Pten regulates neuronal soma size: a mouse model of Lhermitte-Duclos disease.

Somatic inactivation of PTEN occurs in different human tumors including glioblastoma, endometrial carcinoma and prostate carcinoma. Germline mutations in PTEN result in a range of phenotypic abnormalities that occur with variable penetrance, including neurological features such as macrocephaly, seizures, ataxia and Lhermitte-Duclos disease (also described as dysplastic gangliocytoma of the cerebellum). Homozygous deletion of Pten causes embryonic lethality in mice. To investigate function in the brain, we used Cre-loxP technology to selectively inactivate Pten in specific mouse neuronal populations. Loss of Pten resulted in progressive macrocephaly and seizures. Neurons lacking Pten expressed high levels of phosphorylated Akt and showed a progressive increase in soma size without evidence of abnormal proliferation. Cerebellar abnormalities closely resembled the histopathology of human Lhermitte-Duclos disease. These results indicate that Pten regulates neuronal size in vivo in a cell-autonomous manner and provide new insights into the etiology of Lhermitte-Duclos disease.

5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers.

Loss of heterozygosity on chromosome 9p21 is one of the most frequent genetic alterations identified in human cancer. The rate of point mutations of p16, a candidate suppressor gene of this area, is low in most primary tumours with allelic loss of 9p21. Monosomic cell lines with structurally unaltered p16 show methylation of the 5' CpG island of p16. This distinct methylation pattern was associated with a complete transcriptional block that was reversible upon treatment with 5-deoxyazacytidine. Moreover, de novo methylation of the 5' CpG island of p16 was also found in approximately 20% of different primary neoplasms, but not in normal tissues, potentially representing a common pathway of tumour suppressor gene inactivation in human cancers.

R-type virus-like particles in avian sarcoma virus-induced rat central nervous system tumors.

Morphologically distinct virus-like particles (VLP), similar to R-type VLP, were observed by electron microscopy in experimental rat central nervous system tumors induced with the B-77-C strain of avian sarcoma virus (ASV). R-type VLP have a characteristic internal radial structure and were observed previously only in hamster cells and in an established bovine cell line. They were not observed in the B-77 ASV inoculum used to induce the rat tumors or in the B-77 induced hamster glioma cells from which the B-77 was rescued. Nevertheless, the genome of an endogenous hamster R-type particle also might have been rescued and carried in the B-77 inoculum. Alternatively, R-type VLP may exist in a number of animal species, including the rat, and may be expressed in certain conditions such as neoplastic transformation.

Selective induction by N-nitrosoethylurea of oligodendrogliomas in fetal forebrain transplants.

Induction of neuroepithelial neoplasms by a single transplacental exposure to N-nitrosoethylurea (NEU) has been widely used as an experimental model for human brain tumors. NEU-induced gliomas are variably composed of neoplastic oligodendrocytes, astrocytes, and ependymal cells. It has remained controversial whether these neoplasms originate from differentiated glia or from pluripotent precursor cells of the subependymal matrix layer. We have taken a novel approach to define the histogenesis of these gliomas based on neural grafting techniques and the extraordinary difference in susceptibility between the fetal and adult brain to neoplastic transformation by alkylnitrosoureas. Pregnant rats received a single i.v. dose of NEU (50 mg/kg) on the 14th day of gestation. One day later, suspensions were prepared from the fetal forebrain and stereotactically injected into the caudoputamen of adult rats. These host animals received additional i.v. injections of NEU (50 mg/kg each) 8 days and 9 weeks posttransplantation. After a mean survival time of 316 days, all animals developed brain tumors within the neural graft. Histopathologically, these neoplasms were classified as oligodendrogliomas, ranging from early neoplastic foci to large, infiltrating malignant tumors. The selective induction of oligodendrogliomas indicates that neoplastic transformation in the nervous system can occur in a differentiated glial cell or in a precursor cell committed to oligodendrocytic differentiation, and that transformation of a pluripotential stem cell is not necessary. Omission of the first (prenatal) dose of NEU led to a much lower tumor incidence, whereas this dose in itself, i.e., without additional postgrafting exposure, did not produce brain tumors in any of the experimental animals. This differential effect of pre- and postgrafting exposure to NEU constitutes the first in vivo evidence of a multistep development of brain tumors.

The cytotoxicity of gamma-L-glutaminyl-4-hydroxybenzene for cells that contain tyrosinase, a study of melanocytes in the hair follicle of the mouse.

The s.c. administration of gamma-L-glutaminyl-4-hydroxybenzene (GHB) to neonatal black mice produced a prompt, generalized, and selective swelling and lysis of the melanocytes of the hair follicles. The findings indicate that this cytotoxic effect was dependent upon the intracellular activation of GHB by tyrosinase. Supportive of this conclusion were: (a) an absence of comparable cytological alterations in adjacent keratinocytes; (b) a lack of response by melanocytes of albino mice; and (c) patterns of deficient pigmentation produced by GHB in juvenile black mice, suggesting that susceptible follicles were those in the tyrosinase-producing growth phase. The administration of GHB also induced condensation, or "apoptosis," of individual follicular keratinocytes of both black and albino mice and in the melanocytes in the latter. This response was apparently independent of tyrosinase. It was transitory and without appreciable effect on hair growth. The findings further characterize the selective cytolytic properties of GHB for mammalian cells that possess tyrosinase and suggest a potential for this natural compound as a chemotherapeutic agent against melanocarcinoma.

Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia.

The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) removes alkyl adducts from the O6 position of guanine. MGMT expression is decreased in some tumor tissues, and lack of activity has been observed in some cell lines. Loss of expression is rarely due to deletion, mutation, or rearrangement of the MGMT gene, but methylation of discrete regions of the CpG island of MGMT has been associated with the silencing of the gene in cell lines. We used methylation-specific PCR to study the promoter methylation of the MGMT gene. All normal tissues and expressing cancer cell lines were unmethylated, whereas nonexpressing cancer cell lines were methylated. Among the more than 500 primary human tumors examined, MGMT hypermethylation was present in a subset of specific types of cancer. In gliomas and colorectal carcinomas, aberrant methylation was detected in 40% of the tumors, whereas in non-small cell lung carcinomas, lymphomas, and head and neck carcinomas, this alteration was found in 25% of the tumors. MGMT methylation was found rarely or not at all in other tumor types. We also analyzed MGMT expression by immunohistochemistry in relation to the methylation status in 31 primary tumors. The presence of aberrant hypermethylation was associated with loss of MGMT protein, in contrast to retention of protein in the majority of tumors without aberrant hypermethylation. Our results suggest that epigenetic inactivation of MGMT plays an important role in primary human neoplasia.

Specific chromosomal abnormalities in malignant human gliomas.

Karyotypic analysis of 54 malignant human gliomas (5 anaplastic astrocytomas, 43 glioblastoma multiformes, 3 gliosarcomas, 2 giant cell glioblastomas, 1 anaplastic mixed glioma) has demonstrated that 12 tumors contained normal stemlines or only lacked one sex chromosome. The 42 tumors with abnormal karyotypes included 38 tumors which could be completely analyzed. Six of these 38 cases had near-triploid or near-tetraploid stemlines and 32 had near-diploid stemlines. Statistically significant numerical deviations in the near-diploid group were gains of chromosome 7 (26 of 32; P less than 0.001), and losses of chromosome 10 (19 of 32; P less than 0.001). Double minutes occurred in 18 of 32 near diploid tumors. The distribution of structural abnormalities was analyzed statistically by comparing the incidence of breakpoint in each chromosomal arm to the expected value based on chromosomal arm length. This analysis demonstrated that structural abnormalities of 9p and 19q were significant statistically (P less than 0.005 and P = 0.02, respectively). Although chromosome 1, 6p, the centromeric region of chromosome 11, 13q, and 15q were also frequently involved in structural abnormalities, the incidence of these breaks did not reach statistical significance. This demonstration of specific chromosomal abnormalities in near-diploid gliomas provides the basis for the investigation of genes which may be quantitatively or qualitatively altered in these neoplasms.

Interstitial taxol delivered from a biodegradable polymer implant against experimental malignant glioma.

Taxol is a novel antitumor agent with demonstrated efficacy against ovarian, breast, and non-small cell lung cancers in Phase II clinical trials, but which has been shown not to cross the blood-brain barrier. To adapt taxol as a therapy for brain tumors, we have incorporated it into a biodegradable polyanhydride matrix for intracranial implantation and evaluated this formulation in a rat model of malignant glioma. Fischer 344 rats bearing intracranial 9L glioma tumors were treated with 10 mg poly[bis(p-carboxyphenoxy)propane-sebacic acid] (20:80) copolymer discs, containing 20-40% taxol by weight, 5 days after tumor implantation. The taxol-loaded polymers doubled (38 days, 40% taxol loading, P < 0.02) to tripled (61.5 days, 20% taxol loading, P < 0.001) the median survival of rats bearing tumor relative to control rats (19.5 days). Drug loadings of 20-40% taxol by weight released intact taxol for up to 1000 h in vitro. In rats followed up to 30 days postimplant, the polymer maintained a taxol concentration of 75-125 ng taxol/mg brain tissue (100-150 microM taxol) within a 1-3-mm radius of the disc. At points more distant from the disc (up to 8 mm away, the size limit of the rat brain), the polymer maintained a taxol concentration of greater than 4 ng taxol/mg brain tissue (5 microM). We conclude that taxol shows promise as a therapy for malignant glioma when delivered interstitially from a biodegradable polymer.

Oncogene amplification in pediatric brain tumors.

Despite a considerable amount of information concerning chromosomal and molecular abnormalities found in gliomas in adults, relatively little is known regarding these abnormalities in pediatric brain tumors. We have analyzed DNA from 37 primary brain tumors and 4 tumor-derived cell lines for oncogene amplification. Probes utilized represent 11 known oncogenes (erbB1, gli, neu, myc, L-myc, N-myc, H-ras, K-ras, N-ras, sis, and src). Of 20 primary medulloblastomas studied, only one tumor was found to have erbB1 amplification. In contrast, of the 4 medulloblastoma cell lines studied, 1 had c-myc amplification, 1 had erbB1 amplification, and 1 had amplification of N-myc. Twelve glial brain tumors were analyzed, and only 1 case with amplification of the erbB1 oncogene was found. Other tumors studied include 1 meningioma, 2 ependymomas, 1 anaplastic ependymoma, and 1 cerebral primitive neuroectodermal tumor, none of which had oncogene amplification. These results suggest that oncogene amplification is relatively uncommon in primary medulloblastomas, but the frequency and diversity of oncogene amplification is greater in tumors that can be established as cell lines. The lower frequency of erbB1 amplification in glial brain tumors in children compared to adults is consistent with the generally lower grade of glial tumor histology seen in pediatric patients. However, the case with amplification of the erbB1 oncogene represented 1 of 2 cases of glioblastoma multiforme we studied, which suggests that pediatric glioblastoma multiforme may have a similar frequency of erbB1 oncogene amplification to glioblastomas seen in adults. Our results suggest that oncogene amplification is a relatively uncommon mechanism of oncogene activation in pediatric brain tumors, and they provide molecular evidence for heterogeneity in tumors classified as medulloblastomas.

Methylation and silencing of the Thrombospondin-1 promoter in human cancer.

Neovascularization is a common feature of many human cancers, but relatively few molecular defects have been demonstrated in genes regulating angiogenesis. Decreased expression of Thrombospondin-1 (THBS1), a P53 and Rb regulated angiogenesis inhibitor, has been observed in some human tumors, including glioblastoma multiforme (GBM). To study whether methylation-associated inactivation is involved in down-regulating THBS1 expression in cancer, we analysed the methylation status of THBS1 in several cell lines and primary tumors. Three cell lines (RKO, CEM and RAJI) were completely methylated at several CpG sites within the THBS1 5' CpG island, and had no detectable expression by RT-PCR. THBS1 expression was readily reactivated using the methylation-inhibitor 5-deoxy-azacytidine in all three lines. Furthermore, THBS1 methylation was present in 33% (14/42) of primary GBMs. Thus, de novo methylation may serve as a potential way to inactivate THBS1 expression in human neoplasms.

Concordant methylation of the ER and N33 genes in glioblastoma multiforme.

Methylation of promoter-associated CpG islands appears to be a potential way by which tumor suppressor genes are inactivated in cancer. Using Southern blot analysis, we have studied the methylation of several genes in glioblastoma multiforme (GBM), trying to determine their contribution to tumorigenesis. Genes studied included the estrogen receptor (ER), N33, the candidate tumor-suppressors P15, P16 and HIC1 and a control gene, c-abl. Hypermethylation of N33, ER, HIC1, P16, P15 and c-abl were found in 61%, 59%, 60%, 5%, 2% and 0% of GBM respectively. HIC1 methylation was detected in normal brain as well, but appeared to be more extensive in tumors. ER and N33 methylation were significantly more frequent in tumors from individuals over the age of 40 (70% and 88% vs 36% and 14%). In addition, there was a strong association between ER and N33 methylation, which were concordant in 81% of the cases (P<0.01). ER and N33 methylation in GBM may therefore appear as a result of shared etiologic factors, which may relate in part to aging cell populations in the brain.

Localization of common deletion regions on 1p and 19q in human gliomas and their association with histological subtype.

Allelic alterations of chromosomes 1 and 19 are frequent events in human diffuse gliomas and have recently proven to be strong predictors of chemotherapeutic response and prolonged survival in oligodendrogliomas (Cairncross et al., 1998; Smith et al., submitted). Using 115 human diffuse gliomas, we localized regions of common allelic loss on chromosomes 1 and 19 and assessed the association of these deletion intervals with glioma histological subtypes. Further, we evaluated the capacity of multiple modalities to detect these alterations, including loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), and comparative genomic hybridization (CGH). The correlation coefficients for detection of 1p and 19q alterations, respectively, between modalities were: 0.98 and 0.87 for LOH and FISH, 0.79 and 0.60 for LOH and CGH, and 0.79 and 0.53 for FISH and CGH. Minimal deletion regions were defined on 19q13.3 (D19S412-D19S596) and 1p (D1S468-D1S1612). Loss of the 1p36 region was found in 18% of astrocytomas (10/55) and in 73% (24/33) of oligodendrogliomas (P < 0.0001), and loss of the 19q13.3 region was found in 38% (21/55) of astrocytomas and 73% (24/33) of oligodendrogliomas (P = 0.0017). Loss of both regions was found in 11% (6/55) of astrocytomas and in 64% (21/33) of oligodendrogliomas (P < 0.0001). All gliomas with LOH on either 1p or 19q demonstrated loss of the corresponding FISH probe, 1p36 or 19q13.3, suggesting not only locations of putative tumor suppressor genes, but also a simple assay for assessment of 1p and 19q alterations as diagnostic and prognostic markers.

Randomized comparison of diaziquone and carmustine in the treatment of adults with anaplastic glioma.

PURPOSE: We conducted a phase III trial comparing intravenous (IV) diaziquone (AZQ) and carmustine (BCNU) as single agents in patients with cerebral anaplastic gliomas who had received surgery and radiotherapy. Its purpose was to compare the efficacy of AZQ with that of BCNU, the standard agent for brain tumor chemotherapy. PATIENTS AND METHODS: Randomization between the two regimens occurred 8 weeks after completion of radiotherapy. A total of 251 patients were randomized to receive either AZQ or BCNU, and there were no significant differences between the two treatment arms in any of the known prognostic variables, including age, histologic grade, and Karnofsky performance status (KPS). RESULTS: There was no significant difference in either time to tumor progression or survival between the two treatment arms. Age and histology were strong predictors of outcome, whereas KPS had relatively less effect. Three groups of patients with distinctly different outcomes could be identified: (1) older age (45+) and glioblastoma/gliosarcoma (GBM/GS) patients had a median survival of 37 weeks after randomization; (2) patients with either older age or GBM/GS had a median survival of 61 weeks; and (3) younger age (< 45) and non-GBM/GS (usually anaplastic astrocytoma) patients had a median survival of 147 weeks. Toxicity was primarily hematologic, although acute gastrointestinal toxicity and chronic pulmonary toxicity were more common with BCNU. Patients randomized to AZQ who had significant hematologic toxicity that required dose reduction after the first treatment cycle had significantly longer time to tumor progression and survival than those who did not require dose reduction (P = .011 and .016, respectively). CONCLUSION: There was no significant difference in efficacy between AZQ and BCNU in patients with anaplastic gliomas as tested in this study, although AZQ was somewhat better tolerated.

Phase II diaziquone-based chemotherapy trials in patients with anaplastic supratentorial astrocytic neoplasms.

We treated 103 patients with histologically confirmed anaplastic supratentorial astrocytic neoplasms with either diaziquone (AZQ) and carmustine (BCNU) or AZQ and procarbazine. There were 74 patients with glioblastoma multiforme (GBM) and 29 patients with anaplastic astrocytoma (AA). AZQ plus BCNU produced partial (PR) or unequivocal responses in seven of 32 (21.9%) patients with GBMs and three of ten (30%) patients with AAs. Two patients with GBMs (6.3%) and five patients with AAs (50%) showed stable disease (SD). AZQ plus procarbazine produced PRs or unequivocal responses in five of 42 (11.9%) patients with GBMs and nine of 19 (47.4%) patients with AAs. Eight patients with GBMs (19%) and one patient with an AA (5.2%) showed SD. In addition to histologic diagnosis, only the Karnofsky performance-status (KPS) rating independently influenced response and survival. Differences in response rates between the two regimens were not significant, although estimated median survival after adjusting for performance status was slightly better with AZQ plus BCNU than with AZQ plus procarbazine (P = .031). Neither age nor prior chemotherapy were significant independent risk factors. Toxicity was mild and primarily hematologic. We conclude that these AZQ-based regimens have activity in patients with recurrent anaplastic gliomas, but that they are not clearly superior to other agents in current use. The histologic diagnosis of GBM is associated with a significantly worse prognosis than AA, and we believe that this important distinction must be recognized in phase II as well as phase III trials.

Phase II treatment of medulloblastoma and pineoblastoma with melphalan: clinical therapy based on experimental models of human medulloblastoma.

We conducted a phase II study of intravenous (IV) melphalan in the treatment of children with recurrent medulloblastoma and in the initial treatment of children with poor-prognosis medulloblastoma and pineoblastoma. There was one complete response (CR) and two partial responses (PRs) among the 12 children with recurrent medulloblastoma. There were three PRs in the four patients initially treated with melphalan for poor-prognosis medulloblastoma or pineoblastoma. Toxicity was limited to severe myelosuppression with marked neutropenia and thrombocytopenia. These results support our laboratory studies demonstrating melphalan activity in human medulloblastoma, suggest that similar activity may be demonstrated against pineoblastoma, and support further trials with this agent (administered prior to radiotherapy) in the treatment of patients with newly diagnosed poor-prognosis medulloblastoma.

Alterations of chromosome arms 1p and 19q as predictors of survival in oligodendrogliomas, astrocytomas, and mixed oligoastrocytomas.

PURPOSE: A recent report suggests that alterations of chromosome arms 1p and 19q are associated with chemotherapeutic response and overall survival in anaplastic oligodendroglioma patients treated with procarbazine, lomustine, and vincristine chemotherapy. We set out to further clarify the diagnostic and prognostic implications of these alterations in a broader set of diffuse gliomas, including astrocytic neoplasms and low-grade oligodendrogliomas. PATIENTS AND METHODS: Fluorescence in situ hybridization (FISH) signals from DNA probes mapping to 1p and 19q common deletion regions were enumerated in 162 diffuse gliomas (79 astrocytomas, 52 oligodendrogliomas, and 31 mixed oligoastrocytomas), collected as part of an ongoing prospective investigation of CNS tumors. RESULTS: The oligodendroglial phenotype was highly associated with loss of 1p (P =.0002), loss of 19q (P <.0001), and combined loss of 1p and 19q (P <.0001). Combined loss of 1p and 19q was identified as a univariate predictor of prolonged overall survival among patients with pure oligodendroglioma (log-rank, P =.03) and remained a significant predictor after adjusting for the effects of patient age and tumor grade (P <.01). This favorable association was not evident in patients with astrocytoma or mixed oligoastrocytoma. CONCLUSION: Combined loss of 1p and 19q is a statistically significant predictor of prolonged survival in patients with pure oligodendroglioma, independent of tumor grade. Given the lack of this association in patients with astrocytic neoplasms and the previously demonstrated chemosensitivity of oligodendrogliomas, a combined approach of histologic and genotypic assessment could potentially improve existing strategies for patient stratification and management.

Nuclear localization and mutation of beta-catenin in medulloblastomas.

The adenomatous polyposis coli (APC) gene, a member of the Wingless/Wnt signal transduction pathway, has been implicated in the development of medulloblastomas in Turcot's syndrome. beta-catenin also functions in this highly conserved signaling pathway and is instrumental in growth and development. Mutations in either APC or beta-catenin can stabilize beta-catenin protein. Stabilized beta-catenin complexes with Tcf/Lef transcription factors and moves from the cytoplasm into the nucleus where it regulates the transcription of c-Myc and other genes. Nuclear localization of beta-catenin therefore implies activation of the signaling pathway. We have analyzed the subcellular localization of beta-catenin in 51 sporadic medulloblastomas and in 1 medulloblastoma arising in a patient with Turcot's syndrome. Nuclear beta-catenin staining was present in 9 of the sporadic tumors (18%) and in the 1 medulloblastoma from a Turcot's patient. The remaining 41 cases did not show nuclear staining. This confirms earlier observations that Wingless/Wnt signaling is involved in a subset of sporadic medulloblastomas. We also examined 48 glial and meningeal CNS tumors, all of which were negative for nuclear beta-catenin. Exon 3 of beta-catenin was sequenced in 6 of the 9 sporadic medulloblastomas with nuclear beta-catenin staining. Five of the 6 tumors sequenced had mutations affecting highly conserved beta-catenin phosphorylation sites involved in protein stability. These data suggest a simple immunohistochemical method to screen for beta-catenin mutations in medulloblastomas.

Apoptosis, neuronal maturation, and neurotrophin expression within medulloblastoma nodules.

Nodular/desmoplastic medulloblastomas are a well-established histopathological subtype containing reticulin-free nodules or "pale islands' that are comprised of cells with round "neurocytic" nuclei and abundant cytoplasm. Significant neuronal maturation occurs within nodules. We used immunohistochemistry to evaluate neuronal differentiation in the nodules of 6 of these tumors. The neuronal markers NeuN, synaptophysin, and MAP-2 were identified in the "pale islands" of all 6 nodular medulloblastomas examined, and high and medium molecular weight nonphosphorylated neurofilaments were detected in 2 of the 6 cases. We also observed collections of apoptotic cells within nodules. Given the known role of neurotrophin signaling in neuronal maturation and apoptosis, we analyzed immunohistochemically the distribution of neurotrophin receptors TrkA and TrkC and their primary ligands NGF and NT3 in 14 nodular medulloblastomas. TrkA and TrkC were detected in 13 and 10 cases, respectively, and were predominantly localized within nodules. NGF and NT3 were distributed diffusely with some nodular accentuation. The localized expression of Trk receptors within nodules of desmoplastic medulloblastomas suggests neurotrophin signaling is involved in the apoptosis and neuronal differentiation in medulloblastomas. We also examined expression of p53 and BCL-2 in these tumors; both were prominent in internodular regions but only weakly expressed within nodules. Trk receptors, p53, and BCL-2 are all expressed during development of the normal cerebellum. Interestingly, the immunohistochemical expression profile of these proteins in the differentiating nodules of medulloblastomas is in many ways similar to their expression in the developing cerebellum. Thus similar signaling pathways may be operational in cerebellar development and medulloblastoma tumor differentiation.

The immunohistochemical application of three anti-GFAP monoclonal antibodies to formalin-fixed, paraffin-embedded, normal and neoplastic brain tissues.

The intermediate filament, glial fibrillary acidic protein (GFAP) has proven to be an important glial marker in diagnostic neuropathology. We report the histochemical application of three monoclonal antibodies (Mab) produced in this laboratory, 1B4, 2E1, and 4A11, which are monospecific to GFAP by radioimmunoassay, immunoblot electrophoresis, and immunoperoxidase histochemistry. The goal of this study was to compare the specificity and sensitivity of these Mab to GFAP on surgical brain biopsy specimens which had been routinely processed for diagnostic neuropathology with that of a high titer, highly specific, reference polyvalent anti-GFAP antiserum. The Mab stained astrocytes specifically in normal brain. When combined in a "cocktail" preparation, the quality of the immunoperoxidase detection of GFAP by these Mab closely approached that of the reference serum in 71 intracranial and intraspinal neoplasms. As these three Mab represent a continuous supply of a well defined, monospecific reagent, the monoclonal "cocktail" represents a standard reagent for large multi-institutional studies and for studies extending over a period of time.

Early and extensive contribution of pericytes/vascular smooth muscle cells to microvascular proliferation in glioblastoma multiforme: an immuno-light and immuno-electron microscopic study.

Although florid microvascular proliferation (MVP) in glioblastoma multiforme (GBM) has long been considered as proliferation of endothelial cells (EC), recent immuno-light microscopic studies demonstrated many alpha-smooth muscle actin (alpha-sm actin)-positive cells in this MVP, suggesting a major contribution of pericytes and/or vascular smooth muscle cells (VSMC). Under certain culture conditions, however, alpha-sm actin expression has also been described in EC. In order to further investigate to what extent pericytes/VSMC participate in MVP in GBM, we performed an immunohistochemical study at both the light and electron microscopic levels with anti-alpha-sm actin, with an antibody against EC (EN-4) and with an antibody recently described to react with "activated" pericytes in conditions with neovascularization (anti-high molecular weight-melanoma associated antigen). In this detailed study of MVP in GBM, two distinct cell types could be recognized on the basis of a consistent ultrastructural localization and immunophenotype: EC and pericytes/VSMC; no transitional forms were found between these two cell types. The contribution of pericytes/VSMC to MVP in GBM was extensive and already present in many delicate tumor capillaries, suggesting not only an essential but also an early role of these cells in this type of tumor angiogenesis.