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)     

Distribution of type VI collagen in human gliomas: comparison with fibronectin and glioma-mesenchymal matrix glycoprotein

The distribution of type VI collagen was examined immunohistochemically in normal tissues and in 24 human gliomas and six medulloblastomas. Its localization in the neoplasms was compared with that of fibronectin and glioma-mesenchymal extracellular matrix (GMEM) glycoprotein. In normal non-neural tissues type VI collagen was demonstrated in the interstitial connective tissue and in some basement membranes. In normal brain it was localized to the vasculature, leptomeninges, and pial-glial membrane. In neoplasms type VI collagen and fibronectin codistributed in the vasculature and stromal connective tissue. The GMEM glycoprotein, as identified by monoclonal antibody (MAb) 81C6, and a related glioma-mesenchymal matrix antigen identified by MAb 2A6, were expressed not only in the tumor vasculature and connective tissue, but also within the tumor parenchyma in association with glioma cells. The staining intensity was variable in 20 malignant gliomas and weak to absent in two pilocytic astrocytomas and six medulloblastomas. An oligodendroglioma and ependymoma both expressed the 2A6 epitope, but staining with MAb 81C6 was weak to absent. The antigens identified by MAb 81C6 and MAb 2A6 represent the only recognized extracellular matrix components, other than proteoglycans, that are associated with glioma cells in vivo. As prominent constituents of the pericellular matrix, they may be involved in recognized matrix functions such as the modulation of cell adhesion and migration.     

Differential expression of glial- and neuronal-associated antigens in human tumors of the central and peripheral nervous system

Summary The immunoreactivity of a panel of poly-and monoclonal antibodies raised against different glial and neuronal antigens was investigated in paraffin-embedded specimens of 116 human tumors of the central and peripheral nervous system. We used antibodies against the HNK-1 epitope, which is shared between natural killer cells and the nervous system, glial fibrillary acidic protein (GFAP), vimentin, neurofilaments, S-100 protein, neuron-specific enolase (NSE) and myelin basic protein (MEP). HNK-1 immunoreactivity was detectable in nearly all neuroectodermal tumors. Especially in those derived from the neuroepithelium, which include the various types of gliomas, we observed a strong staining with this antibody. The only exceptions were the choroid plexus papillomas and individual ependymomas. In tumors derived from the neural crest HNK-1 reactivity was more variable and less intense. In other tumors of the nervous system HNK-1 was not detectable, except for two out of four malignant lymphomas. In addition to its reactivity with human lymphocytes HNK-1, therefore, seems to be a useful marker for neurogenic tumors in general. GFAP expression was prominent in all astrocytomas and the astrocytic cells within mixed gliomas and gangliogliomas. Immunoreactivity was more variable in glioblastomas and ependymomas, while only isolated GFAP-positive cells were present in oligodendrogliomas, medulloblastomas, one plexus papilloma, and some neurinomas. Vimentin immunoreactivity was found in tumor cells of nearly all tumors of the central nervous system with the exception of oligodendrogliomas, most plexus papillomas, neuronal tumors and most medulloblastomas. Meningeomas, neurinomas and malignant melanomas were always strongly vimentin positive. Besides the tumor cells the vimentin antibody usually stained vascular elements within each tumor. Sarcomatous components in glioblastomas and desmoplastic areas in medulloblastomas were also labeled. Neurofilament expression was restricted to neuronal tumor cells in two gangliogliomas and to individual tumor cells in one medulloblastoma. The NSE antiserum showed more widespread and sometimes diffuse reactivity and, therefore, seems to be less valuable as an indicator for neuronal differentiation than neurofilaments. S-100 expression was demonstrable in numerous tumors including most gliomas and all tumors derived from the neural crest. MBP antibodies never showed reactivity with oligodendroglioma or neurinoma tumor cells. This antibody labeled only myelin sheaths and their remnants within these tumors. Based upon our results we can conclude that, despite the fact that most of the investigated antigens showed a widespread distribution among different tumors, each of them and especially their differential expression might be useful in the classification and differential diagnosis of human tumors of the nervous system.Supported by the Deutsche Forschungsgemeinschaft, SFB 200Dedicated to Prof. F. Seitelberger on the occasion of his seventieth birthday     

Estrogen receptors in brain tumors

We examined the cytosolic estrogen receptor (ER) level in tumor tissue from 77 patients: 36 meningiomas, 20 gliomas (12 glioblastomas, 2 cerebellar astrocytomas, 2 ependymomas, and 4 medulloblastomas), 8 neurinomas, 7 pituitary adenomas (2 prolactin-producing adenomas, 1 growth hormone-producing adenoma, and 4 nonfunctioning adenomas), and 6 metastatic brain tumors (1 from breast cancer, 4 from lung cancers, and 1 from colon cancer). Nuclear ER levels were assayed in 11 meningiomas and 2 glioblastomas. ER was determined by the dextran-coated charcoal method and calculated by Scatchard analysis. Cytosolic ER was detected in 100% of the pituitary adenomas, 50% of the meningiomas, 50% of the metastatic brain tumors, 25% of the neurinomas, and 15% of the gliomas. In gliomas, only medulloblastomas had ER activity. Nuclear ER was found in three premenopausal women with meningioma. The dissociation constant of the ER complex was, in each case, less than 10(-9) M. These observations suggest that some brain tumors may be responsive to estrogen via the cellular ER.     

Notch家族成员在星形细胞瘤及髓母细胞瘤中的表达

目的 检测Notch家族成员Notchl～4蛋白在人脑星形细胞瘤及髓母细胞瘤中的表达,探讨其在肿瘤生成中的作用.方法 应用组织芯片免疫组化染色及Western Blot检测正常脑组织、不同级别大脑星形细胞瘤、小脑髓母细胞瘤中Notch 1、2、3、4蛋白的表达.结果 正常脑组织中Notch 1、2、3、4蛋白均无表达;星形细胞瘤中Notch 1、3、4蛋白的阳性表达率及表达强度随肿瘤病理级别的增高而增高,Notch 2无表达;髓母细胞瘤中Notch 1、3、4低或无表达,Notch 2呈高表达.结论 Notch 1、2、3、4在星形细胞瘤及髓母细胞瘤中表达有所差异,可能与Notch家族成员在脑发育过程中的作用不同有关.     

bcl-2 protein expression in tumors of the central nervous system

bcl-2 protein (BCL-2) expression was immunohistochemically studied in 140 varied central nervous system tumors. The protein was most frequently expressed in neuronomas and ependymomas, and in normal ependymal cells and Schwann cells. Most pituitary adenomas could be classified into one of two subgroups, diffusely positive or diffusely negative tumors, while BCL-2 localized heterogeneously in normal pituitary glands. Although the protein was not detected in normal astrocytes, it was positive in reactive hypertrophic astrocytes observed in various pathological conditions. Similarly, astrocytic tumor cells often expressed BCL-2. Since lowgrade astrocytomas more often exhibited the protein than malignant gliomas, the degree of BCL-2 expression appeared to be related to the degree of malignancy of the gliomas. On the other hand, 7 out of 17 recurrent gliomas and medulloblastomas showed an increase in the frequency of protein expression compared with specimens from initial treatments. One recurrent astrocytic tumor which demonstrated anaplastic change showed a decrease in the frequency of BCL-2-positive cells. It is concluded that the frequency of BCL-2 expression in CNS tumors is increased when the non-neoplastic counterparts of the tumors exhibit the protein. Although it has been reported that overexpression of BCL-2 protects cells from damage by radiation and/or chemotherapy, we could not find any significant relationship between the degree of BCL-2 expression and the length of survival of patients with glioblastomas or medulloblastomas.     

Affinity cytochemistry of vascular endothelia in brain tumors by biotinylated Ulex europaeus type I lectin (UEA I)

Summary The vascularization of 50 tumors of the central nervous system (CNS) including 17 meningeomas, 25 neuroectodermal tumors, i.e., astrocytomas, oligodendrogliomas, mixed gliomas, glioblastomas, medulloblastomas, seven metastatic carcinomas, and one malignant hemangioendothelioma were investigated using biotinylated Ulex europaeus type I lectin (UEA I) in an indirect avidinbiotin-peroxidase procedure. The cytochemical staining pattern of UEA I on paraffin sections was compared with that of biotinylated Dolichos biflorus lectin (DBA), and with the immunocytochemical staining of factor VIII related antigen (F VIII/RAG) by polyclonal antisera using the PAP technique. UEA I visualized the endothelia of blood vessels with equal intensity, sensitivity, and reliability in normal brain and in tumor tissue with neovascularization. While large, medium, and small vessels were equally well demonstrated by UEA I and antibodies against FVIII/RAG, capillaries and endothelial sprouts were stained more consistently and intensely by UEA I. No reliable cytochemical staining could be obtained by DBA regardless of tissue or cell type investigated.It is concluded that UEA I is a highly useful cytochemical marker for the identification of vascular endothelia in paraffin sections of human brain tumors.Dedicated to o. Professor emeritus Dr. Dres. h. c. Hubert Meessen on the occasion of his 75th birthday     

Glia-specific antigen in the intracranial tumors. Immunofluorescence study

Summary 26 gliomas and 14 non-glial tumors were examined for the presence of nervous system specific antigen (CGSA) to assess the antigenic properties of neoplastic tissue in relation to histogenesis and degree of differentiation of tumors. Double layer immunofluorescence (IMF) technique was used for the cellular localization of the antigen.CGSA was found in the cytoplasm of normal, reactive and neoplastic neuroglial cells. Well differentiated astrocytomas showed the stronges IMF reactions and largest number of IMF-positive cells. Tumors with histological signs of anaplasia displayed foci of IMF-negative cells irregularly distributed in the sections. There were no completely negative astrocytomas owing to a marked affinity of the specific antisera to the astrocytic cell line.In the oligodendrogliomas a smaller amount of the antigen was found than in the astrocytomas. Histological evidence of malignancy in these tumors was accompanied by strikingly small number of positive cells and weaker IMF reactions as compared to the well differentiated oligodendrogliomas. Anaplastic gliomas showed only traces of CGSA and non-gial tumors were entirely negative.The results suggest a deficiency of normal antigenic material in the neoplastic glia, particularly of oligodendrogliomas and anaplastic gliomas.Supported by NIH-PL Res. Agreement No. 05-013-1.     

Survival of children with brain tumors: SEER Program, 1973-1980

Eight hundred eighty-seven children with brain tumors were identified by the SEER registries (1973-1980). Twenty-five percent were low-grade supratentorial astrocytomas, medulloblastomas were 23%, cerebellar astrocytomas 12%, high-grade supratentorial astrocytomas 11%, brainstem gliomas 9%, and ependymomas 8%. The worst survivals were in children less than 2 years of age, and the best were in those aged 10 to 14 years. Five-year survivals of children with cerebellar astrocytomas were 91%, low-grade supratentorial astrocytomas 71%, high-grade supratentorial astrocytomas 35%, medulloblastomas 39%, ependymomas 28%, and brainstem gliomas 18%.     

Expression of α-, β-, and γ-synuclein in glial tumors and medulloblastomas

alpha-, beta- and gamma-synuclein are highly homologous proteins that are found predominantly in neurons. Abnormal accumulation of synucleins has been associated with diseases of the central nervous system particularly Parkinson's disease. Immunoreactivity of alpha-synuclein is demonstrated in brain tumors with neuronal differentiation and in schwannomas, whereas gamma-synuclein has been demonstrated in breast and ovarian carcinomas. The immunoreactivity of synucleins has not been described in glial tumors. Immunoreactivity of synucleins in glial cells in culture and in pathological conditions, however, suggests that synucleins may be expressed by glial tumors. We studied the expression of alpha-, beta-, and gamma-synuclein in 84 human brain tumors (24 ependymomas, 31 astrocytomas, 8 oligodendrogliomas, and 21 medulloblastomas) by immunohistochemistry. Our study demonstrates immunoreactivity for gamma-synuclein in high-grade glial tumors; immunoreactivity is found in all anaplastic ependymomas but in only 33% of ependymomas and 16% of myxopapillary ependymomas. Immunoreactivity for gamma-synuclein is noted in 63% of glioblastomas but not in other astrocytic tumors. Of medulloblastomas, 76% have immunoreactivity for either alpha- or beta-synuclein or both; no immunoreactivity for gamma-synuclein is seen in medulloblastomas.     

Short-term culture of pediatric brain tumors

Three short-term culture systems were applied to ten pediatric brain tumors and two samples each of normal child's brain and fetal brain. Growth in monolayer occurred in nine of the ten tumors but normal child's brain also produced cellular growth in these conditions. Six of nine tumors tested also formed colonies in suspension in semisolid or liquid conditions which did not permit the growth of normal glial cells. Astrocytomas and medulloblastomas have distinctive growth patterns both in monolayer and in suspension culture. Fetal brain from midtrimester abortions contained cells which, like tumor, formed colonies in suspension. The semisolid culture system was then used to test the sensitivity of brain tumor stem cells to drugs and radiation in vitro. Survival curves representative of our experience with the medulloblastomas and astrocytomas are presented. It is concluded that the system may have value in screening new agents for activity against children's brain tumors but because of the relative rarity of these diseases, a cooperative effort would be required to produce useful results.This work was supported by grants from the National Cancer Institute of Canada and the Medical Research Council of Canada (W.J.M.)     

Chlorotoxin,a scorpion-derived peptide,specifically binds to gliomas and tumors of neuroectodermal origin

Highly migratory neuroectodermal cells share a common embryonic origin with cells of the central nervous system (CNS). They include enteric, parasympathetic, sympathoadrenal, and sensory neurons of the peripheral nervous system, Schwann cells, melanocytes, endocrine cells, and cells forming connective tissue of the face and neck. Because of their common embryologic origin, these cells and the tumors that derive from them can share genetic and antigenic phenotypes with gliomas, tumors derived from CNS glia. We recently discovered that chlorotoxin (ClTx), a 4-kD peptide purified from Leiurus quinquestriatus scorpion, is a highly specific marker for glioma cells in biopsy tissues (Soroceanu et al. Cancer Res 58:4871-4879, 1998) that can target tumors in animal models. We report on the specificity of ClTx as a marker for tumors of neuroectodermal origin that include peripheral neuroectodermal tumors (PNET) and gliomas. Specifically, we histochemically stained frozen and paraffin tissue sections of human biopsy tissues from 262 patients with a synthetically manufactured and biologically active ClTx bearing an N-terminal biotin. The vast majority (74 of 79) of primary human brain tumors investigated showed abundant binding of ClTx with greater than 90% ClTx-positive cells in each section. By comparison, 32 biopsies of uninvolved brain used for comparison were largely ClTx-negative, with only a few isolated reactive astrocytes showing some ClTx binding. However, as with gliomas, the vast majority of PNETs examined showed specific ClTx binding (31 of 34). These include medulloblastomas (4 of 4), neuroblastomas (6 of 7), ganglioneuromas (4 of 4), melanomas (7 of 7), adrenal pheochromocytomas (5 of 6), primitive PNET (1), small cell lung carcinoma (2 of 3), and Ewing's sarcoma (2 of 2). Under identical staining conditions, normal tissues from brain, skin, kidney, and lung were consistently negative for ClTx. These results suggest that chlorotoxin is a reliable and specific histopathological marker for tumors of neuroectodermal origin and that chlorotoxin derivatives with cytolytic activity may have therapeutic potential for these cancers.     

Immunohistochemical determination of protein kinase C expression and proliferative activity in human brain tumors

Summary Protein kinase C (PKC), the major receptor for phorbol ester tumor promotors, is a phospholipid- and calcium-dependent phosphorylating enzyme which plays an important role in the intracellular signal transduction necessary for a variety of basic cellular functions including the control of cell proliferation. To determine the expression of PKC in human neurogenic tumors we investigated 121 tumors of the human nervous system by means of immunohistochemistry using the monoclonal antibody C5. The results were compared with immunohistochemical staining for intermediate filament proteins, desmoplakins, and the proliferation-associated nuclear antigen Ki-67. Besides strong staining of normal and reactive astrocytes, C5 immunoreactivity was consistently observed in tumor cells of all types of gliomas. However, the fraction of C5 positive tumor cells varied between the different tumor types with astrocytomas and subependymomas demonstrating the strongest immunoreactivity. In the other gliomas, especially those of higher malignancy, a considerable heterogeneity in C5 expression could be observed. There was a tendency for the percentage of C5 immunostained tumor cells being lower in high-grade gliomas compared to low-grade ones and comparison with Ki-67 staining frequently revealed an inverse relationship between proliferative activity and C5 immunoreactivity. Besides the gliomas we found 3 of 7 neurinomas and 6 of 18 meningiomas which were partially C5 positive. All other tumors investigated including medulloblastomas and metastatic carcinomas were C5 negative. Our results thus indicate that immunohistochemistry for PKC using the monoclonal antibody C5 could be an useful aid for histopathological tumor classification in neurooncology.Supported by the Deutsche Forschungsgemeinschaft, SFB 200     

Expression of tubulin beta II in neuroepithelial tumors: reflection of architectural changes in the developing human brain

Tubulin beta II (Tub-II) is widely distributed in the developing neuronal axons and dendrites. Recent studies have demonstrated that Tub-II is also important in the early development of the human brain, and Tub-II represents a marker for progenitor and neural stem cells. To elucidate the correlation between the developing brain and neuroepithelial tumors (NETs), the present study assessed Tub-II expression by NETs and normal brain tissue using immunohistochemical and immunoblot analyses. In the gliomas, decreased numbers and staining intensities of Tub-II-positive cells tended to be associated with increased differentiation. Conversely, neuronal neoplasms displayed high percentages and strong staining intensities among the Tub-II-positive cells, irrespective of differentiation. In neuronal neoplasms and neoplasms with neuronal differentiation, Tub-II staining was far more intense and more homogeneous than Tub-II staining in gliomas. These results indicate that the expression of Tub-II in NETs may reflect architectural changes in the developing brain and may support the hypothesis that neuroepithelial tumors originate from glioneuronal progenitor cells capable of generating astrocytic, and neuronal cell types.     

Cyclin D1 expression in normal oligodendroglia and microglia cells: Its use in the differential diagnosis of oligodendrogliomas

Cyclin D1 regulates G1–S progression. In many carcinomas it is overexpressed and it might even correlate with prognosis. However, the amplification of CCND1 contributes to the loss of cell cycle control only in a small fraction of malignant gliomas. Cyclin D1 can be immunohistochemically demonstrated by DCS‐6 mAb. In astrocytic gliomas the fraction of tumor cells with positive nuclei is almost null in well differentiated tumors and increases with the increase of proliferation rate that occurs in anaplasia. The correct evaluation of this fraction is hindered by the positive staining of normal oligodendrocytes and microglia cells. The cyclin D1‐positive staining of normal oligodendrocytes and microglia cells has been studied in a series of 20 oligodendrogliomas, five diffuse astrocytomas and five oligoastrocytomas and in 10 samples of normal cortex and white matter, using cyclin D1 DCS‐6 mAb, Feulgen reaction and CR3.43 mAb for microglia cells. As well as microglial nuclei, the nuclei of normal oligodendrocytes of the cortex and white matter, including peri‐neuronal satellites and pericapillary cells, were immunostained by DCS‐6 mAb. In infiltrative areas of oligodendrogliomas, normal, cyclin D1‐positive oligodendrocytes and cyclin D1‐negative tumor cells coexisted. In anaplastic oligodendrogliomas, cycling tumor oligodendrocytes may regain the capacity to express cyclin D1, which is thus positive in some tumor cells. The occurrence of positive oligodendrocytes in the peripheral parts of tumors can be useful in distinguishing astrocytomas from oligoastrocytomas.     

P53蛋白在脑胶质细胞瘤中的表达

用SP免疫组化方法检测79例脑胶质细胞瘤中P53蛋白的表达,总阳性率为21.52%.其中星形细胞瘤及室管膜瘤阳性率分别为26%及25%,且Ⅲ、Ⅳ级肿瘤阳性率及表达量均显著高于Ⅰ、Ⅱ级肿瘤.表明P53蛋白的表达与这两型肿瘤的恶性程度密切相关.但临床上恶性程度很高的髓母细胞瘤反而未见表达.     

Galectin‐3: A useful biomarker for differential diagnosis of brain tumors

Galectin‐3 (gal‐3) is a 31 kDa β‐galactoside‐binding lectin that is immunohistochemically expressed in macrophages, lymphocytes, and endothelial cells, and also in some neoplastic cells. Gal‐3's expression in and significance to brain tumors has not been fully addressed. Here, we investigated its immunohistochemical expression in 409 cases of surgically resected primary brain tumors, including various glioneuronal tumors, pituitary adenomas, meningiomas and Schwannomas, among others. In normal brain tissues, gal‐3 was robustly expressed in normal astrocytes, endothelial cells and macrophages. It showed consistent and diffuse positivity in 100% of the pilocytic astrocytomas, pleomorphic xanthoastrocytomas (PXA), Schwannomas, meningiomas, capillary hemangioblastomas, as well as in ependymomas, but it was completely negative in the diffuse astrocytomas, anaplastic astrocytomas, both low‐ and high‐grades of the oligodendrogliomas, central neurocytomas, and medulloblastomas. Definitely positive but heterogeneous expression was found in various tumors including subependymal giant cell astrocytomas (SEGA), classic glioblastoma multiforme, anaplastic oligoastrocytomas, CNS primitive neuroectodermal tumors (CNS PNETs), and hemangiopericytomas. Eighty percent of small cell glioblstomas were completely negative, but 20% showed heterogeneous positivity for gal‐3. Focal positivity for gal‐3 was also found in dysembryoplastic neuroepithelial tumors (DNTs) and gangliogliomas, in which the positive cells were the astrocytic component. On the basis of our immunohistochemical data in conjunction with previous reports, we therefore conclude that gal‐3 is differentially expressed in various brain tumors, and thereby, is a helpful biomarker in making differential diagnoses, especially in cases where a morphological diagnosis is controversial.     

Expression of prostaglandin H synthase-2 in human brain tumors

Numerous studies have demonstrated that prostaglandin H synthase-2 (PHS-2) is involved in gastrointestinal carcinogenesis, and that nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit PHS, can reduce the risk of colon cancer. In brain tumors, elevated prostaglandin production and its correlation to anaplastic grade of gliomas have been demonstrated. To determine whether the increased prostaglandin production is due to enhanced expression of PHS-2 and whether the up-regulation of PHS-2 has any correlation to histopathological findings in brain tumors, we evaluated the profile of PHS expression in several human glioma cell lines and surgical specimens from patients with various types of brain tumors. In glioma cell lines, five out of six cell lines showed constitutive expression of PHS-2, whereas PHS-1 was weakly expressed in all of them. All surgical specimens, except an ependymoma, which expressed both isozymes equally, expressed PHS-2 mRNA predominantly. Immunohistochemistry of various types of brain tumors, including six glioblastomas, nine astrocytomas, six meningiomas, five medulloblastomas, four craniopharyngiomas, three ependymomas, three neurinomas, two oligodendrogliomas, two malignant lymphomas, two dysembryoplastic neuroepitherial tumors and one metastatic brain tumor showed PHS-2 staining in most cases. In gliomas, astrocytomas (grade 2 and 3) were strongly stained, but the staining intensity of glioblastomas was relatively weak. Meningiomas and a metastatic brain tumor were also strongly stained. Our data thus suggest that most brain tumors express PHS-2, which may also play a role in tumorigenesis in the brain.