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.

Determination of gangliosides in six human primary medulloblastomas.

The ganglioside composition of six human medulloblastomas was analyzed. The characterization was performed by thin-layer chromatography, sialidase hydrolysis, and immunological staining with a panel of characterized antiganglioside monoclonal antibodies. The total ganglioside content ranged from 60 to 1,130 nmol of ganglioside sialic acid/g wet weight. Neuronal gangliosides (gangliotetraose series) were found in varying amounts in all medulloblastomas. Gangliosides of the neolactotetraose series (3'-LM1 and LD1) were present in all specimens, and the lactotetraose series ganglioside 3'-isoLM1 was found in all cases showing astrocytic differentiation. This supports our previous findings that 3'-isoLM1 is a marker for proliferating astroglial cells.

Dibutyryl cyclic AMP induces vimentin and GFAP expression in cultured medulloblastoma cells.

Evidence for the astrocytic lineage in medulloblastomas rests largely on the detection of the glial fibrillary acidic protein (GFAP) from which intermediate filaments (IF) specific for astrocytes are assembled. Astrocyte progenitor cells from the mouse neopallium however express another IF protein, vimentin, before they acquire GFAP in vivo and in vitro. The purpose of the current study was to determine if cells obtained from a focally GFAP-positive posterior fossa medulloblastoma previously shown to acquire GFAP in response to dibutyryl cyclic AMP (dBcAMP), also express vimentin before expressing GFAP. More than 80% of cells in the tumor section contained vimentin while fewer than 1% of cells contained GFAP; occasional clusters of malignant GFAP-positive cells and clusters of cells negative for both vimentin and GFAP were also identified in the tumor. One hundred per cent of cultured cells in the first 10 passages from the tumor contained vimentin and no cells expressed GFAP. When cells were cultured in the presence of dBcAMP. Western immunoblotting showed an increase in vimentin which reached maximal values within 24 h followed by an increase in GFAP which reached maximal values at 72 h. The increase in vimentin followed by that of GFAP in cultured medulloblastoma cells has not previously been reported and suggests that most astrocyte progenitors which are derived from medulloblastoma and cultured in vitro may be at a developmental stage which corresponds to the proastroblast stage in the developing mouse brain.(ABSTRACT TRUNCATED AT 250 WORDS)

[Melanotic medulloblastoma. Ultrastructural and histochemical study of a case]. / Meduloblastoma melanótico. Estudio ultraestructural e inmunohistoquímico de un caso.

A electron microscopic and immunohistochemical study of a Melanotic medulloblastoma is reported. The cerebellar tumor was located in the vermis of a 6-year-old boy, dead 11 months after diagnosis. The tumor consisted of medulloblastoma-like areas with focal differentiation and pseudoepithelial structures pigmented with melanin. Electron microscopy showed melanosomes and tight junctions in pigmented areas. On immunohistochemistry, the cytoplasm of melanotic cells were positive to S-100 protein and the differentiated glial cells to GFAP. The tumor histogenesis, its relationship with other pigmented tumors of the CNS and their low frequency is commented on.

Expression of major histocompatibility complex on human medulloblastoma cells with neuronal differentiation.

Medulloblastomas are among the most common malignant brain tumors in children. These tumors consist of immature bipotential cells that may differentiate into neuronal and glial cells. We have established two cell lines for human medulloblastoma. One was derived from a 2-year-old girl with a cerebellar tumor (designated as ONS-76) and another from a 9-year-old girl with a metastatic tumor in the right frontal lobe (ONS-81). The in vitro population-doubling times were 18.6 and 19.2 h, respectively. Immunohistochemical studies showed that both cells possessed neurofilament protein (Mr 145,000 and 200,000) and neuron-specific enolase, without glial fibrillary acidic protein or S-100 protein. Human gamma-interferon enhanced class I major histocompatibility complex antigens on these medulloblastoma cells. Class II major histocompatibility complex antigens were also induced by human interferon-gamma. We here report for the first time the expression of class II major histocompatibility antigens, which play an important role in immune response, on human medulloblastoma cells with neuronal differentiation.

Cerebellar desmoplastic medulloblastomas. A further immunohistochemical characterization of the reticulin-free pale islands.

We studied by immunohistochemistry the features of differentiation in 24 desmoplastic and 16 classic medulloblastomas (median patient ages, 18 and 6.5 years, respectively) with the use of a panel of cytoskeletal and synaptosomal markers. A distinctive pattern of immunoreactivity with a series of monoclonal antibodies (Mabs) was documented in the polar tumor cells forming the reticulin-free pale islands of the desmoplastic variant, denoting overt neuritogenesis. These comprised the following: (1) Mab Tp-NFP1A3 recognizing an epitope in the high-molecular-weight (Mr) isoform of neurofilament protein; (2) Mab AP18 to the high-Mr microtubule-associated protein 2; (3) Mab TUJ1 recognizing the class III beta-tubulin isotype (human h beta 4); and (4) Mab SY38 to synaptophysin. Immunoblot analysis confirmed the expression of h beta 4 in three medulloblastomas, yielding strong single bands in two desmoplastic medulloblastomas and a considerably weaker band in one classic medulloblastoma. Glial fibrillary acidic protein-positive tumor cells frequently formed an integral component of the pale islands. Oligodendrogliallike areas in one classic and in three desmoplastic medulloblastomas were immunopositive for the Mabs to synaptophysin, microtubule-associated protein 2, and h beta 4, indicating a neuroblastic nature. We propose that the reticulin-free structures of desmoplastic medulloblastomas constitute neoplastic foci with features of predominantly neuronal and, to a lesser degree, astroglial differentiation.

[Expression of insulin-like growth factor I receptors in human brain tumors: comparison with epidermal growth factor receptor by using quantitative autoradiography].

By using quantitative autoradiographic techniques, receptors for insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) were analyzed in 13 samples of human brain tumors (4 low grade astrocytomas, 7 glioblastomas, 1 anaplastic ependymoma and 1 medulloblastoma). High number of specific binding sites for IGF-I and EGF were homogeneously present in tissue sections derived from glioblastoma. In low grade astrocytoma, relatively high numbers of binding site for EGF were observed, but there was no significant difference in concentrations of IGF-I binding sites between tumors and control cortex. In medulloblastoma, only IGF-I binding sites were present. These observations might indicate that both IGF-I and EGF are involved in the growth modulation of human gliomas possibly through paracrine or autocrine mechanisms. Antagonists to growth factors or monoclonal antibody against those receptors could have the way for therapeutic application for gliomas.

[Establishment and biological characterization of human medulloblastoma cell lines].

Two cell lines of human medulloblastoma (ONS-76 and ONS-81) were established, and their biological characteristics were investigated. The cell line, ONS-76, was established from a tumor specimens obtained from a large cerebellar tumor of a 2-year-old girl. The pathological diagnosis was a typical medulloblastoma. The other cell line, ONS-81, was derived from a metastatic tumor in right frontal lobe of a 9-year-old girl. The tumor specimens were minced into fragments approximately 1 mm in diameter and cultured in plastic culture flasks in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 50% patients serum. The cells growing as a monolayer were subcultured in RPMI 1640 supplemented with 10% FCS and initially with L-glutamine, sodium pyruvate, and nonessential amino acid. Microscopically, both cultured cells exhibited various morphological appearances, and this morphological heterogeneity seemed to be specific for medulloblastoma cells. The in vitro population doubling time of ONS-76 and ONS-81 were 18.6 and 19.2 hr, respectively. The ONS-76 and ONS-81 cells formed subcutaneous tumors in nude mice as serial transplantable xenograft, and these tumors had a microscopic appearance similar to that of the original medulloblastoma. Ultrastructurally++, the cultured cells showed primitive, undifferentiated appearance, and no neuronal or glial structures were not seen. Immunohistochemical studies showed that both cells expressed neuron-specific enolase (NSE) and neurofilament protein (NFP 200 K, 145 K), but glial fibrillary acidic protein (GFAP) and S-100 protein were not detected. The NFP immunoreactivities of both cultured cells were demonstrated as abnormal perinuclear deposits.(ABSTRACT TRUNCATED AT 250 WORDS)

N-myc gene expression and oncoprotein characterisation in medulloblastoma.

Although medulloblastoma and neuroblastoma share many common biological, histological and immunological features, the frequency of N-myc amplification differs markedly between the two tumours. In this study, Southern blot analysis revealed that the N-myc gene was not amplified in any of the nine medulloblastoma samples analysed. In contrast, over-expression of the gene was found in six of 11 samples as determined by immunocytochemistry and/or Western blot analysis, using an antiserum raised against a synthetic peptide representing a sequence unique to the N-myc gene product. The specificity of this reagent was demonstrated by studies on a variety of cell lines expressing N-myc and/or c-myc oncoproteins. Of the 12 medulloblastoma samples collected over a two-year period and analysed in the course of this project, a trend towards longer disease-free survival was noted in the patients having low levels of the N-myc protein in their tumour.

[Immunohistochemical study of c-myc oncogene product in human brain tumors].

C-myc oncogene is widely distributed in eukaryotic cells and is supposed to play an important role in the cellular proliferation and differentiation. Enhanced expression of this oncogene is reported in many kind of tumors, which is often associated with increased malignancy. It seems, therefore, important to study the expression of this oncogene in analyzing the cell biologic features of brain tumors. In the present paper we investigated the distribution of this oncogene product in paraffin-embedded tissue of various kind of brain tumors with a monoclonal antibody to synthetic c-myc peptide. The results demonstrated that c-myc product was detectable in most of the astrocytoma lineage. The immunoreaction within the cell nuclei was more intense in grade 3 and grade 4 astrocytomas than in grade 2 tumors. The expression in grade 4 tumors was, however, rather weaker that in grade 3 tumors. In benign, non-glial tumors like meningiomas and neurinomas, the nuclear immunoreaction was usually absent or only weak, although it was enhanced in a case of acoustic and spinal neurinomas associated with von Recklinghausen's disease.

High-performance liquid chromatographic separation of gangliosides, combined with immunological detection and fast atom bombardment mass spectrometric characterization.

A complete strategy for the isolation of individual mono- and disialogangliosides has been elaborated. We have used straight-phase silica gel chromatography or partitioning to obtain a crude ganglioside fraction. This fraction was then peracetylated and run through a second silica gel column. After anion-exchange chromatography the gangliosides were separated by straight-phase high-performance liquid chromatography with chloroform-methanol-water mixtures as eluting solvents. The method is suitable for preparative isolation of gangliosides and subsequent structural characterization by thin-layer chromatography-enzyme-linked immunosorbent assay, fast atom bombardment mass spectrometry and/or gas chromatography-mass spectrometry, which is demonstrated by several examples, including the separation of GalNAc-II3NeuAc-GgOse4Cer from GalNAc-isoII3NeuAc-GgOse4Cer.

Prognostic importance of DNA ploidy in medulloblastoma of childhood.

The deoxyribonucleic acid (DNA) content of 53 medulloblastomas was analyzed by means of flow cytometry and compared with the clinical and histological findings in the host patients. Analysis of DNA showed that about half of the tumors were diploid and the other half were aneuploid. More diploid tumors were found among patients of a young age, but the difference was without statistical significance. Cellular differentiation of the tumor did not correlate with DNA ploidy. No correlation was found between Chang's T staging system and the DNA ploidy, whereas the M staging correlated with the ploidy; diploid medulloblastomas had a greater tendency to metastasize than aneuploid medulloblastomas (p = 0.0003). Four-year survival was compared with the extent of resection and DNA ploidy. The patients with total resection and aneuploid medulloblastoma had a better prognosis than those with subtotal resection and diploid tumor (p = 0.001). There was only one survivor among eight patients with subtotally resected diploid medulloblastomas, while all of the seven patients with totally resected aneuploid medulloblastomas survived. Comparison of the G0/G1 phase fraction and S phase fraction in the surviving group and the deceased group offered no significant information.

GD3 expression by cultured human tumor cells of neuroectodermal origin.

Seven monoclonal antibodies (mAbs) reactive with ganglioside II3(NeuAc)2-LacCer (GD3) were generated; four of these mAbs (DMAb-21, DMAb-22, DMAb-23, and DMAb-24) by immunizing mice with GD3 adsorbed to Salmonella minnesota and the remaining three (DMAb-7, DMAb-8, and DMAb-17) with melanoma line SK-MEL 28, which contains 1.4 nmol sialic acid of GD3 per mg protein. The specificities of the mAbs were defined by high-performance thin-layer chromatography (HPTLC) immunostain and solid-phase radioimmunoassay (SP-RIA) with a panel of purified gangliosides. DMAb-7 and DMAb-8 reacted with GD3, IV3(NeuAc)2nLcOse4Cer(3',8'-LD1), and very weakly with IV3(NeuAc)2II3NeuAcGgOse4Cer (GT1a), but not with II3NeuAc-LacCer (GM3), II3NeuAcGgOse3Cer(GM2), II3NeuAcGgOse4Cer (GM1), II3NeuAc, IV3NeuAcGgOse4Cer (GD1a), II3(NeuAc)2GgOse3(GD2), II3(NeuAc)2GgOse4Cer (GD1b), IV3NeuAcII3(NeuAc)2, GgOse4Cer(GT1b), suggesting the binding epitope to be a terminal tetrasaccharide NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4(Glc or GlcNAc). DMAb-7 and DMAb-8 were used to investigate the expression of GD3 on cultured human tumor cells of neuroectodermal origin. Thirteen of 19 gliomas, 3 of 5 medulloblastomas, 5 of 5 neuroblastomas, 2 of 2 melanomas, and 1 of 3 teratomas were shown to react with DMAb-8 and/or DMAb-7 by cell surface-RIA (CS-RIA) and immunofluorescence (IF) assays. HPTLC and densitometric analysis confirmed these results, as positive immunostains in the GD3 region were obtained with oligoganglioside fractions from 9 glioma, 1 medulloblastoma, 2 neuroblastoma, 1 melanoma, and 1 teratoma cell line. Glioma cell line U-105 MG and medulloblastoma cell line Daoy contain GD3 as shown by HPTLC immunostain analysis of extracts, although GD3 was undetectable on the cell surface as determined by CS-RIA and IF.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation in medulloblastomas: immunohistochemical study of twenty-one cases.

Twenty-one cases of medulloblastoma (MB), 20 corresponding to the classical type and 1 to the desmoplastic variant (DPM), were studied immunohistochemically with glial fibrillary acidic protein (GFAP), S-100 protein (S-100P), neuron-specific enolase (NSE) and 200-kd neurofilaments (NF) in order to assess their differentiation potential. Their respective positivities in neoplastic cells were: 42% for GFAP, 47% for S-100P, 61% for NSE and absence of NF. Isolated neoplastic cells (e.g., cells lacking processes and with scanty cytoplasm), appeared positive for neuroglial markers (GFAP and S-100P). Most cases, however, showed positive reactive neuroglial cells. NF were never found in neoplastic cells, but in trapped neuronal processes. In most cases, large numbers of cells had high positivity to NSE. Cells of lucent islands of DPM were positive for GFAP and S-100P, while Homer-Wright type rosettes showed positive only for NSE. The above results support a neuroglial differentiation in MB. NSE was considered a nonspecific marker for neuroblastic cells.

[A simple method for the isolation of GFAP and its use for the study of brain tumors]. / Eine einfache Methode zur Isolierung von GFAP und ihre Anwendung zur Untersuchung von Hirntumoren.

A simple method is described in this paper for the production of a polyclonal antiserum against GFAP. The antiserum was tested on 212 primary brain tumours which had been selected from biopsy and autopsy material of the Institute of Pathological Anatomy at the Medical Academy of Erfurt, GDR. 52 of 81 astrocytomas (64%) and 26 of 47 glioblastomas (55%) gave GFAP-positive results. GFAP-negative responses were primarily recorded from tumours with severe anaplasia. GFAP was found in all 22 ependymomas tested. Epithelioid ependymomas, however, exhibited lower immunological reactions than tanycytic variants. Isomorphic oligodendrogliomas, meningiomas, medulloblastomas, and brain metastases of carcinomas were GFAP-negative. The possibility is discussed in some detail of falsely negative results on account of too little biopsy material or insufficient fixation of tumour tissue.

Differentiation in medulloblastomas: correlation between the immunocytochemical demonstration of photoreceptor markers (S-antigen, rod-opsin) and the survival rate in 66 patients.

Biopsy specimens of 66 medulloblastomas were investigated by means of S-antigen and rod-opsin immunocytochemistry. The patients were operated between 1969 and 1988 and the medical records were retrospectively evaluated to correlate the immunocytochemical features of the tumors to the course of the disease. S-antigen- and rod-opsin-immunoreactive tumor cells were found in 19 out of 66 cases. Since in the normal non-neoplastic state immunoreactive S-antigen and rod-opsin are restricted to retinal photoreceptors and a class of pinealocytes derived from photoreceptor cells, the occurrence of these proteins in certain tumor cells of medulloblastomas suggests a differentiation of these cells along the photoreceptor cell lineage and allows the identification of a special subtype of medulloblastoma displaying photoreceptor-specific characteristics. This subtype appears to be closely related to retinoblastomas and pineal cell tumors. The incidence of this subtype corresponds to approximately 30% of all medulloblastomas. Correlation between the demonstration of immunoreactive S-antigen and rod-opsin and the course of the disease revealed a 10-year survival rate of 50.6% for patients with medulloblastomas displaying photoreceptor-specific characteristics and maximally 11% for patients suffering from medulloblastomas devoid of these markers. Although the statistical evaluation does not provide a significant result, the estimated P-value of 0.085 indicates a distinct trend toward a better prognosis for patients suffering from medulloblastomas with photoreceptor-specific features. The validity of this trend needs to be proven in further studies with a greater number of patients.

[Glial fibrillary acidic protein and neurofilament protein in medulloblastoma].

Medulloblastoma is the most common primitive neuroectodermal tumor (PNET) with the potential to differentiate along glial or neuronal lines. Thirty cases of medulloblastoma were tested by the peroxidase-antiperoxidase (PAP) method with anti-GFAP serum (DAKO) and by the avidin-biotin peroxidase complex (ABC) method with 68kd subunit of anti-NF antibody. All the cases were classified into three subtypes based on these immunohistochemical findings and were analyzed in relation to clinico-pathological features. Fifteen of thirty medulloblastomas contained GFAP positive cells, seventeen showed cells reacting to NF. The reactions for both proteins were present in eight medulloblastomas (PNET-BD, bipotential differentiation). Seventeen medulloblastomas reacted to only one protein (PNET-MD, monopotential differentiation). No reaction for either was found in five cases (PNET-NOS, not otherwise specified). The two year survival rate was 12.5% for PNET-BD compared to 49.2% for PNET-MD and 53.3% for PNET-NOS. Nine variables, i.e. age, tumor stage, metastatic stage, operation, radiotherapy, chemotherapy, histology, GFAP and NF, were analyzed using Cox's proportional hazard model. This revealed that the significant factors were tumor stage (p = 0.0002), GFAP (p = 0.0008) and operation (p less than 0.05). In conclusion, GFAP is the most important histological factor for prognosis and medulloblastoma without glial differentiation has a much better prognosis than one with glial differentiation.

Immunohistochemical and ultrastructural observations on Homer Wright (neuroblastic) rosettes and the "pale islands" of human cerebellar medulloblastomas.

A combined immunohistochemical and ultrastructural study of 20 cerebellar medulloblastomas has demonstrated features of early neuronal differentiation. The differentiation features are primarily encountered in the Homer Wright rosettes and in the reticulin-free "pale islands," or "follicles," of the desmoplastic variant. They consist of parallel arrays of aggregated neurite-like processes containing longitudinally oriented microtubules (immunoreactive for polyvalent antisera to tubulin and gamma-enolase, but nonreactive for a monoclonal antibody to the 150/200 kD subunits of neurofilament protein) and junctional adhesion plaques. We consider the inherent property of self-aggregation of the neurite-like processes with adhesion plaques a significant mechanism in the formation of Homer Wright rosettes. Further differentiation and elongation of these cell processes may lead to the formation of "pale islands" in the desmoplastic medulloblastoma. A meshwork of astroglial cells, coexpressing glial fibrillary acidic protein and S-100 protein immunoreactivity, forms an integral part of the "pale island." The histogenetic significance of these astrocytes and their relationship to tumor cells expressing early neuronal differentiation remains to be defined.

Enhanced melphalan cytotoxicity following buthionine sulfoximine-mediated glutathione depletion in a human medulloblastoma xenograft in athymic mice.

The effect and therapeutic consequences of buthionine-(SR)-sulfoximine (BSO)-mediated depletion of glutathione in the human medulloblastoma-derived cell line, TE-671, growing as s.c. xenografts in athymic nude mice were examined. The glutathione content of the s.c. xenografts was 1.11 +/- 0.15 mumol/g (7.79 +/- 1.61 nmol/mg of protein). Administration i.p. to tumor-bearing mice of D,L-BSO (two doses at 12-h intervals; 5 mmol/kg) depleted the glutathione content of the xenografts to 25.7% of control. Administration of a 30 mM solution of L-BSO in drinking water for 96 h depleted the glutathione content to 17.4% of control. Depletion of glutathione with these regimens resulted in a significant increase in the s.c. tumor growth delay over that produced by melphalan alone: 17.2 days versus 12.6 days for D,L-BSO (i.p.) plus melphalan versus melphalan and 22.9 days versus 16.6 days for L-BSO (p.o.) plus melphalan versus melphalan. These studies demonstrate the increased cytotoxicity of melphalan resulting from BSO-mediated depletion of glutathione in human medulloblastoma and support further efforts to modulate the chemosensitivity and radiosensitivity of this tumor by modulation of glutathione.

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.