In addition, one tumour contained ganglioid cells and this was associated with focal immunoreactivity for neurofilament protein, suggesting that some central neurocytomas may, at least focally, continue to differentiate towards the formation of mature neurons. Two of the tumours expressed glial fibrillary acidic protein in a considerable percentage of neoplastic cells which demonstrates a capacity for bipotential, i.e. glial and neuronal differentiation. We conclude that the central neurocytoma can be reliably diagnosed using antibodies to neuron-specific enolase and synaptophysin, and that histogenetically, this neoplasm is derived from a neuroectodermal precursor cell capable of both, neuronal and glial differentiation. The hypothesis is proposed that the central neurocytoma originates from the subependymal plate of the lateral ventricles, an embryonal matrix cell layer which postnatally maintains a limited proliferative potential.

In a genome-wide screen using differential methylation hybridization (DMH), we have identified a CpG island within the 5' region and untranslated first exon of the secretory granule neuroendocrine protein 1 gene (SGNE1/7B2) that showed hypermethylation in medulloblastomas compared to fetal cerebellum. Bisulfite sequencing and combined bisulfite restriction assay were performed to confirm the methylation status of this CpG island in primary medulloblastomas and medulloblastoma cell lines. Hypermethylation was detected in 16/23 (70%) biopsies and 7/8 (87%) medulloblastoma cell lines, but not in non-neoplastic fetal (n=8) cerebellum. Expression of SGNE1 was investigated by semi-quantitative competitive reverse transcription-polymerase chain reaction and found to be significantly downregulated or absent in all, but one primary medulloblastomas and all cell lines compared to fetal cerebellum. After treatment of medulloblastoma cell lines with 5-aza-2'-deoxycytidine, transcription of SGNE1 was restored. No mutation was found in the coding region of SGNE1 by single-strand conformation polymorphism analysis. Reintroduction of SGNE1 into the medulloblastoma cell line D283Med led to a significant growth suppression and reduced colony formation. In summary, we have identified SGNE1 as a novel epigenetically silenced gene in medulloblastomas. Its frequent inactivation, as well as its inhibitory effect on tumor cell proliferation and focus formation strongly argues for a significant role in medulloblastoma development.

PURPOSE The aim of the present study was to evaluate survival and factors influencing long-term outcome of patients with AFP/beta-HCG secreting (non-seminomatous) central nervous system germ cell tumors (secCNSGCT), who were prospectively collected in the cooperative MAKEI (German: maligne Keimzelltumoren) 89 protocol. PATIENTS AND METHODS Between January 1989 and January 1994, 28 patients with secCNS GCT were registered and treated according to the MAKEI 89 protocol. The protocol recommended, after a clinically or histologically proven diagnosis and cisplatin-based chemotherapy, a resection of residual tumor and craniospinal irradiation (30 Gy) with a tumor boost (20 Gy). RESULTS The estimated (Kaplan-Meier) event-free survival (EFS) of protocol patients is 0.57 +/- 0.09 (n = 28) and the relapse-free survival (RFS) is 0.67 +/- 0.10 (at five and ten years). With respect to long-term survival, the combination of marked neurological symptoms at diagnosis along with primary tumor resection seem to be the main negative prognostic risk factors (Fisher exact test p < 0.05). CNS dissemination at diagnosis can also be considered as a negative risk factor as 3 of 5 patients with primary dissemination died of the disease. CONCLUSION Cisplatin-based three agent chemotherapy followed by resection of the residual tumor and craniospinal irradiation (CSI) with tumor boost is a successful and well-tolerated treatment for secCNSGCTs. The possibility of a clinical diagnosis based on MRI and tumor markers together with the use of modern neurosurgical techniques gives us the chance to postpone or even avoid major surgery. This gives an additional chance to reduce acute morbidity and further decrease late effects.

The sonic hedgehog (Shh) and the Wnt signalling pathways are involved in the development of medulloblastomas (MBs), the most frequent malignant brain tumours in children. Components of these two developmental and cancer-associated pathways, including (Patched) PTCH, SMOH, adenomatous polyposis coli (APC), beta-catenin and AXIN1 show somatic mutations in sporadic MBs. In this study we analysed SUFU (human Suppressor of Fused), which acts as a negative regulator of both the Shh and Wnt signalling pathways and therefore represents a putative tumour suppressor gene, to find out if it is also involved in the pathogenesis of sporadic MBs. We screened 145 primitive neuroectodermal tumours (PNETs) including 90 classic MBs, 42 of the desmoplastic variant and two medullomyoblastomas as well as 11 MB cell lines for mutations using single-strand conformational polymorphism (SSCP) and sequencing analysis. 18% of the MBs exhibited allelic losses on chromosome 10q. In contrast to a previous report, in which truncating mutations of SUFU have been identified in 9% of MBs, we were not able to identify somatic mutations of SUFU in our large tumour panel. We uncovered single nucleotide polymorphisms (SNPs) in exon 4, 8, 11 and in intron 2 in the SUFU gene. Expression analysis by competitive reverse transcription-polymerase chain reaction (RT-PCR) revealed no difference in SUFU mRNA levels of both MB subtypes and normal foetal or adult cerebellar tissues. Our results indicate that genetic alterations of the SUFU gene, do not contribute significantly to the molecular pathogenesis of MBs.

Comparative investigation of immunoglobulin (Ig) heavy chain gene rearrangements and DNA sequence analyses of a primary lymphoma of the CNS (PCNSL) and its recurrence revealed that both tumors used the same Ig gene segment. In addition to shared somatic mutations, the primary and the recurrent PCNSLs harbored somatic mutations unique to each tumor. Clonal evolution rather than subclone selection appears to underlie the development of tumor recurrence in this case.

Focal epilepsies in young patients are frequently associated with differentiated glioneuronal tumours. Dysplastic neurones represent a characteristic neuropathological feature of gangliogliomas, the most common entity encountered in this group. Here, we have analysed two major components of the reelin pathway involved in neuronal migration and cortical development, that is, p35 and disabled-1 (dab1), in gangliogliomas. Genomic structures of human dab1 and p35 were identified 'in silico' using the HTGS databank, NCBI BLAST 2.1. DNA sequence analysis was carried out in gangliogliomas obtained from 29 epilepsy patients vs. peripheral blood DNA from non-affected control individuals (n = 100). Gene expression of dab1 and p35 was determined by real-time RT-PCR (reverse transcriptase polymerase chain reaction) in gangliogliomas (n = 14) vs. non-neoplastic central nervous system tissue (n = 20). The human dab1 gene contains 13 coding exons and is located on chromosome 1p31-32. A single coding exon constitutes the human p35 gene, which is located on chromosome 17q11.2. A novel homologueous genomic region on chromosome 2 has to be taken into account for future studies on p35. One ganglioglioma patient showed a unique polymorphism in the p35 gene. The single base exchange (C to A) at nucleotide 904 of the p35 cDNA (GenBank X80343, start ATG, codon 302) results in a leucine-isoleucine amino acid substitution. No mutations of the dab1 and p35 genes in gangliogliomas were observed. However, significantly lower levels of dab1 and p35 gene transcripts were detected in gangliogliomas compared to controls (dab1 28.24%, t-test P0.001; p35 21.28%, t-test P0.001, in gangliogliomas vs. controls). Our data suggest that mutational events of dab1 and p35 are not involved in the molecular pathogenesis of gangliogliomas. A potential functional role of these developmentally regulated genes for the formation of epileptogenic glioneuronal lesions remains to be elucidated.

Quality management will have an increasing impact on the field of clinical neurosciences. The neuropathological examination of surgical or autopsy samples obtained from the central nervous system (CNS) and related structures, cerebrospinal fluid, peripheral nerve or skeletal muscle serves three major purposes: to identify a structural correlate of the disease, provide a reliable basis for treatment strategies and to investigate underlying pathogenic mechanisms. In order to achieve histopathological diagnosis at a consistent quality, quality management and control have to be implemented at different levels. At the local site, these concern neuropathological services, such as acquisition of tissue specimens, handling and staining procedures as well as diagnostic examination. Training programs offered in slide seminars and diagnostic workshops are important tools to highlight new developments and maintain high standards. National reference centers for brain tumors, neurodegenerative disorders, prion or muscle and nerve diseases assist with a second opinion in difficult cases and support epidemiological as well as clinical therapy trials. An international panel of neuropathologists has established guidelines for the classification of brain tumors, recently updated during a WHO 2000 consensus conference. Finally, the standardized histopathological and cytological diagnosis of CNS disorders plays also an important role in providing quality control for associated fields of the clinical neurosciences, such as neuroradiology, neurology, psychiatry and neurosurgery.

A considerable potential for neurogenesis has been identified in the epileptic rat hippocampus. Here, we explore this feature in human patients suffering from chronic mesial temporal lobe epilepsy. Immunohistochemical detection of the neurodevelopmental antigen nestin was used to detect neural precursor cells, and cell-type specific markers were employed to study their histogenetic origin and potential for neuronal or glial differentiation. The ontogenetic regulation of nestin-positive precursors was established in human control brains (week 19 of gestation-15 years of age). A striking increase of nestin-immunoreactive cells within the hilus and dentate gyrus could be observed in a group of young patients with temporal lobe epilepsy (TLE) and surgical treatment before age 2 years compared to adult TLE patients and controls. The cellular morphology and regional distribution closely resembled nestin-immunoreactive granule-cell progenitors transiently expressed during prenatal human hippocampus development. An increased Ki-67 proliferation index and clusters of supragranular nestin-immunoreactive cells within the molecular layer of the dentate gyrus were also noted in the group of young TLE patients. Confocal studies revealed colocalization of nestin and the betaIII isoform of tubulin, indicating a neuronal fate for some of these cells. Vimentin was consistently expressed in nestin-immunoreactive cells, whereas cell lineage-specific markers, i.e., glial fibrillary acidic protein, MAP2, neurofilament protein, NeuN, or calbindin D-28k failed to colocalize. These findings provide evidence for increased neurogenesis in pediatric patients with early onset of temporal lobe epilepsy and/or point towards a delay in hippocampal maturation in a subgroup of patients with TLE.

Medulloblastoma (MB) represents the most frequent malignant brain tumor in children. Most MBs appear sporadically; however, their incidence is highly elevated in two inherited tumor predisposition syndromes, Gorlin's and Turcot's syndrome. The genetic defects responsible for these diseases have been identified. Whereas Gorlin's syndrome patients carry germ-line mutations in the patched (PTCH) gene, Turcot's syndrome patients with MBs carry germ-line mutations of the adenomatous polyposis coli (APC) gene. The APC gene product is a component of a multiprotein complex controlling beta-catenin degradation. In this complex, Axin plays a major role as scaffold protein. Whereas APC mutations are rare in sporadic MBs, a hot-spot region of beta-catenin (CTNNB1) mutations was identified in a subset of MBs. To find out if Axin is also involved in the pathogenesis of sporadic MBs, we analyzed 86 MBs and 11 MB cell lines for mutations in the AXIN1 gene. Using single-strand conformation polymorphism analysis, screening for large deletions by reverse transcription-PCR, and sequencing analysis, a single somatic point mutation in exon 1 (Pro255Ser) and seven large deletions (12%) of AXIN1 were detected. This indicates that AXIN1 may function as a tumor suppressor gene in MBs.

Gangliogliomas constitute the most frequent tumour entity in patients with temporal lobe epilepsy. The characteristic histopathological admixture of glial and neuronal elements, the focal nature and their differentiated phenotype and benign biological behaviour suggest an origin from a developmentally compromised or dysplastic precursor lesion. The present study analysed TSC1 and TSC2 genes as potential candidates involved in the pathogenesis of this intriguing neoplasm. Recent data suggest that both genes play a role in cortical differentiation and growth control. DNA sequence analysis of TSC1 and TSC2 was studied in 20 patients with gangliogliomas. Fifteen of these tumours (75%) carried polymorphisms in the TSC2 gene. The frequency of these polymorphisms was significantly increased in intron 4 (12.5%) and exon 41 (15%) compared to control individuals (8.1 and 6.5%, respectively, n = 100). A somatic mutation in intron 32 of the TSC2 gene was encountered in one patient. In the TSC1 gene, seven polymorphisms occurred as a combination of base exchanges in exon 14 and intron 13. No mutations were observed in this gene. Laser microdissection and harvesting of individual neuronal and glial elements identified the intron 32 mutation within the glial portion but not in dysplastic neurones of the tumour. The data demonstrate numerous polymorphisms as well as a novel TSC2 mutation in gangliogliomas from patients with chronic epilepsies. The selective detection of the TSC2 mutation within the glial component of a ganglioglioma suggests that the glioma portion has undergone clonal evolution in this case.

After adoptive transfer of pre-activated lymphocytes into the operation cavity of glioma patients, tumor regression and improved survival have been reported in some patients. Results were most impressive when bispecific antibodies with tumor x CD3 specificity were also applied. In this study, we attempted to avoid time-consuming pre-activation procedures for adoptively transferred cells by using a combination of bispecific antibodies directed to the EGF receptor (EGFR) on tumor cells and to CD3 and CD28 on T cells. Eleven patients with high-grade malignant glioma received 3 injections of 2 bispecific antibody fragments (EGFR x CD3 and EGFR x CD28) together with freshly isolated autologous lymphocytes via an Ommaya reservoir. Intracavitary fluid aspirated during immunotherapy was examined for markers of T-cell activation. Increased levels of soluble IL-2 receptor and TNF-alpha were detected in the intracavitary fluid of all patients tested. Two of the 11 treated patients experienced a beneficial response to therapy as defined by a transient contrast enhancement in subsequent MRI scans and prolonged survival. Side effects were transient and consisted of fever, nausea, headache and aggravation of pre-existing neurologic deficits. These adverse effects were most likely due to the antibody construct containing anti-CD3 specificity. Two patients developed cerebral edema and required steroid treatment.

The rationale of the HIT-LGG protocol is to delay standard radiotherapy through administration of chemotherapy for children with progressive low grade glioma at an age under 5 years and in older children upon individual decision.Until October 10th., 1999, 402 patients from 69 hospitals were registered. 130 children with progressive tumors were treated after a median observation time of 5 months: 46 patients received primary radiotherapy and 84 primary chemotherapy. A ten week induction period with weekly Vincristine and pulses of Carboplatin at weeks 1, 4, 7 and 10 is followed by consolidation with simultaneous application of both drugs every 4 weeks until week 53.Of 84 patients in the chemotherapy arm of the study (49 male, 35 female, 23 NF I, median age 2.99 years) 36 received treatment at diagnosis and 43 after a median observation time of 19.7 months. 94.3% achieved a clinical and neuroradiological response according to protocol criteria (5 CR, 30 PR/OR, 31 SD) after a median of 5.1 months. 4 tumors showed primary progression (9 too early, 5 not known). Only 6 of 84 children received radiation therapy for progressive disease during (2) or after termination (4) of chemotherapy, after a median delay of 25.6 months at a median age of 6.0 years. At a median observation time of 21.0 months, 6 children are in CR, 11 in PR, 48 have SD, 4 tumors are progressive and 3 children died of their tumor. (9 too early, 3 not known). PFS is at 72% after 36 months. 24 of 27 children experiencing allergic reactions to Carboplatin had to interrupt chemotherapy prematurely.Combination therapy with Carboplatin and Vincristine can effectively delay the start of radiotherapy in children with progressive low-grade glioma. The high rate of hypersensitivity reactions has to prompt future modifications of treatment.

Selective hippocampal cell loss and altered neurotransmitter receptor expression have been proposed as pathogenic mechanisms in the development of chronic mesial temporal lobe epilepsy (TLE). Studies in animal models point to metabotropic glutamate receptors (mGluRs) as modulators of hippocampal epileptogenesis. In addition, mGluRs may constitute specific targets for the development of novel anticonvulsive drugs. As mGluR4 represents an inhibitory class III mGluR associated with the reduction of intracellular cyclic AMP levels and calcium influx, we have analyzed the regional and cellular expression of mGluR4 in surgical hippocampal specimens obtained from patients with TLE by using immunohistochemistry and in situ hybridization. Although the hippocampi of control specimens (n = 11) were almost devoid of mGluR4 immunolabeling, all TLE specimens (n = 35) showed a striking up-regulation of mGluR4 immunoreactivity, in particular within the dentate gyrus. Immunoelectron microscopy localized the receptor protein to the periphery of presynaptic and postsynaptic membranes. In situ hybridization revealed increased transcript levels of mGluR4 in dentate granule cells and residual CA4 neurons of TLE specimens compared with controls. Our results suggest a potential role of mGluR4 in counteracting excitatory hippocampal activity and in modulating seizure-associated vulnerability of hippocampal neurons. These data may also provide a basis for pharmacological studies of mGluR4 agonists as potential novel drugs in the treatment of TLE.

Marked neovascularization and vascular endothelial proliferation are characteristic features of malignant gliomas. Vascular endothelial growth factor (VEGF), an angiogenic protein secreted by glioma cells, appears to play a crucial role for induction of neoangiogenesis. The VEGF receptors fms-like tyrosine kinase-1 (Flt-1)/VEGFR-1 and kinase insert domain-containing receptor (KDR)/ VEGFR-2 are up-regulated on the surface of endothelial cells (ECs) in gliomas. Both receptor genes contain an Ets-responsible element in their promoters. The proto-oncogene ets-1 encodes a transcription factor that has been associated with blood vessel formation in vivo under physiological and pathophysiological conditions including tumor neovascularization. Ets-1 is induced by VEGF in cultured ECs. In vitro data also point to a role of Ets-1 as a transcriptional activator of Flt-1. These properties prompted us to investigate Ets-1 expression in 32 human astroglial tumors of WHO grades I-IV and to correlate the data with the expression pattern of VEGF, Flt-1, and KDR. By in situ hybridization, high ets-1 mRNA levels were found in the glioma microvasculature with particularly prominent signals in glomeruloid vascular endothelial proliferations of glioblastomas (WHO grade IV). Semiquantitative reverse transcription-PCR identified the full-length ets-1 transcript but none of three known splice variants encoding isoforms with different functional domains. Immunohistochemical staining demonstrated Ets-1 protein preferentially in the nucleus of those ECs with an epithelioid morphology consistent with an activated state, whereas quiescent flat-shaped ECs predominantly displayed cytosolic immunoreactivity. This observation proposes nuclear translocation of Ets-1 during neoangiogenesis. VEGF synthesis by glioma cells was accompanied by Ets-1 expression in adjacent microvascular ECs. Furthermore, a highly significant correlation was observed between Ets-1 and Flt-1 (but not KDR) expression in ECs of the glioma microvasculature. Our data suggest that VEGF secreted by glioma cells induces Ets-1 in adjacent microvascular ECs, which subsequently transactivates the VEGF receptor Flt-1. This cascade may crucially promote neoangiogenesis in human gliomas.

VEGF (vascular endothelial growth factor), one of the most potent angiogenic factors, has recently been identified as an inducer of neoangiogenesis in many tumors including gliomas. VEGF itself appears to be regulated through different pathways. Since malignant gliomas frequently show EGF receptor amplification and express IL-1, a pivotal regulatory cytokine involved in angiogenesis, we analyzed interactions between EGF/EGF receptor and IL-1/IL-1 receptor and VEGF in the established glioblastoma cell lines U-87 MG and A-172. Basal VEGF expression was an order of magnitude higher in U-87 MG compared to A-172. IL-1 caused a fast and strong increase of VEGF secretion in U-87 MG which appeared to harbor an intracellular VEGF pool for enhanced exocytosis. The IL-1 receptor antagonist (IL-1-ra) reversed this effect suggesting an IL-1 receptor-associated mechanism. In contrast, VEGF secretion could not be increased by exogenous IL-1 exposure in A-172, which apparently lacked an intracellular VEGF pool for augmented exocytosis. However, IL-1-ra treatment alone caused a significant reduction of basal VEGF secretion in both U-87 MG and A-172. This suggests that baseline secretion of VEGF involves IL-1 receptor activation by endogenously produced IL-1. EGF also stimulated the secretion of VEGF into the cell supernatant. However, this effect, observed in both U-87 MG and A-172, was delayed and only occurred following replenishment of the intracellular VEGF pool. EGF upregulated the amount of VEGF mRNA. In general, the effects of IL-1 and EGF on VEGF were additive, suggesting independent mechanisms. Since IL-1 appears to be involved in VEGF secretion in glial tumors through an autocrine/paracrine mechanism, recombinant human IL-1-ra may evolve as a new agent for anti-angiogenic glioma therapy.

The induction of neural tumors by N-ethyl-N-nitrosourea (EtNU) in inbred strains of rats has evolved as a valuable model system of developmental stage- and cell type-dependent oncogenesis. Tumor yield and latency times are strongly influenced by genetic background. Compared with BDIX rats, BDIV rats are relatively resistant to the induction of brain tumors by EtNU, with a lower tumor incidence and latency periods prolonged by a factor of 3. To characterize genetic abnormalities associated with impaired tumor suppressor gene function in neuro-oncogenesis, losses of heterozygosity (LOHs) and microsatellite instability (MI) were investigated in brain tumors induced by EtNU in (BDIV x BDIX) F(1) and F(2) rats. The polymerase chain reaction was used to amplify 55 polymorphic microsatellite markers spanning the entire rat genome. The tumors displayed different histologies and grades of malignancy, corresponding to part of the spectrum of human gliomas. MI was not observed in any of the tumors. LOH of rat chromosome 1q was predominantly detected in oligodendrogliomas and mixed gliomas, with a 30% incidence in informative cases. 11p15.5, the human genome region syntenic to the consensus region of LOHs observed on rat chromosome 1, has been shown to be involved in the formation of gliomas in humans. Furthermore, rat brain tumors of different histologies often showed allelic imbalances on chromosome 17p. In both cases of LOH, there was a clear bias in favor of the parental BDIV allele, suggesting the involvement of tumor suppressor genes functionally polymorphic between the two rat strains.

In addition to functionally affected neuronal signaling pathways, altered axonal, dendritic, and synaptic morphology may contribute to hippocampal hyperexcitability in chronic mesial temporal lobe epilepsies (MTLE). The sclerotic hippocampus in Ammon's horn sclerosis (AHS)-associated MTLE, which shows segmental neuronal cell loss, axonal reorganization, and astrogliosis, would appear particularly susceptible to such changes. To characterize the cellular hippocampal pathology in MTLE, we have analyzed hilar neurons in surgical hippocampus specimens from patients with MTLE. Anatomically well-preserved hippocampal specimens from patients with AHS (n = 44) and from patients with focal temporal lesions (non-AHS; n = 20) were studied using confocal laser scanning microscopy (CFLSM) and electron microscopy (EM). Hippocampal samples from three tumor patients without chronic epilepsies and autopsy samples were used as controls. Using intracellular Lucifer Yellow injection and CFLSM, spiny pyramidal, multipolar, and mossy cells as well as non-spiny multipolar neurons have been identified as major hilar cell types in controls and lesion-associated MTLE specimens. In contrast, none of the hilar neurons from AHS specimens displayed a morphology reminiscent of mossy cells. In AHS, a major portion of the pyramidal and multipolar neurons showed extensive dendritic ramification and periodic nodular swellings of dendritic shafts. EM analysis confirmed the altered cellular morphology, with an accumulation of cytoskeletal filaments and increased numbers of mitochondria as the most prominent findings. To characterize cytoskeletal alterations in hilar neurons further, immunohistochemical reactions for neurofilament proteins (NFP), microtubule-associated proteins, and tau were performed. This analysis specifically identified large and atypical hilar neurons with an accumulation of low weight NFP. Our data demonstrate striking structural alterations in hilar neurons of patients with AHS compared with controls and non-sclerotic MTLE specimens. Such changes may develop during cellular reorganization in the epileptogenic hippocampus and are likely to contribute to the pathogenesis or maintenance of temporal lobe epilepsy.

Ependymal tumors are heterogeneous with regard to morphology, localization, age at first clinical manifestation, and prognosis. Several molecular alterations have been reported in these tumors, including allelic losses on chromosomes 10, 17, and 22 and mutations in the NF2 gene. However, in contrast to astrocytic gliomas, no consistent molecular alterations have been associated with distinct types of ependymal tumors. To evaluate whether morphological subsets of ependymomas are characterized by specific genetic lesions, we analyzed a series of 62 ependymal tumors, including myxopapillary ependymomas, subependymomas, ependymomas, and anaplastic ependymomas, for allelic losses on chromosome arms 10q and 22q and mutations in the PTEN and NF2 genes. Allelic losses on 10q and 22q were detected in 5 of 56 and 12 of 54 tumors, respectively. Six ependymomas carried somatic NF2 mutations, whereas no mutations were detected in the PTEN gene. All six of the NF2 mutations occurred in ependymomas of WHO grade II and were exclusively observed in tumors with a spinal localization (P = 0.0063). These findings suggest that a considerable fraction of spinal ependymomas are associated with molecular events involving chromosome 22 and that mutations in the NF2 gene may be of primary importance for their genesis. Furthermore, our data suggest that the more favorable clinical course of spinal ependymomas may relate to a distinct pattern of genetic alterations different from that of intracerebral ependymomas.

The etiology and pathogenesis of epilepsy-associated local lesions remain largely unknown. Histopathologically, the most frequent lesions comprise gangliogliomas and glioneuronal malformations, i.e., hamartias or hamartomas, with a preferred location in the temporal lobe of young patients. A characteristic histopathological admixture of glial and neuronal elements, the focal appearance and the benign clinical behaviour suggest a malformative nature. So far, no molecular genetic alterations specifically involved in the pathogenesis of these glioneuronal lesions have been identified. However, immunohistochemical analysis revealed distinct distribution patterns of oncofetal antigens. The embryonic form of the neural cell adhesion molecule is present within glioneuronal hamartias, indicating an early migrational disorder. Recently, we have observed immunoreactivity for the stem cell marker CD34 in the majority of gangliogliomas and glioneuronal hamartomas. Based on these findings, we propose a common origin of gangliogliomas and glioneuronal hamartomas from a bipotent precursor that undergoes abnormal glioneuronal development.

Limbic P300 potentials can be recorded within the mesial temporal lobes of patients with temporal lobe epilepsy (TLE). To delineate possible mechanisms of their generation and pathological alteration, we analysed limbic P300s in 55 TLE patients with and 29 without Ammon's horn sclerosis (AHS) and correlated their amplitudes with neuronal cell counts in 30 histopathological specimens. Limbic P300 amplitudes were reduced on the side of the epileptogenic focus only in patients with AHS. Moreover, in AHS patients, limbic P300 latencies were prolonged bilaterally; and in patients with left-sided AHS, amplitudes were reduced bilaterally. Both findings suggest bilateral functional deficits in TLE with unilateral AHS. Limbic P300 areas correlated significantly with neuronal densities of dentate gyrus granule cells but not hippocampal pyramidal cells in the CA1-4 (cornu ammonis) subfields. This finding points to a potential mechanism for the bilateral effects of unilateral AHS as both dentate gyri exhibit strong reciprocal contralateral connectivity.

The gene mutated in ataxia telangiectasia (A-T) patients (ATM) is located on chromosome 11q22-23, a region frequently altered in mammary tumors. Patients homozygous for ATM mutations are prone to develop a variety of different neoplasms. Female heterozygotes have been reported to carry a 5- to 8-fold increased risk of breast cancer. However, germline mutations in the ATM gene are rare in women with sporadic breast carcinomas. Most of the alterations described in A-T patients result in a functionally inactive ATM protein. Moreover, it has been suggested that mutations of the ATM gene in A-T patients influence the amount of ATM mRNA and that this may affect the severity of the disease. In the present study, we have analyzed ATM transcripts in a series of 39 breast carcinomas, 14 benign breast lesions and 12 normal breast tissue samples. ATM mRNA levels were determined by semiquantitative competitive RT-PCR. Competitor RNA molecules for the ATM gene and the housekeeping gene beta-2-microglobulin (B2M) were generated by PCR mutagenesis. Low concentrations of ATM transcripts were detected in breast carcinomas, intermediate levels in benign lesions and highest levels in normal breast tissue specimens (F-test, p = 0.0013). Our results indicate that reduced expression of the ATM gene may contribute to the development and/or malignant progression of breast carcinomas.

Both surgical and molecular neuropathologists have recently achieved remarkable progress in the histogenetic classification and molecular characterization of human gliomas. Major histopathological achievements in the revised WHO classification include the introduction of immunohistochemical reagents for glial fibrillary acidic protein and for the proliferation-associated antigens, the definition of glioblastoma multiforme as an astrocytic neoplasm and the recognition of the pleomorphic xantho--astrocytomas as a novel clinico-pathological entity. In molecular neuropathology, alterations of oncogenes and tumor suppressor genes and their potential functions have been identified, microsatellite analyses have revealed novel loci for putative tumor suppressor genes and distinct molecular pathways for different tumor entities are beginning to emerge. Mutations in cell cycle regulatory genes are present in most glioblastomas and may account for their striking growth potential. Autocrine and paracrine growth factors and their respective protein tyrosine kinase receptors appear to contribute both to glial and endothelial cell proliferation. In our contribution, we would like to focus on astrocytic gliomas. Findings with potential diagnostic relevance include changes associated with malignant progression of low grade astrocytomas, patterns of genetic alterations which allow to further differentiate histopathological entities such as the glioblastoma multiforme into genetically distinct subsets and mechanisms of tumor angiogenesis in malignant gliomas. One of the major tasks ahead is to establish correlations and relationships between histopathological, molecular and clinical data. This will require a long-term collaboration between molecular neuropathologists, neurosurgeons and clinical neuro-oncologists.

Although clinical and electrophysiological evidence indicates that the amygdaloid body plays an important role in the pathogenesis of temporal lobe epilepsy, there are very few detailed data on histopathological changes in this nucleus in epilepsy patients. In the present study we have examined the lateral nucleus of the amygdaloid body in 70 surgical specimens from patients with temporal lobe epilepsy and in 10 control specimens with respect to neuronal density and gliosis. The results were compared to the neuronal loss in the hippocampal formation. Our goal was to examine the pathological alterations of the amygdaloid body and their correlation with other morphological changes in temporal lobe epilepsy. In epilepsy patients with Ammon's horn sclerosis or focal lesions of the temporal lobe, the neuronal density of the lateral amygdaloid nucleus was significantly decreased as compared to normal controls (P0.001). Overall, the mean volumetric density in epilepsy patients was reduced to 59% of that in normal individuals. There was no correlation between the neuronal density in the lateral amygdaloid nucleus and that in the different segments of the hippocampal formation or to the age at onset or the duration of epilepsy. The neuronal loss of the amygdaloid nucleus correlated well with the presence of fibrillary gliosis. Our findings demonstrate that the amygdaloid body is severely altered in most patients with temporal lobe epilepsy and that these changes are independent of those in the hippocampus. The presence of neuronal loss and gliosis in the amygdaloid nucleus of patients with focal lesions but no Ammon's horn sclerosis is compatible with an involvement of the amygdala in secondary epileptogenesis.

Inactivating mutations in the PTCH gene, a human homologue of the Drosophila segment polarity gene patched, have been identified recently in patients with nevoid basal cell carcinoma syndrome. These patients are predisposed to various neoplasias including basal cell carcinomas and medulloblastomas (MBs). To determine the involvement of PTCH in sporadic MBs, which represent the most frequent malignant brain tumors in children, we screened for PTCH alterations in an unselected panel of 64 biopsy samples from 62 patients and four continuous MB cell lines, all derived from patients with sporadic MBs. Using single-strand conformational polymorphism analysis, we screened exons 2-22 and detected nonconservative PTCH mutations in 3 of 11 samples from sporadic cases of the desmoplastic variant of MB but none in 57 MBs with classical (nondesmoplastic) histology. In two of the tumors with mutations and in two additional desmoplastic cases, loss of heterozygosity was found at 9q22. These findings suggest that PTCH represents a tumor suppressor gene involved in the development of the desmoplastic variant of MB.

In the normal brain, low levels of cytokines are observed, whereas inflammatory disorders of the central nervous system are characterized by an up-regulation of cytokine production. The cellular sources for cytokines in the central nervous system are largely undefined. In the present study, we have analyzed intracerebral cytokine production in normal and Toxoplasma gondii-infected mice using immunohistochemistry, in situ hybridization, flow cytometry of brain-derived leukocytes, and reverse transcriptase polymerase chain reaction detection in various subpopulations of inflammatory cells. In the normal brain, neurons and choroid plexus epithelia expressed interleukin (IL)-1 beta and IL-10. Microglia/macrophages produced IL-1 beta, IL-10, and tumor necrosis factor-alpha In Toxoplasma encephalitis, these cell types exhibited increased levels of the respective cytokines. In addition, microglia/macrophages showed a de novo expression of inducible nitric oxide synthase. CD4+ and CD8+ T cells, which were recruited to the brain, produced IL-2, IL-10, tumor necrosis factor-alpha, and interferon-gamma. IL-4 was exclusively detectable in CD4+ T cells, whereas CD8+ T cells showed expression of IL-1 beta. As chronic Toxoplasma encephalitis was not associated with neuronal degeneration and an up-regulation of neurotrophic factors, some cytokines may also exert neurotrophic and/or neuroprotective properties.

Toxoplasma gondii may cause severe infections in immunocompromised patients including fetuses and those with AIDS. Among the factors mediating protection against T. gondii, IFN-gamma has gained special attention. To analyze the role of IFN-gamma in the early phase of toxoplasmosis, IFN-gamma receptor-deficient (IFN-gamma R0/0) mice were orally infected with low-virulent toxoplasms. IFN-gamma R0/0 mice died of the disease up to day 10 postinfection, whereas immunocompetent wild-type (WT) mice developed a chronic toxoplasmosis. Histopathology revealed that in IFN-gamma R0/0 mice, the parasite multiplied unrestrictedly in the small intestine, the intestinal lymphatic tissue, the liver, and the spleen. Ultimately, animals died of a necrotizing hepatitis. In WT mice, the same organs were effected, but multiplication of the parasite was effectively limited. Compared with WT mice, immunohistochemistry and flow cytometry demonstrated that in IFN-gamma R0/0 mice, macrophages were only marginally activated in response to the infection, as evidenced by a reduced expression of major histocompatability complex class II antigens. In addition, immunohistochemistry and RT-PCR showed a reduced production of the macrophage-derived cytokines tumor necrosis factor-alpha, inducible nitric oxide synthase, and IL-1 beta in the liver of IFN-gamma R0/0 mice. In contrast, activation of T cells, recruitment of immune cells to inflammatory foci, and anti-T. gondii IgM antibody production were unaffected by the mutation of the IFN-gamma R. Moreover, induction of IL-2, IL-4, and IL-10 mRNA transcripts in the liver was normal in IFN-gamma R0/0 mice. Adoptive transfer experiments revealed that the immune T cells of WT animals did not protect IFN-gamma R0/0 mice from lethal infection with highly virulent toxoplasms, whereas WT mice were significantly protected by the adoptive transfer. Based on these studies, we conclude that IFN-gamma is absolutely required for an efficient activation of macrophages. Macrophages are of critical importance in toxoplasmosis, and insufficient macrophage activation cannot be compensated by other immune mechanisms.

Medulloblastoma is a primitive neuroectodermal tumor of the cerebellum with poorly understood pathogenesis. Previous studies have reported loss of heterozygosity (LOH) on chromosome arms 17p, 11p and 9q and cytogenetic abnormalities of chromosome 1 in medulloblastoma. We have used the polymerase chain reaction to amplify 10 microsatellites on the short arm and 8 microsatellites on the long arm of chromosome 1 to assess allelic loss in 22 medulloblastomas. Loss of heterozygosity (LOH) on chromosome 1 was found in 9 cases. Eight medulloblastomas (36%) showed an interstitial LOH on chromosome 1q. The common region of overlap was mapped between D1S 1604 and D1S237 and included the locus F13B in the chromosomal region 1q31-q32.1. An additional tumor had LOH in a proximal region of 1p, but did not exhibit LOH on 1q. None of the medulloblastomas exhibited LOH of the telomeric portion of chromosome 1p, which has been associated with several other human malignancies. Our data suggest the presence of a putative tumor suppressor gene located near the locus F13B on chromosome arm 1q that appears to be involved in the pathogenesis of medulloblastoma.

Although medulloblastoma (MB) represents the most frequent malignant brain tumour in children, the molecular pathogenesis of this tumour is still poorly understood. Microsatellite and RFLP studies have revealed allelic losses on the short arm of chromosome 17 in the region 17p13 in approximately 50% of MBs. A candidate for this putative MB suppressor on chromosome 17p13.3 is the recently cloned LIS-1 gene which codes for a regulatory subunit of the intracellular form of the platelet activating factor (PAF) acetylhydrolase. PAF is involved in signal transduction pathways during cerebral development and in the process of neuronal cell migration and differentiation. In this study, 42 medulloblastomas including seven tumour cell lines and 35 primary tumours were analysed for LIS-1 mRNA expression by RT-PCR and Northern blot analysis. Twenty-four paired blood and MB DNA samples were examined by PCR analysis with a panel of microsatellites located on chromosome 17p to detect loss of heterozygosity (LOH). Finally, the single-strand conformation polymorphism (SSCP) technique and DNA sequencing were employed to detect mutations or small deletions in the coding region of the LIS-1 gene. Of 24 MBs, 13 exhibited loss of heterozygosity on chromosome 17p13.3 for at least one of the microsatellite markers. LIS-1 mRNA expression was detected in all MB tumours and MB cell lines. None of the tumours showed a somatic mutation in the LIS-1 coding region. A novel polymorphism in the 3'UTR of LIS-1 was found in MB patients, but also in healthy control persons. These data indicate that the LIS-1 gene located at 17p13.3 is not involved in the molecular pathogenesis of MBs.

Oral infection with a low-virulence strain of Toxoplasma gondii (Tg) induced a persisting encephalitis in resistant strains of mice. In the present study we have examined transcripts of various cytokines during acute and chronic stages of murine Tg encephalitis. In the brain of infected animals, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), IL-6, IL-10 and IL-12 mRNA were induced to a significant extent, but only low levels of IL-4 mRNA were detectable. A similar cytokine profile was observed in the spleen. However, in contrast to the brain, the increase of IL-2 mRNA was particularly pronounced in the spleen, whereas the opposite was found for IFN-gamma and TNF-alpha mRNA. Thus, cytokines involved in T-cell proliferation were more prevalent in the spleen, but in the brain, where Tg actively multiplies, the effector molecules IFN-gamma and TNF-alpha were preferentially up-regulated. In addition, a detailed analysis of cytokine mRNA levels in major histocompatibility complex (MHC)-congenic strains of BALB and B10 mice revealed that the genetically regulated susceptibility to Tg was correlated with the amount of intracerebrally produced cytokine mRNA for IFN-gamma, TNF-alpha and IL-6. Mice with a strong increase of these cytokine mRNA were significantly better protected against Tg. This indicates that the outcome of toxoplasmosis may be critically dependent on an adequately regulated intracerebral immune response.

In recent years there has been considerable progress in brain tumor neuropathology. Several new diagnostic entities have been recognized, subclassification schemes have been modified, and new concepts on the histogenesis and cell biology of brain tumors have emerged. In 1993, a revised WHO classification of brain tumors was published by an international committee. This article summarizes the pertinent new aspects. As novel tumor entities, the central neurocytoma, the dysembryoplastic neuroepithelial tumor (DNT), desmoplastic infantile ganglioglioma (DIG) and pleomorphic xanthoastrocytoma (PXA) have been included. Several histopathological variants of meningiomas have been added of which only the papillary meningioma and the atypical meningioma are characterized by an increased rate of recurrence. Meningeal hemangiopericytomas and hemangioblastomas are classified as tumors of non-meningothelial origin. The glioblastoma multiforme, which had previously been listed as an embryonal tumor, is now recognized as an astrocytic glioma. Immunohistochemistry has greatly advanced the practical diagnosis and classification of brain tumors. There are specific markers for all normal and neoplastic cell types except for oligodendroglioma cells. The prognosis of and therapeutic approaches to brain tumors greatly depend on histopathological grading. The WHO proposes four tumor grades, i.e., I, II, III, and IV. As a rule, grades I and II tumors are viewed as benign or semi-benign neoplasms and grades III and IV tumors as malignant. There are attempts to use new biological parameters for the grading of brain tumors. Antibodies to proliferation-associated proteins reflect tumor growth. Molecular genetic approaches to tumor-associated genes and gene loci are particularly promising new tools for the future.

Human glioblastomas were evaluated for overexpression of the epidermal growth factor receptor (EGFR) in a therapeutic trial with the anti-EGFR antibody EMD 55900. A total of 55 cases were examined by immunohistochemistry using 4 different monoclonal antibodies on frozen or on paraffin sections: EGFR-1 (Amersham), E 62 (Merck), E 30 (Merck), and EMD 55900 (MAB 425, Merck). Definition for inclusion in clinical trials of EMD 55900 was an immunohistochemical overexpression of grade 4+ or 3+ in a scale of 4 grades of staining quality. The use of the 4 different antibodies gave essentially equal results. In 21 cases, the immunohistochemical results were supplemented by molecular genetic analysis of EGFR amplification on the corresponding locus of chromosome 7, using frozen tissue from the same blocks after screening for vital tumor areas. Since no other material was available, the differential polymerase chain reaction technique was applied. Interferon-gamma (IFN-gamma) served as a reference gene. In a preliminary experimental series with cases of known EGFR amplification, a densitometric EGFR/IFN-gamma ratio higher than 3 was determined as indicator for amplification of the EGFR gene. With this experimental approach we were able to identify an amplified EGFR gene in 13 specimens including 2 from recurrent glioblastomas in the same patients. All of these showed an increased immunoreactivity for EGFR protein. The degree of EGFR amplification (EGFR/IFN-gamma ratio as measured by DNA densitometry) showed a positive correlation with the grade of immunohistochemical protein expression, both in regard to the fraction of positive cells and to the overall staining intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

The surgical treatment of chronic epilepsies is increasing rapidly. A review of 279 surgical specimens of patients with chronic pharmaco-resistant epilepsies from the University of Bonn Medical Centre revealed specific histopathological findings in 89% of all well-preserved specimens. The most frequent focal lesions were low-grade brain tumours such as gangliogliomas, pilocytic astrocytomas and dysembryoplastic neuroepithelial tumours. Other common findings were glioneuronal and vascular malformations and Ammon's horn sclerosis. This suggests that chronic intractable epilepsies are frequently caused by structural alterations.

Loss of heterozygosity (LOH) indicates the existence of tumor suppressor genes (TSG) in the affected chromosomal loci. In order to uncover the involvement of such genes, we analyzed LOH in different chromosomal regions of sporadic breast carcinomas.47 breast cancer patients were screened for LOH with microsatellite markers on 18 different loci. DNA fragments were amplified by PCR from tumor and reference tissue. The PCR products were run on 8% denaturing polyacrylamide gels and visualized by silver straining.The following LOH-rates were found for the different loci: D6S497 (6p21, WAF-Region): 0%, D7S495: 9%, D7S522: 13%, D7S523: 22%, D11S488 (11q24-25): 38%, D13s321 and D13s765 (13q13-14, Rb-Region): 30% and 17%, D13S260 and D13S267 (13q12.3, BRCA2-Region): 28% both, D16S539 (16q22-24, E-Cadherin-Region): 35%, D17S5 (17p13.3): 17%, TP53 (17p13.1): 32% D17S250 (17q11-12): 22%, D17S855 (17q21 within the BRCA1 gene): 25%, D17S579 (17q21 telomer from BRCA1): 13%, D17S846 (centromere from BRCA1): 17%, 17q24 (SSTR 2): 9%, D22S684 (22q12, NF2-Region): 20%. Overall 66% of the tumors exhibited LOH. Lymphnode positive tumors showed significantly higher LOH rates than lymphnode negative tumors.Highest LOH-rates were found on chromosomes 11, 13, 16 and 17 indicative of relevant TSG's in the examined loci. In addition the findings indicate prognostic relevance of multiple LOH's in breast cancer.

Medulloblastoma (MB) represents the most frequent malignant brain tumor of childhood but only a few cell lines and animal models of this primitive neuroectodermal tumor (PNET) have thus far been established. Using specific cell culture conditions, we were able to derive four human MB cell lines (MHH-MED-1-4) as well as a cell line from a spinal PNET (MHH-PNET-5). The four MB cell lines grew in suspension as floating cell aggregates or as slightly adherent cells. They consisted of undifferentiated cells that did not express markers of late neuronal or glial lineages such as neurofilaments or glial fibrillary acidic protein. They also lacked expression of major histocompatibility complex class I or II antigens on the cell surface. All four MB lines were positive for vimentin and neuron-specific enolase, whereas synaptophysin, neural cell adhesion molecule, galactocerebroside, GD2, GD3, and the A2B5 antigen were expressed inconsistently. In contrast, MHH-PNET-5 grew as adherent monolayer and expressed major histocompatibility complex class I antigen. By cytogenetic analysis, the lines were near diploid with clonal aberrations. The MB lines showed no losses of chromosome arm 17p by either cytogenetic or microsatellite analyses. The cell line MHH-MED-2 exhibited double minute chromosomes, amplification of the c-myc gene, and overexpression of c-myc mRNA and protein. N-myc, p53, and Rb protein expression were unaltered. All four continuously passaged MB cell lines and the MHH-PNET-5 line were xenotransplanted s.c. into athymic mice; three of four MB lines and the spinal PNET line gave rise to tumors. These cell lines will be useful tools for biological and preclinical studies on PNETs.

The p53 tumor suppressor gene encodes a nuclear phosphoprotein which is involved in the regulation of cell proliferation (Boyd and Barrett 1990). The wild-type p53 gene acts as a tumor suppressor gene, whereas some p53 mutations occurring within highly conserved regions not only cause loss of tumor suppressor function but may activate p53 to an oncogene in a dominant negative fashion (Finlay et al. 1989; Eiyahu et al. 1989). A variety of human tumors have been shown to contain either a loss of both alleles of the p53 gene, a loss of one allele of the p53 gene, and one p53 allele with an associated point mutation, insertion, or deletion of the remaining allele or an inactivation of the p53 gene in one allele but a normal (wild-type) sequence in the other. The rapidly accumulating data on p53 genetic alterations indicate that it may constitute the gene most frequently involved in human oncogenesis (Hollstein et al. 1991; Levine et al. 1991). In this review, we summarize all the available data on p53 mutations in human nervous system tumors.