Immunohistochemical study of fibronectin in human glioma and meningioma

The presence of fibronectin (FN) and glial fibrillary acidic protein (GFAP) in two astrocytomas, 17 glioblastomas, and five meningiomas was studied by indirect immunoperoxidase staining of formalin-fixed and paraffin-embedded surgical specimens. Angiogenesis in tumor was scored by the microscopic angiogenesis grading system (MAGS), and plasma FN levels were measured by single radial immunodiffusion. In astrocytomas and glioblastomas, GFAP-positive tumor cells had no FN expression and FN was confined to proliferating vessel walls and the leptomeninges, showing a mutually exclusive FN and GFAP expression. GFAP-positive tumor cells were occasionally surrounded by a network of FN-positive matrix produced by cells derived from the leptomeninges or blood vessels. In meningiomas, FN expression was found in vessel walls and meningioma cells including whorl formations and psammoma bodies. In general, deep immunoperoxidase staining for FN was shown in the endothelial cells and the psammoma bodies. Plasma FN levels were correlated significantly not to the degree of leptomeningeal proliferation but to the MAGS scores in gliomas.     

DNA content and marker expression in human glioma explants

Summary Immunohistochemical studies of astrocytoma tissue have predominately shown fibronectin (FN) positivity restricted to vessels and glial fibrillary acidic protein (GFAP) positivity in the parenchyma. Cultured glioma cell lines, however, express both FN and GFAP. We measured the DNA content of explants of gliomas to determine if the ploidy of the FN-positive and GFAP-positive cells differed. Thirty-three explants from four high grade gliomas were cultured on slides. FN and GFAP markers were determined by double immunofluorescence. The slides were stained by the Feulgen method, the explants relocated and the DNA content measured by microdensitometry using the CAS-100 instrument. Human leukocytes applied to the slides were used as a diploid standard. Eleven GFAP-positive explants were hyperdiploid and one hypodiploid. Five FN-positive explants were diploid, three hypodiploid and ten hyperdiploid. One FN-positive explant was biclonal with aneuploid subpopulations. Two hyperdiploid explants, each of which had monoclonal histogram patterns, expressed both FN and GFAP. We conclude that most FN-positive cells, in addition to GFAP-positive cells, from cultured gliomas represent neoplastic cells. These may be present in the tumor in low numbers or may result from marker switching in culture.     

Herpes simplex virus antigen detection in human acute encephalitis:

Summary The distribution of type I, III, IV and V collagen in 35 gliomas and 20 meningiomas was studied by indirect immmunofluorescence staining. In addition, the presence of fibronectin (FN) and laminin (LN) is also reported. In gliomas expression of type IV collagen and LN was found in the vessel walls and associated with the endothelial glomerulus-like proliferations. FN and type V collagens were located in proliferating vessel walls in a pattern corresponding both to the basement membrane and the perivascular matrix around the vessels. In the extracellular matrix of grade III and IV gliomas occasional faint intercellular fluorescence was also observed with both FN and type V collagen. Type I and III collagens were localised in the vessel walls and in the perivascular connective sheet. Glioma cells did not express any of the antigens investigated. In meningiomas, type IV and V collagens, LN and FN were found in vessel walls, whorls formations and psammoma bodies. These stainings support the hypothesis of a vascular origin of these psammoma bodies which were only found in syncytial and transitional meningiomas. Both type I and III collagens were detected in the perivascular connective tissue. In general, meningioma cells and extracellular matrix did not express any of these molecules, except in transitional meningiomas where occasional fluorescence was observed in extracellular matrix with type V collagen and FN.     

Expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor receptor-β (PDGFR-β) in human gliomas

The growth of solid tumors is highly dependent on vascular proliferation. Vascular endothelial growth factor (VEGF), the main mediator of angiogenesis, and platelet-derived growth factor receptor-β (PDGFR-β), receptor for the potent mitogen PDGF, are two indicators of the angiogenic potential of human gliomas. We studied a series of 57 surgical biopsies of astrocytic neoplasms by immunohistochemistry to elucidate the relationship between tumor proliferation, quantified as Ki67-LI, and the expression of these two proteins. Ki67-LI increases throughout histological malignancy, although staining in endothelial cells has rarely been recorded. Elevated amounts of VEGF-positive tumor cells (VEGF-LI) were found in anaplastic astrocytomas and glioblastomas, mainly around areas of necrosis, cysts, or edema. Endothelium of blood vessels was consistently stained. PDGFR-β positivity was found in glomeruloid formations and in tumor cells, excluding pilocytic astrocytomas. Multinucleated giant cells and perivascular tumor cells were positive in glioblastomas. In addition, peritumoral microglia-like cells were also stained in some cases. Statistical correlation was only found between PDGFR-β and Ki67 LIs. In conclusion, VEGF as permeability factor is involved in the development of secondary neoplastic changes, whereas PDGFR-β is directly correlated to proliferation indexes. Strong expression of VEGF and PDGFR-β found in endothelium and tumor cells would seem to support a combined role in tumoral neoangiogenesis     

Flow-through fluorocytophotometry of different brain tumours

Summary The flow-through cytophotometric method was used to investigate the single-cell DNA content in 60 tumours of the CNS and allied structures (9 meningiomas, 5 ependymomas, 15 astrocytomas, 11 anaplastic astrocytomas, 8 glioblastomas, 7 medulloblastomas, 2 oligodendrogliomas, 1 anaplastic oligodendroglioma, and 2 neurinomas). The cytophotometric parameters were correlated with morphological and clinical data of the tumours. The results are summarised in Tables 1–7. With regard to the DNA content of their cell nuclei, the gliomas present a behaviour similar to tumours in other organs, but in the present study the cytophotometric signs of malignancy in gliomas are not so evident as in carcinomas or sarcomas. The information obtained by flow cytophotometric methods may be helpful in diagnosing the degree of malignancy in brain tumours.     

Morphological changes in basement membrane associated with endothelial proliferation in astrocytic tumors--an immunohistochemical study of laminin

Morphological changes of the basement membrane associated with endothelial proliferation in astrocytic tumors are studied in this report. Laminin is known to be a specific glycoprotein of basement membranes. We applied this characteristic of laminin to enable us to observe various characteristics of the basement membrane. The presence of laminin in 13 glioblastomas, 15 anaplastic astrocytomas, 7 astrocytomas, and 6 pilocytic astrocytomas was examined by peroxidase-antiperoxidase (PAP) staining of formalin-fixed and paraffin-embedded surgical specimens. White matter from five normal cerebral hemispheres obtained during autopsy and subsequently embedded using the same method, were used as a control. Laminin was observed at the glioma-mesenchymal junction in astrocytic tumors, and the deposits of laminin made the tumor vasculature come into intense relief. The destructive changes of the basement membrane, including disruption, thickening, disconnection, dissociation, winding, and conjunction, became greater with progressive endothelial proliferation in astrocytic tumors. Those changes were seen to be most remarkable in glioblastoma. In addition, there was a marked variety of morphological change in the basement membrane in different areas of glioblastomas, although the changes were almost constant in other astrocytic tumors. We present a schematic hypothesis of the stages of angiogenesis in glioblastoma based on the above morphological changes of the basement membrane and discuss it in this report.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical demonstration of serum proteins in human cerebral gliomas

Summary The leakage of different serum proteins, including immunoglobulins, into human cerebral gliomas was studied by use of the unlabeled peroxidase-antiperoxidase (PAP) method on cryostat and paraffin sections. Our series of 50 tumour biopsies included 21 isomorphic astrocytomas and oligodendrogliomas (grade II), 19 anaplastic astrocytomas and oligodendrogliomas (grade III), and 10 glioblastomas (grade IV). The immunohistochemical staining of the serum proteins was similar on paraffin and cryostat sections and graded with respect to occurrence, distribution, and intensity. Serum proteins of a small hydrodynamic radius with a low serum concentration (prealbumin) or with a high serum concentration (albumin) were diffusely present in the interstitial spaces of all glioma types. Serum proteins with a medium molecular size and variable serum concentrations, i. e. IgG, IgA, and ceruloplasmin, were detected preferentially in anaplastic gliomas and in glioblastomas (grade III and IV) displaying comparable distribution patterns but different intensities. Alpha-2-macroglobulin a serum protein with a large hydrodynamic radius was also demonstrated in grade III and IV gliomas, whereas IgM and beta-lipoprotein being the largest serum proteins tested were almost restricted to blood vessels and tumour necroses. In addition, most serum proteins occurred with high intensities in those areas of isomorphic grade II gliomas that showed a macro-or microcystic or mucinous tissue degeneration. The varying immunohistochemical staining results for the serum protiens studied indicate that the blood-brain barrier within isomorphic and anaplastic gliomas is not completely disturbed. It appears that the vascular permeability is preferentially increased for small-sized serum proteins, whereas the leakage of larger serum proteins into the glioma interstitium seems to depend on the tumour type and on increasing malignancy.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Characterisation of molecular alterations in microdissected archival gliomas

Classification of gliomas according to their molecular characteristics may be important in future histopathological diagnosis. However, gliomas frequently display heterogeneity at the histological, biological and molecular level. In this study of archival diagnostic gliomas, precision microdissection was used to enrich samples in the most malignant cells or to investigate intratumoural histological heterogeneity. Analysis of tumour samples microdissected from the most aggressive regions, representative of the histopathological diagnosis, revealed PTEN mutations in 4/14 anaplastic astrocytomas, 4/13 glioblastomas and 1 gliosarcoma, but not in 19 low-grade gliomas. Using a novel PCR procedure and direct sequence analysis of the entire coding sequence, TP53 mutations were detected in 1/3 pilocytic astrocytomas, 3/13 astrocytomas, 4/14 anaplastic astrocytomas, 5/13 glioblastomas and 1 gliosarcoma. All but one of the tumours with TP53 mutation showed p53 immunopositivity, but 5 low-grade and 10 high-grade gliomas had p53 protein nuclear accumulation in the absence of detectable mutation. p53 status was unrelated to p21 expression. Neither PTEN nor TP53 mutations influenced the proliferative index or microvessel density of high-grade astrocytomas. Unusual findings include: TP53 mutation in a juvenile pilocytic astrocytoma; TP53 and PTEN mutations in a de novo glioblastoma, a gliosarcoma with identical mutations in gliomatous and sarcomatous components, and an infratentorial anaplastic astrocytoma with an earlier supratentorial grade II astrocytoma bearing the same TP53 mutation but not the PTEN mutation or loss of heterozygosity (LOH) of 10q23. Similarly, the transition to high-grade histology was associated with acquisition of PTEN mutations and 10q23.3 LOH in two de novo high-grade tumours with regions of low-grade histology.     

Immunohistochemical study of human brain tumors with vimentin and astroprotein (GFAP)

Distributions of two different subclasses of intermediate filaments, vimentin and glial filaments, were studied immunohistochemically in human brain tumors using specific antiserum to each protein subunit, vimentin and astroprotein (GFAP), Surgical specimens (5 meningiomas, 4 ependymomas, 5 benign astrocytomas, 5 anaplastic astrocytomas and 7 glioblastomas) were fixed in 95% ethanol or ethanol-acidic acid (95:5) and embedded in paraffin Avidin biotin peroxidase-complex (ABC) method (Vectastain) was carried out on 6 microns-thick paraffin sections. All meningioma cells were negative for astroprotein (GFAP) and positive for vimentin. Ependymoma cells showed various patterns of immunoreaction for astroprotein (GFAP) but were invariably positive for vimentin. In benign astrocytomas, many cells (or cell body and processes) were positive for astroprotein (GFAP). Immunoreaction for vimentin was, however, less frequent and intense. In anaplastic astrocytomas, population of astroprotein (GFAP)-positive cells decreased and vimentin-positive cells increased. Astroprotein (GFAP)-positive cells were further decreased in glioblastomas and the population of vimentin-positive cells varied among tissues. The present study suggests that the anaplastic change of astrocytoma cells were associated with decreased expression of glial filaments and increased expression of vimentin filaments. It was also suggests that the expression of both intermediate filaments may be suppressed in highly-malignant glial tumor cells.     

Differential expression of tenascin-C and tenascin-X in human astrocytomas

Tenascins (TNs) are a family of extracellular matrix glycoproteins. The first member of this family to be recognized, tenascin-C (TN-C), is known to be expressed in various tumors including human astrocytomas. Tenascin-X (TN-X) is the latest member of the TN family to be reported, and its expression in tumor tissues has not yet been examined. In this study, we found expression of TN-X in glioma cell lines and human astrocytomas by immunoblot analysis using anti-mouse TN-X antibodies. We also examined the expression of TN-C and TN-X immunohistochemically in a series of 32 human astrocytomas and tissue from 5 normal brains. Expression of TN-X was up-regulated to a higher degree in low-grade astrocytomas than in high-grade astrocytomas. TN-X was mainly localized in the perivascular stroma around tumor vessels, and weakly expressed in the intercellular spaces among tumor cells. In contrast, TN-C was more strongly expressed in the intercellular spaces and in tumor vessels in high-grade astrocytomas (anaplastic astrocytomas and glioblastomas) than in low-grade astrocytomas. In the tissues expressing both TNs, the distribution of TN-X was often reciprocal to that of TN-C. These findings indicate that the expression of TN-C and TN-X in astrocytomas is different, and that these glycoproteins could be involved in neovascularization in different manners. Received: 7 June 1996 / Revised: 25 October 1996 / Accepted: 30 October 1996     

Immunohistochemical localization of fibronectin, laminin and fibronectin-receptor in human malignant gliomas--in relation to tumor invasion]

In order to examine a role of extracellular matrix (ECM) components in the process of glioma cell invasion, we investigated the immunohistochemical localization of fibronectin (FN), laminin(LN) and FN-receptor (FN-R) in human malignant gliomas. The surgical specimens were obtained from 15 patients with malignant gliomas. Tumor tissue and adjacent brain tissue including tumor infiltration were frozen at -80 degree C immediately after the resection. Ten microns thick frozen tissue was cut out on a cryostat and divided into three different parts on the histology stained with HE, ie, the tumor region(T), brain tissues with tumor infiltration(I), and the border region between these two parts(B). These sections were air-dried, and fixed with cold acetone (-4 degrees C) for 5min. Adjacent sections were immunohistochemically stained by ABC method, using monoclonal antibody for FN-R and polyclonal antibodies for FN and LN. FN, LN and FN-R were all stained at the vascular and pial-glial basement membranes intensely in all gliomas. In immunostain for FN, fine networks of FN were observed in the extracellular space in all three parts. Some tumor cells were clustered around such networks of FN in brain tissues with tumor infiltration. Immunostain for LN demonstrated that the vascularity in the border between the tumor and the brain with tumor infiltration was much higher than that in other parts. LN was not stained in the extracellular space in all these gliomas. FN-R was expressed in some tumor cells, especially in the clustered tumor cells in the brain with tumor infiltration.(ABSTRACT TRUNCATED AT 250 WORDS)     

p53 protein expression in central nervous system tumors: an immunohistochemical study with CM1 polyvalent and DO-7 monoclonal antibodies

Summary Formalin-fixed, paraffin-embedded surgical specimens from 137 primary central nervous system tumors, including 26 astrocytomas (21 fibrillary, 1 protoplasmic, 1 gemistocytic and 3 pilocytic), 26 anaplastic astrocytomas, 9 glioblastomas, 1 gliosarcoma, 8 oligodendrogliomas, 4 ependymomas, 1 anaplastic ependymoma, 2 subependymomas, 3 paragangliomas, and 57 meningiomas, were immunostained with the CM1 polyclonal (pAb) and the DO-7 monoclonal (mAb) antibodies against the p53 protein, using the streptavidin/peroxidase method. In addition, two series of 17 and 9 medulloblastomas were also immunostained with the above pAb and mAb, respectively. p53 protein expression was observed in 7 fibrillary astrocytomas, 17 anaplastic astrocytomas, 5 glioblastomas, 1 gliosarcoma, 1 oligodendroglioma, 1 anaplastic ependymoma, and 4 meningiomas with the CM1 pAb. An additional 10 cases (i.e., 3 anaplastic astrocytomas and 7 meningiomas) were found to be p53 protein positive with the DO-7 mAb. Of the medulloblastomas, 8 (of the 17) and 4 (of the 9) were found to express p53 protein with CM1 pAb and DO-7 mAb, respectively. Our results indicate that p53 protein is expressed in a number of central nervous system neoplasms, and suggest that in astrocytic tumors a possible association may exist between p53 protein expression and tumor progression through increasing histological grades of malignancy.     

Mutations and immunohistochemistry ofp53 and proliferation markers in astrocytic tumors of childhood

Thirty cases of hemispheric astrocytic tumors of childhood, consisting of 11 pilocytic astrocytomas, 2 fibrillary astrocytomas, 9 anaplastic astrocytomas, and 8 glioblastomas, were studied for the presence ofp53 mutations and for immunohistochemical demonstrations ofp53 and proliferation markers PCNA and Ki-67 MIB-1. The study was performed using polymerase chain reaction (PCR)-as-sisted single-strand conformation polymorphism analysis of exons 5–8 and direct sequence analysis of PCR products. For immunohistochemistry, DO1 and PAb 1801 were used. No mutation and no positivity forp53 protein were found in pilocytic astrocytomas. Mutations (at codons 144, 202, and 245) were found in 2 out of 8 glioblastomas and in 1 out of 9 anaplastic astrocytomas, whereas positive staining was found in 11 out of 17 malignant gliomas. Cases with mutations showed the highestp53 labeling index and also PCNA and MIB-1 labeling indices. The negative results in pilocytic astrocytomas are in line with the benign course of these tumors, whereas for malignant gliomas no difference seems to exist in comparison with adult cases.     

Immunocytochemical studies in canine neuroectodermal brain tumors

Summary Seventy-four canine neuroectodermal tumors were examined immunocytochemically for the presence of glial fibrillary acidic protein (GFAP). Eleven oligodendrogliomas were examined for the presence of myelim basic protein (MBP) and myelin-associated glycoprotein (MAG). Twenty-three tumors, including ten astrocytomas, one ependymoma, two glioblastomas, one case of gliomatosis, and nine poorly differentiated gliomas were positive for GFAP. Two astrocytomas, eleven oligodendrogliomas, eight ependymomas, four choroid plexus papillomas, two medulloblastomas, one glioblastoma, nine poorly differentiated gliomas, six cases of gliomatosis, and three unclassified tumors were GFAP-negative. In six tumors (including four that were classified as astrocytoma) GFAP staining was equivocal. All oligodendrogliomas were MBP-negative but three expressed MAG. It was concluded that many canine gliomas are not only morphologically but also mmunocytochemically similar to human gliomas, but that a larger proportion of canine neuroctodermal growths are undifferentiated tumors.Supported by the Swiss National Science Foundation (grant no, 3.809.81)     

Amplification of the platelet-derived growth factor receptor-A (PDGFRA) gene occurs in oligodendrogliomas with grade IV anaplastic features

Activation of the platelet-derived growth factor (PDGF) signaling system has been implicated in the development and malignant progression of diffuse gliomas. Overexpression of PDGF system components, particularly the alpha subtype receptor (PDGFRA), is common in glial tumors, and PDGFRA gene amplification has been reported in glioblastomas. In order to address the incidence of PDGFRA gene amplification in a broad set of diffuse gliomas, we used Southern and fluorescence in situ hybridization (FISH) analyses to examine 167 astrocytic gliomas (20 grade III and 147 grade IV), 41 anaplastic oligodendrogliomas, and 29 anaplastic oligoastrocytomas. PDGFRA gene amplification was identified in 4 anaplastic oligodendrogliomas and in a single case of anaplastic oligoastrocytoma, but in none of the malignant astrocytomas. Each of the 5 tumors with PDGFRA amplification displayed features generally associated with grade IV malignancy in astrocytic tumors. Consequently, our data indicate that this gene alteration is restricted to tumors having oligodendroglial differentiation and highly anaplastic features.     

The incidence of intracranial gliomas in southern Finland

146 intracranial gliomas were found in an epidemiological study of adults in southern Finland 1978-1980. The mean adult population was 896,000 in the study area. The age- and sex-adjusted incidence was 5.6/100,000/year for all gliomas and 4.7 for the histologically verified ones. The incidence was 6.4 for men and 4.9 for women. Of the histologically verified 126 (86%) neoplasms 41 (32%) were glioblastomas, 29 (23%) anaplastic astrocytomas, 30 (24%) benign astrocytomas and 26 (21%) oligodendrogliomas, mixed oligo-astrocytomas, ependymomas, medulloblastomas or gangliogliomas; 20 (14%) were without histological verification.     

Activation of STAT3, MAPK, and AKT in malignant astrocytic gliomas: correlation with EGFR status, tumor grade, and survival

Diffuse astrocytic gliomas are the most common human glial tumors with glioblastoma being the most malignant form. Epidermal growth factor receptor (EGFR) gene amplification is one of the most common genetic changes in glioblastoma and can lead to the activation of various downstream signaling molecules, including STAT3, MAPK, and AKT. In this study, we investigated the activation status of these 3 signaling molecules as well as wild-type (EGFRwt) and mutant (EGFRvIII) EGFR in 82 malignant astrocytic gliomas (55 glioblastomas and 27 anaplastic astrocytomas) using immunohistochemistry. The presence of EGFRwt, but not EGFRvIII, immunopositivity correlated significantly with prevalent EGFR gene amplification in glioblastomas. STAT3 and AKT activation correlated significantly with EGFR status, although the correlation for p-STAT3 was attributed exclusively to EGFRvIII. The distribution of these 3 activated molecules varied significantly with tumor grade; although activation of STAT3 was essentially identical between anaplastic astrocytomas and glioblastomas, an increase in the activation of MAPK and AKT appeared to correlate with the progression of anaplastic astrocytoma to glioblastoma. Finally, activated STAT3 and AKT were marginally predictive of improved and worse prognosis, respectively. Taken together, these findings begin to elucidate the interrelationship between these signaling pathways in astrocytic gliomas in vivo.     

Molecular tunnels and boundaries for growing axons in the anterior commissure of hamster embryos

We have analyzed the immunohistochemical expression of chondroitin sulfate proteoglycan (CSPG), fibronectin (FN), laminin (LN), tenascin (TN), and glial fibrillary acidic protein (GFAP) along the anterior commissure (AC) of hamster embryos (n = 175; from embryonic day (E)12 to E16). Frozen sections were cut at different planes from embryonic brains between E12 and E16, treated for immunohistochemistry, and observed under epifluorescence microscopy. During the pre-crossing stage (E12–E13), CSPG was expressed as a sagittal stratum between the interhemispheric fissure and the prospective AC region. TN appeared rostral to the third ventricle and along the medial subventricular zone of the lateral ventricles. LN and FN both presented a faint expression, and GFAP was not detected. Although AC axons started crossing the midline region (E13.5–E14), CSPG, FN, LN, and, much less intensely, GFAP circumscribed the AC bundle, forming a tunnel through which AC fibers elongate. TN was no longer seen at the midplane but remained visible laterally. During the post-crossing stage (E14.5–E16), CSPG and TN were no longer seen at the midline, although both could be observed between the AC limbs, seeming to form boundaries for AC lateral growth. LN and FN were then absent near the AC bundle. During this late stage, GFAP expression became most intense, forming a distinct tunnel around the AC. We have shown that the expression of extracellular matrix molecules and GFAP follow a time- and space-regulated course related to AC development, plausibly representing influential factors for growth and guidance of commissural fibers. J. Comp. Neurol. 399:176–188, 1998. © 1998 Wiley-Liss, Inc.