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Recent advances in molecular tumor biology and gene technology have provided the possibility to treat patients with malignant brain tumors by altering gene expression in tumor cells. Tumor development and progression involves alterations in a wide spectrum of genes, therefore a variety of gene therapy approaches for malignant gliomas have been proposed. In this review article, we discuss some principles of current gene therapeutic strategies that are under investigation in laboratories and in clinics. In addition, some general issues that remain to be resolved for clinical application of gene therapy in patients with malignant gliomas will be addressed.

The Far Upstream Element [FUSE] Binding Protein 1 (FUBP1) regulates target genes, such as the cell cycle regulators MYC and p21. FUBP1 is up-regulated in many tumours and acts as an oncoprotein by stimulating proliferation and inhibiting apoptosis. Recently, FUBP1 mutations were identified in approximately 15% of oligodendrogliomas. To date, all reported FUBP1 mutations have been predicted to inactivate FUBP1, which suggests that in contrast to most other tumours FUBP1 may act as a tumour suppressor in oligodendrogliomas.As no data are currently available concerning FUBP1 protein levels in gliomas, we examined the FUBP1 expression profiles of human glial tumours by immunohistochemistry and immunofluorescence. We analysed FUBP1 expression related to morphological differentiation, IDH1 and FUBP1 mutation status, 1p/19q loss of heterozygosity (LOH) as well as proliferation rate.Our findings demonstrate that FUBP1 expression levels are increased in all glioma subtypes as compared with normal central nervous system (CNS) control tissue and are associated with increased proliferation. In contrast, FUBP1 immunonegativity predicted FUBP1 mutation with a sensitivity of 100% and a specificity of 90% in our cohort and was associated with oligodendroglial differentiation, IDH1 mutation and 1p/19q loss of heterozygosity (LOH). Using this approach, we detected a to-date undescribed FUBP1 mutation in an oligodendroglioma.In summary, our data indicate an association between of FUBP1 expression and proliferation in gliomas. Furthermore, our findings present FUBP1 immunohistochemical analysis as a helpful additional tool for neuropathological glioma diagnostics predicting FUBP1 mutation.

Snow tussock (Chionochloa macra and C. flavescens ssp. brevis) population structures and seedling regeneration were investigated in 10 montane-subalpine stands on formerly forested sites in the Harper-Avoca catchment, Canterbury, subjected to different histories of sheep grazing and browsing by European hares. Population structures were assessed by assigning individuals to four “age-states” based on basal diameter, number of tillers, height, and percent crown death: “seedlings”, “juvenile tussocks”, “mature tussocks”, and “senescent tussocks”. On areas subjected to c. 80 years of grazing by sheep, most snow tussocks had been destroyed. Remaining tussocks were predominantly senescent and seedlings were infrequent, suggesting a continuing decline in abundance. In contrast, stands retired from sheep-grazing for 34 or 21 years were characterised by low proportions of senescent tussocks and high proportions (>60%) of seedlings and juveniles, suggesting the onset of increases in tussock abundance. Popu...

Traditionally, prognosis of intracranial tumors is considered to be correlated with histology. Today a four grade scale is increasingly used, in which the grades II through IV are attributed to gliomas depending upon their cellular and tissue pleomorphism. These tumors will regrow after one to five years. Tumors which will not relapse after successfull operation are considered to be of grade I. This grading, which is part of the WHO histological typing of intracranial tumors, can be supplemented by the proliferation markers. These may allow better evaluation of the prognosis. Traditionell wird die Prognose intrakranieller Tumoren an der Histologie abgelesen. Heute ist eine IV-Grad-Skala gebrauchlich, bei der die Gliome II-IV aufgrund der zellularen und geweblichen ,,Polymorphie'' unterteilt werden. Diese Tumoren rezidivieren nach 1 bis 5 Jahren. Tumoren, die nach gegluckter Entfernung nicht rezidivieren, besitzen den Grad I. Diese Einteilung, die auch in die Klassifikation der intrakraniellen Tumoren der WHO aufgenommen worden ist, wird heute erganzt durch Proliferationsmarker, die moglicherweise der Tumorproliferation naherstehen und die Prognose noch besser einzuschatzen gestatten.

Two proliferation markers, silver stained nucleolar organizer regions (AgNOR) and immunoreactivity with Ki-67, were used to assess the proliferative activity in 80 smear preparations from neurosurgically removed intracranial tumours. These included 45 gliomas, 18 meningiomas, 8 metastases and 9 others. We found a remarkably close correlation between the results obtained with both methods. Increasing malignancy, as determined by conventional grading, was paralleled by an increase in the growth fraction and the number of nucleolar organizer regions. Linear regression analysis yielded the following equation: AgNORs/cell = 0.35 x L1 (Ki-67) + 3.24, with a correlation coefficient of rs = 0.53 (Spearman rank correlation test, p less than 0.0001). Thus, both the Ki-67 L1 and the AgNOR technique appear suitable for estimating the proliferative potential in smear preparations of human intracranial neoplasms. The AgNOR technique may be particularly useful for application to stereotaxic biopsies since it can easily be performed on minute tumour samples.

Glial cell cytoplasmic inclusions were identified in a case of spinocerebellar ataxia type 2. These have not been reported before. The inclusions were found in low frequency in the dentate nucleus, cerebellar white matter, pontine transverse fibres, and the inferior olivary nucleus. They were of variable size and shape and expressed ubiquitin, thus resembling glial cytoplasmic inclusions in multiple system atrophy. However, their immunohistochemical profile was different as they did not show immunoreactivity for either tau protein or alpha-synuclein. There was no evidence of expanded polyglutamine tracts in these inclusions, which also failed to label with silver stains. As in many other neurodegenerative diseases, in spinocerebellar ataxia type 2 there may be pathogenic contributions of glial cells other than the common astrogliotic response to neuronal damage.

Vascular endothelial growth factor (VEGF) is a regulator of angiogenesis, vasculogenesis and vascular permeability. In this contribution, molecular and biological properties of VEGF are described. Furthermore, this article focuses on the evidence that angiogenesis in brain tumors is mediated by VEGF. Among the topics discussed are expression patterns of VEGF and its receptors in different brain tumors, possible regulatory mechanism involved in the VEGF-driven tumor angiogenesis and the involvement of VEGF in the genesis of peritumoral edema. Finally, anti-angiogenesis approaches to target VEGF/VEGF receptors are discussed.

Aim of this study was to develop and characterize an applicable in vivo model to investigate angiogenesis of human gliomas. An established glioblastoma spheroid model was used to investigate the neovascularization of a standardized avascular solid tumor mass. Spheroids of two human glioma cell lines were labeled with an in vivo fluorescent dye. Single spheroids were implanted into the cortex of athymic rats. After 1, 3, 7, 14, and 21 days, brain sections containing the spheroid were immunostained for endothelial cells or vascular endothelial growth factor (VEGF). The dye-stained glioma spheroid and the endothelial cells were visualized by confocal microscopy. Two distinct mechanisms of tumor vascularization could be observed. (1) "Classical" angiogenesis with new vessels sprouting from existing host vessels into the spheroid was seen. (2) Individual endothelial cells were found to migrate towards and into the center of the spheroid where they coalesced to form new vessels. This process occurred as early as 24 hr after spheroid implantation. Spheroid vascularization was accompanied by an increase of VEGF expression, which peaked 7 days after implantation and returned to normal patterns by 14-21 days. Besides the "classical" angiogenesis by angiogenic blood vessels, the recruitment of individual endothelial cells seems to be an additional mechanism in early glioma vascularization. Our model proves to be a reliable, reproducible system to study in vivo angiogenesis of human gliomas.

It has previously been suggested that in human brain tumours, endothelial cell proliferation during angiogenesis is regulated by a paracrine mechanism involving vascular endothelial growth factor (VEGF) and its receptors (VEGF receptor 1 and VEGF receptor 2). The mechanism of growth factor up-regulation is based on hypoxic activation of mRNA expression and mRNA stabilization and genetic events, leading to an increase of growth factor gene expression. The role of the other newly discovered VEGF family members with a high specificity for endothelial cells in the pathogenesis of glial neoplasms is unknown. To investigate which other members of the VEGF family are overexpressed in human brain tumours, the mRNA levels of placenta growth factor (PlGF), VEGF-A, and VEGF-B genes were determined by northern blot analysis in surgically obtained human meningiomas. In the 16 meningiomas examined, the mRNA for PlGF was highly expressed in four tumours and VEGF-A mRNA was highly abundant in three tumour samples. There was no close correlation between PlGF mRNA levels and VEGF-A expression levels. VEGF-B mRNA was abundantly expressed in all tumour samples at uniform levels. In a PlGF-positive tumour sample, immunoreactive VEGFR-1 and VEGFR-2 were detected in endothelial cells of the blood vessels. PlGF protein was detectable in most but not all capillaries of the tumour. PlGF is thus highly up-regulated in a subset of human meningiomas and may therefore have functions, in some tumour vessels, connected to endothelial cell maturation and tube formation. These findings suggest that PlGF, in addition to VEGF-A, may be another positive factor in tumour angiogenesis in human meningiomas.

The vascular system is responsible for supplying all organs in a body with blood, oxygen and nutrients and for removing waste products. As a reflection of this highly important function, the vascular system is the first organ system to be formed in a mammalian embryo. Thereafter, the growth of all organs is accompanied by a similar expansion of the vascular system.

Vascular endothelial growth factor (VEGF) is a hypoxia-inducible angiogenesis and vascular permeability factor which is expressed in high amounts in perinecrotic palisading cells in human glioblastomas. In vitro VEGF gene expression is enhanced approximately ten times by hypoxia. Current evidence suggests, that hypoxia is also the driving force for VEGF gene expression in glioblastoma cells in vivo and represents the most important trigger for tumor angiogenesis and edema. Our approaches to inhibit tumor angiogenesis and edema formation in glioblastoma patients will concentrate on the disruption of VEGF/VEGF receptor signal transduction pathway in vivo.

Glioblastomas are rapidly growing tumours with large hypoxic areas featuring necrosis. These hypoxic tumour compartments are most likely responsible for the relative resistance of glioblas-tomas to radiotherapy. In vivo studies suggest that perinecrotic palisading represents the most hypoxic tumour compartment in glioblastomas. Current evidence suggests that hypoxia has several deleterious effects for glioblastoma patients, since it leads to growth factor expression in glioma cells, induces tumour angiogenesis, vascular permeability and genetic instability, and selects for apoptosis-resistant cells. In addition, hypoxia impairs significantly the efficacy of the anti-oedema drug dexamethasone. Taken together, these findings suggest that on a physiological basis, hypoxic tumour cells are the most malignant cell clones in glioblastoma and thus represent a challenging target for novel therapies, for example by targeting heterologous gene expression to hypoxic tumour cells.

Vascular endothelial growth factor (VEGF) and its tyrosine kinase receptors VEGFR-1 (flt-1) and VEGFR-2 (flk-1/KDR) are key mediators of physiological and pathological angiogenesis. They are expressed in most tissues during embryonic development but are down-regulated in the adult, when angiogenesis ceases. Up-regulation of VEGFR-2 and of VEGF are observed in many pathological conditions under which angiogenesis is reinduced. A major regulator of VEGF expression is hypoxia. Although the temporal expression pattern of VEGFR-2 parallels VEGF expression to a high extent, little is known about its regulation. Here, we show that VEGFR-2 is highly expressed in early postnatal mouse brain but is down-regulated commencing at postnatal day 15 (P15) of mouse brain development and is hardly detectable in P30 mouse brain. Using P30 mouse brain slices, we observed that hypoxia up-regulates VEGFR-2 in the slices but not in human umbilical vein endothelial cells, suggesting the presence of a hypoxia-inducible factor in the murine neuroectoderm that up-regulates VEGFR-2. To identify the factors involved, normoxic P30 cerebral slices were cultured with growth factors that are either hypoxia-inducible (e.g., PDGF-BB, erythropoietin, and VEGF) and/or are known to act on endothelial cells (e.g., PDGF-BB, VEGF, and PIGF). Exogenously added recombinant VEGF led to an up-regulation of VEGFR-2 expression, which could be inhibited by preincubation with a neutralizing anti-VEGF antibody. Addition of PDGF-BB, PIGF, and erythropoietin had no effect on VEGFR-2 expression. Our results suggest a differential but synergistic regulation by hypoxia of VEGF and VEGFR-2: a direct induction of VEGF that subsequently up-regulates VEGFR-2 in endothelial cells. This autoenhancing system may represent an important mechanism of tumor angiogenesis.

Up-regulation of vascular endothelial growth factor (VEGF) expression is a major event leading to neovascularization in malignant gliomas. Hypoxia is believed to be the crucial environmental stimulus for this up-regulation. To critically assess this hypothesis, we asked whether the mechanisms defined previously for hypoxia-induced VEGF expression in vitro are similarly involved and sufficient for up-regulation of VEGF gene expression in vivo, using a lacZ reporter gene under the control of VEGF regulatory sequences in an experimental glioma model. Inclusion of the binding site for hypoxia-inducible factor 1 (HIF 1) in the 5' regulatory sequences used in the hybrid gene produced weak beta-galactosidase staining in a special tumor cell subtype, the so-called perinecrotic palisading (PNP) cells that flank necrotic regions within the tumor. Deletion of the HIF 1 binding site abolished reporter gene expression in the PNP cells, indicating that transcriptional activation of VEGF expression in gliomas is mediated by HIF 1. Inclusion of 3' untranslated sequences from the VEGF gene in the reporter constructs resulted in an increased beta-galactosidase staining in the PNP cells, suggesting that mRNA stabilization also contributes to VEGF up-regulation in glioblastoma cells growing as solid tumors. Combination of the 5' flanking region including the HIF 1 site along with 3' untranslated sequences produced increased levels of beta-galactosidase expression in PNP cells. EF 5 immunostaining for regions of low oxygen partial pressure covered the same PNP cells that were stained for beta-galactosidase. Collectively, the data provide experimental evidence that VEGF gene expression is activated in a distinct tumor cell subpopulation, the perinecrotic palisading cells of gliomas, by two distinct hypoxia-driven regulatory mechanisms.

Glioblastoma, one of the best vascularized tumours in humans, appears well suited for an antiangiogenic therapy. VEGF (vascular endothelial growth factor), the most important angiogenesis factor identified to date, is highly expressed in glioblastoma. VEGF is particulary upregulated in palisading cells adjacent to necroses and has subsequently been shown to be hypoxia-inducible in glioma cells in vitro. VEGF-receptor tyrosine kinases, VEGF-R1 (flt-1) and VEGF-R2 (flk-1), are induced in a tumour stage dependent manner during glioma progression and are exclusively expressed in tumour vascular endothelial cells. These observations suggest that VEGF-receptors are promising targets for tumour endothelial cell specific therapy. The ability to block VEGF-signalling by the VEGF-R2 dominant-negative mutant identifies the VEGF/VEGF-R2 system as a major regulator of glioma angiogenesis. Several experimental approaches demonstrate that in rat gliomas tumour growth can be prevented by the inhibition of angiogenesis. These findings are of pivotal importance for the development of anti-angiogenic therapies in glioblastoma patients.

Hemangioblastomas are highly vascularized tumors of not well-defined histological origin which are frequently associated with cysts. They arise preferentially in cerebellum, medulla and spinal cord and are histologically indistinguishable from vascular lesions in the retina (so-called angiomatosis retinae). Hemangioblastomas are the most frequent manifestations of the von Hippel-Lindau (VHL) disease, an autosomal-dominant inherited cancer syndrome but also occur as sporadic non-hereditary tumors. The VHL tumor suppressor gene has recently been cloned and enormous progress has been made towards the understanding of molecular biology and biological function of the VHL gene. Germline mutations in VHL patients, as well as somatic mutations in different tumors, including hemangioblastomas, have been identified, its ability to act as a tumor suppressor in vivo has been confirmed, and interaction with transcription factors Elongin B and C leading to inhibition of transcriptional elongation has been demonstrated. The mechanism underlying neovascularization and cyst formation in hemangioblastomas and how this is linked to inactivation of the VHL tumor suppressor gene is not known. However, the finding of dramatic up-regulation of vascular endothelial growth factor (VEGF), a potent endothelial cell growth factor with vascular permeability-inducing activity, in stromal cells and the corresponding receptors, VEGFR-1 and VEGFR-2, in tumor endothelial cells suggests that angiogenesis and cyst formation in hemangioblastomas may be regulated by this signaling pathway via a paracrine mechanism.

Angiogenesis, the sprouting of new blood vessels from existing vessels, occurs in many physiological and pathological processes, including embryonic development, wound healing, and tumor growth. It is required for tumor growth because new blood vessel formation is necessary for tumors to expand beyond a minimum volume. Several growth factor receptor tyrosine kinases have been implicated in angiogenesis, including receptors for epidermal, fibroblast, and platelet-derived growth factors, as well as the receptors Flk-1/KDR, Flt-1 Tek/Tie-2, and Tie-1. Endothelial cells in the vessels of tumors express Flk-1/KDR, a receptor for vascular endothelial growth factor. Flk-1 was previously shown to play a role in angiogenesis and tumor formation of s.c. xenografts of C6 glioma cells using dominant-negative methodology. We now demonstrate that Flk-1 seems to be generally involved in the growth of a wide range of solid tumors, including mammary, ovarian, and lung carcinoma, as well as glioblastoma. Furthermore, survival times in rats bearing intracerebral tumors were prolonged using the same dominant-negative methodology. The involvement of Flk-1 in a variety of tumor types suggests an important role for Flk-1 in tumor angiogenesis.

Capillary hemangioblastoma is the most frequent manifestation of the autosomal dominantly inherited von Hippel-Lindau (VHL) disease but also presents as a nonfamilial, sporadic vascular tumor. Hemangioblastomas are characterized by a dense network of capillaries in association with cysts. To investigate the mechanisms underlying neovascularization and cyst formation, we analyzed eight VHL disease-associated and five sporadic hemangioblastomas. Histologically, both tumor types showed a similar phenotype. The capillaries expressed the endothelial cell markers von Willebrand factor and CD31 antigen. We investigated the expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen which is also known to induce vascular permeability in vivo, and its high affinity tyrosine kinase receptors flt-1 and KDR. Northern blot and in situ hybridization analysis revealed significant up-regulation of VEGF and VEGF receptor expression in VHL disease-associated and sporadic hemangioblastomas compared to normal brain and tumor stromal cells as sites of abundant VEGF transcription. Endothelial cells did not express detectable amounts of VEGF mRNA but coexpressed flt-1 and KDR. By immunohistochemistry, VEGF protein was detectable in the tumor interstitium and was found to be concentrated around capillaries. Performing reverse transcription-PCR, we demonstrated that VEGF121 and VEGF165 were the splice variants predominantly expressed, whereas mRNA encoding VEGF189 was present at smaller amounts. Our findings suggest that, in VHL disease-associated and sporadic hemangioblastomas, VEGF121 and VEGF165 are secreted by stromal cells and interact with the corresponding VEGF receptors expressed on tumor endothelial cells. This paracrine mechanism may mediate neovascularization and cyst formation in capillary hemangioblastomas.

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.

A family with von Hippel-Lindau disease (vHLD) is presented. Three family members suffered from typical cerebellar hemangioblastomas. Another family member presented with a cerebellar neoplasm of different histology. Detailed histological analysis revealed a primitive neuroectodermal tumor (PNET) with neuronal, glial, myoid and ependymal differentiation. This is apparently the first description of the association of vHLD and a cerebellar PNET with multipotent differentiation.

Endothelial cells proliferate during brain development, are quiescent in normal adult brain but proliferate again under pathologic conditions such as glioma growth. The vascular phenotype of low grade glioma is comparable to normal brain, however high grade gliomas are focally highly vascularized and there is associated prominent endothelial cell proliferation. The mechanisms of this change in vascular phenotype are unknown but there is evidence that growth factors play an important role in this process as well as in normal angiogenesis and vascular differentiation.To investigate whether endothelial cells become activated during tumorigenesis and progression of human gliomas by a platelet-derived growth factor (PDGF) dependent pathway, we analyzed platelet-derived growth factor receptor-beta (PDGFR-beta) expression by in situ hybridization and immunocytochemistry in normal human brain, astrocytoma (grade II), anaplastic oligo-astrocytoma (grade III), and glioblastoma multiforme (grade IV).PDGFR-beta mRNA was not detectable in the vessels of normal human brain, but was expressed in the vasculature of low and high grade gliomas, particularly in endothelial cell proliferations in glioblastomas. The expression of the receptor in the tumor microvessels, was confirmed by double immunofluorescence in which the staining appeared to be in the endothelial cells. Primary cultures of endothelial cells derived from glioblastoma multiforme maintained receptor expression for 2 days in vitro, whereas it was not detectable in vitro in endothelial cells derived from normal brain. Tumor cells in all grades of glioma expressed very little PDGFR-beta mRNA in situ.Our results indicate that the malignant phenotype in human glial tumors is associated with an upregulation of the PDGFR-beta on endothelial cells of vessels which vascularize the tumor. These findings may contribute to our understanding of the mechanisms that regulate vessel growth and differentiation in normal and pathologic states.

The silver staining of interphase nucleolar organizer regions (NORs) is a recently developed method to measure cell proliferation in tissue specimens. The major silver staining protein is nucleolin, a 92 kd nucleolar protein, which probably controls rDNA transcription. Nucleolin itself is under control of p34cdc2 kinase, which is a subunit of M phase kinase. The specific silver staining of NORs measures ribosomal gene activity and is therefore useful in measuring cell proliferation via nucleolar biosynthetic activity. Although human tumors of different malignancy grades usually can be distinguished by their AgNOR number, there is considerable overlap between different grades which significantly hampers their use in individual cases. For routine application of the AgNOR technique in histopathology we propose a standardised staining protocol with use of internal control cells and the use of image analysis for AgNOR enumeration.

Two cases of saccrococcygeal chordoma that were diagnosed on the basis of smear preparations are presented. Only one case showed typical physaliferous cells. In both cases the final diagnosis was greatly facilitated by applying peroxidase-antiperoxidase immunocytochemistry techniques to the cytologic specimens. Chordomas coexpress epithelial markers, such as intermediate filaments of the cytokeratin type, epithelial antigens (such as tissue polypeptide and epithelial membrane antigen), intermediate filaments of the vimentin type and S-100 protein. This antigenic spectrum may greatly facilitate the differential diagnosis of chordoma from filum terminale ependymoma, chondroma and chondrosarcoma, metastases of clear cell-type carcinomas and schwannomas, and neurofibromas, even when the only specimens available are from aspiration cytology.

Traditionally, prognosis of intracranial tumors is considered to be correlated with histology. Today a four grade scale is increasingly used, in which the grades II through IV are attributed to gliomas depending upon their cellular and tissue pleomorphism. These tumors will regrow after one to five years. Tumors which will not relapse after successful operation are considered to be of grade I. This grading, which is part of the WHO histological typing of intracranial tumors, can be supplemented by the proliferation markers. These may allow better evaluation of the prognosis.

Eighty-one meningiomas (63 grade I, 9 grade II and 9 grade III) and 2 meningeal sarcomas (grade IV) were investigated by a simple one-step silver staining for nucleolar organizer region (NOR)-associated proteins (AgNOR technique) and by DNA cytometry. The number of NORs per cell and the NOR area per cell were correlated with the histopathologic grading, as were the 5c exceeding rate and the 2c deviation index (2cDI) obtained by DNA cytometry. The differences in NOR parameters were only significant (at P less than .001) between grades I and II; P was less than .05 for the 2cDI between grades I and II. No significant differences between grades II and III were found. Among recurrent tumors, the AgNOR technique revealed the proliferative potential in 8 of 11 tumors studied, whereas DNA cytometry failed to recognize malignant features in 8 of 10 tumors investigated.

The value of immunocytochemistry and nucleolar organizer regions (NORs) for the histogenetic identification and the estimation of the proliferative potential of brain tumors was assessed by the investigation of imprint smears of 51 neurosurgical tumor specimens. A panel of five monoclonal antibodies was used to cover a broad range of immunohistochemical markers. For the assessment of NORs, a silver staining technique (AgNOR) was used. NORs were enumerated and measured by means of an interactive image analysis system. The immunocytochemical results were similar for the smears and paraffin-embedded sections for 95.6% of the investigations performed and for 76.2% of the cases. Glial fibrillary acidic protein (GFAP) was positive in 9 of 17 tumors of glial origin, but was negative in 9 metastatic tumors. Vimentin was positive in 10 of 10 and fibronectin in 9 of 10 meningiomas investigated. The number of NORs increased steadily with the increasing grade of malignancy. Especially in glioblastomas, the number of NORs per cell exhibited a wide range, which might reflect the heterogeneity of these neoplasms. Metastases revealed a higher number of NORs per cell than did glioblastomas. In the cytologic differential diagnosis of these tumors, an absence of GFAP expression combined with a high NOR count is suggestive of a metastatic tumor.

The silver staining of nucleolar organizer regions (AgNORs) was evaluated in a total of 697 tumours from ten different tissues. By means of qualitative light microscopy, type, duration and delay of fixation proved to be influential in determining the AgNOR result. For standardization of the AgNOR technique, staining time series in 30 tissue blocks of the ten types of tissue were evaluated using digital image analysis. The silver incubation time which rendered the most distinct diagnostic difference in the AgNOR content of benign and malignant tissue varied considerably. Accordingly, staining time has to be adjusted to the individual argyrophilia of each tissue block or tissue section, for which the use of internal staining standards such as lymphocytes or connective tissue was found to be mandatory. For routine purposes, the appropriate silver incubation time is achieved if AgNORs are visible as black dots mainly within the nucleoli of proliferating cells.

Since the introduction of the WHO-classification of tumours of the central nervous system in 1979, meningiomas are subdivided in 8 histological types and graded I to IV. The basis for this classification were histopathological investigations combined with clinical follow-up data. Since the introduction of immunocytochemistry as a diagnostic tool in neurooncology, morphological methods have gained renewed interest among neurosurgeons and neurologists. Some of the more recent developments, as well as the classical methods of the neuropathologist, are discussed here, and the results of the impact of morphology on prognosis is discussed on 139 own cases. Beside the histopathological grading, the application of so called proliferative markers as Ki-67 and BUdR seems to be most promising in the prediction of recurrences. Clinical parameters as patients age, preoperative physical status, tumor size and location on the other hand are of particular importance concerning postoperative mortality.

Summary The purpose of this investigation is to give an introduction to a novel method in tumor pathology, namely the Ag-NOR technique. The basis of this method is the argyrophilic staining of intranucleolar, non-histon proteins which are specifically associated with transcriptionally active sites of ribosomal DNA. They can therefore be considered as a marker for the protein synthesis and thus the proliferation rate of a given cell. The morphologic basis ofthe argyrophilic reaction is presented by metaphasic and interphasic tissue culture cells. The applicability of Ag-NOR technique to tumor pathology is exemplified by main results of three studies dealing with tissue sections of 65 meningiomas, whole organ sections of 50 renal carcinomas, and cytospin preparations of 30 urinary washout specimens. These studies document the considerable value of the Ag-NOR content for both, malignancy diagnosis and tumor grading. With the help of image analysis it can be shown that besides the mean number of Ag-NORs the mean area per Ag-NOR dot is of diagnostic significance. In conclusion the Ag-NOR technique is a simple inexpensive and accurate method which can be applied both to formalin fixed, paraffin-embedded tissue and cytologic specimens. As a marker of malignancy it is an invaluable new tool for the diagnostic pathologist.