Transplantation resistance to mouse ependymoblastoma following immunization with dehistonized syngeneic tumor chromatin.

Female C57BL/J6 mice pretreated with dehistonized ependymoblastoma chromatin rejected 5 x 10(4) or 10(5) s.c.-implanted syngeneic ependymoblastoma cells. Control mice that received Freund's adjuvant containing dehistonized chromatin from normal mouse brain developed palpable tumor nodules in 90% and 35 to 45% of the animals, respectively. Increased binding of spleen lymphocytes from mice pretreated with dehistonized tumor chromatin to Sepharose 4B coupled with components of dehistonized tumor chromatin was observed. This provides initial evidence for the presence of "committed" lymphocytes in mice pretreated with dehistonized chromatin from this mouse ependymoblastoma.

Ganglioside composition of an experimental mouse brain tumor.

The ganglioside composition of an experimental ependymoblastoma was examined in C57BL/6 mice. This tumor was produced by Dr. H. Zimmerman in 1949 from methylcholanthrene implantation in the brain and has been maintained in serial transplants through many generations. The influence of tumor environment on ganglioside composition was determined by studying the tumor growing intracerebrally and s.c. (over the skull and in the flank). The ganglioside composition of this tumor is markedly different from that of adult mouse brain. The total ganglioside sialic acid content (micrograms/100 mg dry weight) of the tumor growing in the cerebrum, s.c. over the skull, and in the flank was 70.4 +/- 3.8 (N = 3), 66.8 (N = 2), and 41.7 +/- 0.7 (N = 3), respectively. These values are about 10-fold lower than the ganglioside content of normal mouse cerebrum. This tumor contained a significant amount of N-glycolyneuraminic acid (NGNA). Histological analysis revealed two basically different cell types. The predominant cell type is densely packed and poorly defined in shape, whereas the minor cell type is less densely packed and fibroblastlike in shape. GM3, which migrates as double bands on thin-layer chromatography, is the predominant ganglioside of this tumor in all three regions of growth. Also present in all regions are gangliosides NGNA-GM3 and GM1. Significant amounts of GD1a, GD1b, GT1b, and GQ1b are present only in the cerebral tumor. These gangliosides therefore represent contaminants from normal brain tissue surrounding the tumor and are not native to the tumor. Ganglioside GD3, however, is a minor component of the tumor. Using a thin-layer chromatography-immunostaining method with anti-GA1 antibody, we found significant amounts of ganglioside with a GA1 oligosaccharide backbone migrating near GD3 and GD2. This tumor is similar to other neural tumors in having elevated amounts of GM3 and reduced amounts of total ganglioside and polysialogangliosides but is unique in having a high content of NGNA-containing gangliosides. The possible origin of the NGNA-containing gangliosides is discussed.

Protein patterns in various malignant human brain tumors by two-dimensional gel electrophoresis.

Two-dimensional gel electrophoresis with silver staining was used to study protein patterns in various malignant human brain tumors obtained at surgery. These samples included 20 high-grade astrocytomas (anaplastic astrocytomas and glioblastomas), one low-grade astrocytoma, six juvenile astrocytomas, four ependymomas, and five medulloblastomas. Histological correlates of the sampled tissue were carefully established prior to micropunch sampling. The molecular weight range of these gels was 14,000 to 100,000, and the isoelectric points ranged from 4.7 to 7.0. Proteins that have been identified include albumin, actin, tubulin, glial fibrillary acidic protein, vimentin, glutamic oxaloacetic transaminase, neuron-specific enolase, and the beta-subunit of the guanine nucleotide regulatory proteins. Each type of tumor was found to have a characteristic protein profile that set it apart from the other tumors studied. By providing a convenient tool for the display of a wide spectrum of tumor markers in a single study, two-dimensional gel electrophoresis protein profiles may be useful as diagnostic and prognostic adjuncts. Furthermore, several protein spots that were not noted in normal human cortex were identified in the various tumor gels. Antibodies can be raised against some of these tumor-associated proteins, and their further characterization could provide valuable insights into the biology of these tumors.

Differential spectrum of expression of neural cell adhesion molecule isoforms and L1 adhesion molecules on human neuroectodermal tumors.

A series of four medulloblastomas, seven neuroblastomas (Nb), two ependymomas, and three gliomas, human neuroectodermal tumors, were screened for their expression of adhesion molecules L1, carcinoembryonic antigen, neural cell adhesion molecule isoforms (N-CAM) and HNK1 epitope by Western blotting and double immunofluorescence labeling. All seven neuroblastomas, whatever their differentiated state, expressed L1, a neural cell surface developmental antigen, whereas all other tumors tested were negative. All tumors expressed N-CAM; however, a large diversity was observed among the isoforms. Low sialylated N-CAM 140 was present, with different intensity, in ependymomas and astrocytomas. High sialylated isoforms were detected by a monoclonal antibody (anti-MenB) specifically recognizing high polymers of alpha 2-8 linked neuraminic acid. They were expressed in all medulloblastomas studied (4 of 4), and in 4 of 7 Nbs examined. Negative cases corresponded to tumors having undergone chemotherapeutic treatment or to ganglioneuroma. The interconversion from high to low sialylated forms might reflect changes which are critical for the conversion of Nbs into benign ganglioneuromas. HNK1 epitope was expressed on a large diversity of molecules by nearly all tumors except two Nbs which were also negative with anti-MenB antibody. This simultaneous loss of carbohydrate epitopes could correlate with higher maturation states of the tumors. None of the tumors expressed carcinoembryonic antigen. Therefore, anti-L1 and anti-MenB antibodies define differentiation-related antigens that could differentiate between Nbs and other tumors and may prove helpful in diagnosis and understanding of the biological nature of neuroectodermal tumors. An immunodot assay was set up and allowed to titrate the presence of polysialic acid units in cerebrospinal fluid from patients presenting meningeal spread of medulloblastomas. It could help to assess metastasis and to monitor the effects of chemotherapeutic treatment on polysialylated N-CAM positive tumors.

Immunohistochemical localization of S100 beta in human nervous system tumors by using monoclonal antibodies with specificity for the S100 beta polypeptide.

The immunohistochemical localization of the calcium-binding protein, S100 beta, in human nervous system tumors has been examined by using a monoclonal antibody with specificity for the S100 beta polypeptide. S100 beta-specific immunoreactivity is detected in astrocytoma, glioblastoma, Schwannoma, ependymoma, and craniopharyngioma, whereas no reactivity is seen in oligodendroglioma, meningioma, neuroblastoma, or medulloblastoma. These data suggest that analysis of S100 beta localization with these monoclonal antibodies may be useful for research or diagnostic purposes.

Ependymoma of the ovary. A clinico-pathologic, ultrastructural and immunohistochemical investigation. A case report.

A case of primary malignant ovarian ependymoma is described. The course of the tumor disease was extremely prolonged with a 50 year history. The diagnosis is supported by immunohistochemical and ultrastructural evidence of ependymal differentiation. The histogenesis and the origin from a possible preexisting ovarian teratoma are discussed.

Estrogen and progestin receptors in an ovarian ependymoma.

Ovarian neoplasms of the pure neuroectodermal type, although quite rare, have recently been described. Lesions of similar histology in the central nervous system have previously been shown to contain estrogen and progestin receptors. A patient with a pure ovarian ependymoma is presented. The tissue was rich in estrogen and progestin receptor protein, containing 17 femtomoles of estrogen receptor per milligram cytosol protein and 80 femtomoles of progestin receptor per milligram cytosol protein. The case is used to illustrate the possible influence of hormonal manipulation in the management of this rare gynecologic neoplasm.

Cytogenetic analysis and flow cytometric DNA measurement of a human tumor with pronounced hypodiploidy.

A case of malignant choroid plexus papilloma of the brain with severe hypodiploidy is presented. The hypodiploidy was estimated by means of flow cytometric measurements of the nuclear DNA content in two investigations with an interval of 21 months. The latter investigation was supplemented with chromosome analyses including quinacrine bonding. A modal chromosome number of 34 to 35 was found with a consistent loss of one chromosome Nos. 2, 3, 4, 5, 10, 13, 14, 15, 17, and 18, and no major structural changes. The corresponding calculated DNA content per nucleus correlated very well with the measured content, which was found to be 75% of the male diploid amount. The paper briefly discusses cell survival in extreme hypodiploidy and provides a comparison with cases from the literature in which banding analysis gives comparable information.

Determination of glial fibrillary acidic protein (GFAP) in human brain tumors.

Brain tumors have been tested for their glial fibrillary acidic protein (GFAP) content by means of the rocket electrophoresis technique. Meningiomas and neurinomas were low in GFAP. Metastases had a low level of GFAP except when contaminated with surrounding tissue. Non-nervous tumors such as myeloma, myeloplaxoma and adenocarcinoma gave negative results. More detailed correlations with histological observations have been looked for in glial tumors. Low levels of GFAP were always associated with signs of malignancy such as mitoses and giant or atypical cells, whereas high levels of GFAP were correlated with the presence of well-preserved astrocytes.

Primary leptomeningeal ependymoblastoma. Case report.

Ependymoblastoma is considered to be a primitive malignant glioma with ependymal differentiation. This rare tumor occurs in very early life and shows rapid growth and a diffuse infiltration through the leptomeningeal space. The tumor cells are highly immature, with numerous mitoses and multilayered ependymal rosettes. The ependymoblastoma described in this report was found in a 17-year-old girl. In spite of detailed clinical and postmortem examinations, no definite primary site was identified in the neuraxis. The lesion spread predominantly throughout the leptomeningeal space. Histological analysis strongly suggested that this tumor originated from a heterotopic glial nest in the subarachnoid space. The absence of immunohistochemical neural tissue markers, glial fibrillary acidic protein, S-100 protein, neuron-specific enolase, and neurofilaments ruled out neuronal or glial differentiation. The authors were unable to find any previous report of primary leptomeningeal ependymoblastoma.

The distribution of nuclear DNA from human brain-tumor cells.

Flow cytometry (FCM) is a technique that measures the quantity of DNA contained in individual nuclei and records a frequency distribution of the DNA content per nucleus in the sampled cell population. Nuclei from a variety of human brain-tumor types were isolated by means of tissue grinding, purified by centrifugation through 40% sucrose (15 minutes at 4000 rpm), fixed with 10% formalin, stained with acriflavin-Feulgen, and analyzed by FCM. Profiles of DNA distribution in histologically benign tumors, such as meningiomas, pituitary adenomas, neuroblastomas, and low-grade astrocytomas, revealed a large diploid population (2C) with a few nuclei in DNA synthesis, as well as a small premitotic population (G2 cells) that contains a 4C DNA complement. In contrast, malignant gliomas, including glioblastomas, consist of more cells in DNA synthesis; these tumor cells show a highly variable distribution of ploidy consisting not only of diploid, and/or aneuploid, but also of triploid, tetraploid, and possibly octaploid populations. Also, a large variability between different regions of each tumor was always observed. In contrast, metastatic brain tumors, despite the fact that they contain a considerable number of cells undergoing DNA synthesis, demonstrate little variability within each individual tumor. The ability to rapidly characterize the cell populations of human brain tumors with FCM may enhance the effectiveness of their clinical management.

In situ cell kinetics studies on human neuroectodermal tumors with bromodeoxyuridine labeling.

Thirty-eight patients undergoing surgical removal of neuroectodermal tumors of the central nervous system were given a 1-hour intravenous infusion of bromodeoxyuridine (BUdR), 150 to 200 mg/sq m, to label tumor cells in the deoxyribonucleic acid (DNA) synthesis phase (S-phase). The excised tumor specimens were divided into two portions: one was fixed with 70% ethanol and embedded in paraffin and the other was digested with an enzyme cocktail to make a single-cell suspension. The paraffin-embedded tissues were stained by an indirect peroxidase method using anti-BUdR monoclonal antibody (MA) as the first antibody. Single-cell suspensions were reacted with fluorescein isothiocyanate (FITC)-conjugated anti-BUdR MA's for flow cytometric analysis. S-phase cells that had incorporated BUdR into their DNA were well stained by both methods. The percentage of BUdR-labeled cells, or S-phase fraction, was calculated in tissue sections by microscopic examination and in single-cell suspensions by flow cytometric analysis. The biological malignancy of the tumors was reflected in the S-phase fractions, which were 5% to 20% for glioblastoma multiforme, medulloblastoma, and highly anaplastic astrocytoma, but less than 1% in most moderately anaplastic astrocytomas, ependymomas, and mixed gliomas. Two juvenile pilocytic astrocytomas and two low-grade astrocytomas from children had high S-phase fraction despite the fairly benign and slow-growing nature of these tumors. These results indicate that the S-phase fraction obtained immunocytochemically with anti-BUdR MA's may provide useful information in estimating the biological malignancy of human central nervous system tumors in situ.

Hormones and tumours in central nervous system (CNS): steroid receptors in primary spinal cord tumours.

On the basis of the studies reported on steroid receptors in brain tumours, cytoplasmic and nuclear estrogen (ER) and progesterone (PR) receptors have been examined in forty primary spinal cord tumours: fifteen neurinomas, three neurofibromas, nine meningiomas, nine ependymomas, two astrocytomas, one oligodendroglioma and one hemangiopericytoma with the exchange method in the presence of sodium thiocyanate for ER and using the synthetic progestin R5020 for PR. Regardless the type of the tumour, ER have been detected with a higher incidence in male than in female patients (78% versus 59%). PR had the same incidence in male and in female patients. The neurinoma was the oncotype more constantly provided with steroid receptors: nuclear ER, in fact, has been found with an incidence of 75% in male and of 43% in female patients. This oncotype is usually provided in both sexes with PR. Ependymomas is spinal tumour with the highest incidence of cytosol ER both in male and in female patients. On the basis of the above results reported it can be assumed that hormonal factors might be involved in the occurrence as well as in the growth of spinal cord tumours. Therefore it can be hypothesized that hormonal treatment might favourably be used as an adjuvant therapy in some selected patients with receptor positive spinal tumours.

Immunocytochemical analysis of intermediate filaments in human ependymal tumors.

The peroxidase anti-peroxidase technique was used for localization of glial fibrillary acidic protein (GFAP) and vimentin (VM) in 19 ependymal tumors in order to determine if a unique pattern of intermediate filament (IF) expression could be demonstrated. Cytokeratin (CK) immunoreactivity was examined in a subgroup of 7 tumors with papillary pattern. Nineteen non-ependymal neuroectodermal tumors were used as controls. Ependymomas, subependymomas and astrocytomas were positive for both IF. Oligodendrogliomas, oligodendroglial portions of mixed gliomas and the majority of medulloblastomas were negative for GFAP and VM. Areas of poor differentiation in all tumors demonstrated little expression of any IF. A composite ependymoma/choroid plexus papilloma showed the presence of GFAP, VM and CK in the papillomatous portion only. Four papillary ependymomas were negative for CK. This study emphasizes the parallel distribution of GFAP and VM in well differentiated ependymomas and other glial tumors and casts doubt upon the concept of VM as a marker for de-differentiation in neuroectodermal neoplasia.

GFAP in brain tumor diagnosis: possibilities and limitations.

Investigation of GFAP (Glial Fibrillary Acidic Protein) in 175 brain tumours showed varying amounts of fibrillary acidic protein in every glioma. In ependymal and oligodendroglial tumours a high number of positive neoplastic elements were detected, GFAP positive were also the peri-vascular cells of a so-called astroblastoma. In pilocytic astrocytomas, Rosenthal fibers were in part GFAP positive, in part negative. In giant cells gliomas, giant cells were GFAP negative or weakly positive. Intraleptomeningeal growing tumour cells presented usually a very strong positivity. In 8 recurring oligodendrogliomas, the number of GFAP positive tumour cells was the same in the primary tumour and in its recurrence. These results demonstrate that GFAP is not a specific astrocytic, but a glial-specific protein. Although GFAP is usually present in greater concentration in differentiated, slow growing gliomas, absolute reliable predictions on biological behaviour of the individual tumour are not possible, because a high GFAP content can be detected also in malignant tumours. GFAP investigation does not seem reliable for solving the pathogenetic problems of undifferentiated tumours: the results obtained in 50 medulloblastomas showed that the investigation of small tumour samples or the positivity of a single cell are inadequate data for a correct evaluation of the findings, especially bearing in mind that GFAP of degenerated astrocytes can be phagocytised by other cells, these findings giving rise to misinterpretations.

Ependymoma of the mediastinum.

A case of primary ependymoma of the mediastinum is reported. The tumor was adherent to the lung and metastasized to adjacent mediastinal lymph nodes. An autopsy showed no evidence of tumor in the central nervous system. The diagnosis of ependymoma was confirmed by the immunohistochemical positivity for glialfibrillary acidic protein. To the best of our knowledge, this is the first reported example of an ependymoma in this location.

Immunocytochemical demonstration of glial fibrillary acidic protein in imprint smears of human brain tumors.

Papanicolaou-destained imprint smears from 24 brain tumors were investigated by means of avidin-biotin-peroxidase complex method (ABC) with the use of monoclonal antibodies against glial fibrillary acidic protein (GFAP). Positive staining reaction to GFAP antibody has been demonstrated in cells from the following tumors: astrocytoma, anaplastic astrocytoma, glioblastoma multiforme, mixed glioma, and ependymoma. The reaction for GFAP was negative for the following tumors: medulloblastoma, neurilemmoma, melanoma, hemangioblastoma, and metastatic tumors. In astrocytoma, the cell bodies and processes were positive with delicate fibrillary patterns; in anaplastic astrocytoma, cytoplasm and the processes were intensively stained. In glioblastoma multiforme, the staining patterns were also mixed, and the short, thickened processes were characteristic. Use of both a smear preparation and the immunoperoxidase staining technique is of great value in diagnosis of tumors of the central nervous system.

[A case of large malignant choroid plexus papilloma in the third ventricle--immunohistochemical and ultrastructural studies].

A case of malignant choroid plexus papilloma (choroid plexus carcinoma) originated in the third ventricle is reported. A 14-month old girl was admitted to our department with two-month history of impaired vision and gait disturbance. Neurological examination on admission disclosed a lethargy, blindness, and left hemiparesis. Computed tomographic (CT) scan and magnetic resonance imaging (MRI) demonstrated a large contrast-enhancing mass, approximately 5 cm in diameter, in the region of third ventricle, extending to the bilateral lateral ventricles. The patient had gross total removal of the tumor via lateral ventricle route, and received 40 Gy of postoperative radiation therapy. Light microscopically, the tumor was composed of epithelial cells showing both papillary and poorly differentiated pattern. There were considerable cellular pleomorphism, frequent mitoses, and occasional necroses. Immunohistochemically, anti-keratin antibody was detected within majority of neoplastic cells. Both neoplastic epithelial cells and stroma showed negative reaction to anti-GFAP antibody. Ultrastructurally, the shape of the nuclei varied from ovale to irregular with many indentations. The chromatin was clumped around the periphery of the nuclei. The neoplastic cells contained numerous free ribosomes, glycogen granules, and rough endoplasmic reticulum. The apical cell surfaces showed various size of club-like or roundish microvilli filled with glycogen granules, and rarely 9 + 2 cilia. Elongated junctional complexes were occasionally seen near the apical ends. The basal portions of the cells had a continuous basement membrane. These immunohistochemical and ultrastructural findings were comparable to the choroid plexus papilloma with malignant features.(ABSTRACT TRUNCATED AT 250 WORDS)

[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.

[Immunohistochemical study on distribution of transthyretin in normal human brain tissue and tumors].

Immunohistochemical examination of transthyretin (TTR), which is known to be synthesized in the epithelial cells of the choroid plexus as well as in the liver cells, was carried out on normal brain tissues and 84 human brain tumors, using a peroxidase-antiperoxidase (PAP) technique. TTR was demonstrated diffusely and strongly in the cytoplasm of normal choroid plexus cells, but not in ependyma and other tissues of normal brain. In all of 10 choroid plexus papillomas, TTR was found within the cytoplasm of tumor cells. In contrast, neither the two papillary ependymomas nor any other brain tumors contained TTR. Among the choroid plexus papillomas, some cases showed clear positive reactions in almost all tumor cells, while others had only a few TTR-positive cells. With these immunohistochemical findings, TTR proved a very useful marker of normal choroid plexus and choroid plexus papilloma.