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Analysis of the DNA content of various solid tumors and hematological malignancies may provide useful prognostic information. To date, however, there has been a striking lack of correlation between DNA content in neoplasms of the central nervous system and clinical behavior. Simultaneous quantitation of DNA content and proliferation-associated nuclear antigen (p105) by flow cytometry was performed on paraffin-embedded tissues representing three major groups of central nervous system neoplasms--1) 21 astrocytic tumors, 2) 13 pituitary tumors, and 3) 19 meningiomas--and the results were correlated with clinical behavior. All 4 well-differentiated gliomas were diploid, while 3 of 9 anaplastic astrocytomas and 1 of 8 glioblastomas had a demonstrable aneuploid peak. Three of 13 pituitary tumors had an identifiable aneuploid peak, while only 2 of 19 meningiomas had an aneuploid DNA content. Cell-cycle analysis of the malignant gliomas revealed a significantly higher proliferative index (PI, %S + G2M) compared with the well-differentiated astrocytomas (P less than 0.05). Within the subgroup of diploid anaplastic astrocytomas, however, extended patient survival appeared to be associated with a higher PI. For diploid pituitary adenomas, the PI was consistently lower in the 3 tumors that recurred than it was in the remaining 8 adenomas. Nuclear antigen quantitation of diploid tumors showed a wide range of p105 expression in G0G1 cells, suggesting that, within each tumor, the cells are heterogeneous with respect to proliferative activity. Aneuploid nuclei of glial tumors showed enhanced expression of p105 relative to diploid cells of the same specimen. In pituitary tumors, the median G2M/G0G1 fluorescence ratio for p105 was significantly higher (P less than 0.05) for the 3 diploid recurrent tumors than for those that did not recur. These data support the assumption that the aggressive clinical course of malignant glial neoplasms may be related to an abnormal DNA stemline and/or an alteration in cell proliferative activity. Cell cycle analysis and measurement of p105 by this technique may provide information useful from both a prognostic standpoint and in directing adjuvant therapy.

Cytosolic assay was used to detect gonadal steroid receptors in brain tumor tissue from 6 dogs and 2 cats. For 4 samples, the maximal number of binding sites and the equilibrium dissociation constant were calculated, using Scatchard analysis. The concentration of receptor protein that was discovered was similar to that detected in hormone-sensitive tumors.

The concentrations of three sex steroids, estradiol, progesterone and testosterone, were analyzed by radioimmunoassay after celite chromatography in brain tumor and breast cancer tissues. The concentrations in malignant gliomas and breast cancers showed interindividual variations, especially evident with regard to estradiol. High estradiol concentrations were recorded in two patients with malignant astrocytoma. The concentrations of 1.00 pg/mg and 3.32 pg/mg were 10 to 30 times as high as in normal female brain. In five of ten astrocytomas the estradiol concentration was higher than the lowest breast cancer value. The distribution of progesterone seemed more even, and the level was significantly lower in brain tumors and breast cancers as compared with female brain, perhaps indicating an increased metabolism. Testosterone levels were somewhat higher in brain tumors, as compared with breast cancers, but not different from values in brain tissue. There were no significant age or sex correlation or differences in the concentrations of steroids in the brain tumors. The results suggest that manipulation of sex steroid metabolism in malignant brain tumors can be of beneficial therapeutic value as has been shown for breast cancer and prostatic carcinoma.

Phospholipids in meningiomas were studied by light and electron microscopy, and by high-performance liquid chromatography. They were microscopically demonstrated in six of the ten cases by Sudan III staining after the fixation with potassium dichromate. However, the conventional ultrastructural fixation with glutaraldehyde and osmium tetroxide failed to confirm phospholipids, as most of them were dissolved during dehydration. In contrast, the specimens pretreated with tannic acid before osmication ultrastructurally retained phospholipids which were represented by multilamellar bodies or ribbon-like rings. Both were found in 23 of the 30 cases within the cytoplasm, among the plasma membranes and in the extracellular matrices. The outermost lamella or ribbon showed a direct continuity from the neighbouring plasma membranes of the cytoplasm or the mitochondria. The multilamellar bodies showed an overall distribution, while the ribbon-like rings were predominantly distributed around the psammoma bodies. Precipitation of hydroxyapatite crystals within the ribbon-like rings resulted in matrix minerals of psammoma bodies. Chromatographical analyses of meningiomas disclosed phospholipids including phosphatidyl ethanolamine, phosphatidyl choline, phosphatidyl serine, sphingomyelin and phosphatidyl inositol in that order. Psammomatous meningiomas contained a higher percentage of phosphatidyl serine than non-psammomatous tumors. Ultrastructural study of synthetic phospholipids adequately treated with tannic acid showed that the multilamellar bodies were similar to phosphatidyl choline, while the ribbon-like rings were similar to phosphatidyl serine. The role of phospholipids in meningiomas is discussed.

Two-thirds of all meningiomas and four-fifths of intraspinal and sphenoidal meningiomas occur in women. Meningiomas frequently enlarge or become symptomatic during pregnancy or during the luteal phase of the menstrual cycle. There is an increased incidence of meningiomas in women with breast carcinoma. In a series of 23 patients with meningiomas, the authors assayed biopsy specimens of the tumor for the presence of estrogen (ER) and progesterone (PR) receptors, using glycerol density gradient centrifugation and dextran-coated charcoal techniques. Significant levels of ER were found in only 17% of the patients, while significant PR levels were detected in 39%. Only one of the 16 tumors from female patients had significant ER levels, whereas three of the seven tumors from men had significant ER levels. Eight of the 16 tumors in women had significant PR levels, whereas only one of the seven tumors in men had a significant PR level. Thus, three out of four tumors with definite ER were from men, whereas eight of nine tumors with definite PR were from women. Of the eight women whose tumors contained PR, three were premenopausal and five postmenopausal. The single tumor with high levels of PR in the male patient was histologically atypical. The results of this series were compared with six published series of sex steroid assays in meningiomas. These seven series were divided into two groups: one group included two reports from the same laboratories in France, and the other the remaining five reports. Much higher percentages of both ER- and PR-positive tumors were reported from the French group. The authors suggest that this discrepancy may be due to the use of preoperative glucocorticoid therapy in the series from the United States. Since meningiomas are known to enlarge during periods when levels of circulating progestins are high, the presence of significant quantities of PR in a high percentage of tumors may have therapeutic implications for recurrent, malignant, or incompletely excised tumors, or for medically fragile patients. Conversely, since meningiomas are not known to enlarge during the proliferative phase of the menstrual cycle or with exogenous estrogen therapy, the small number of tumors positive for ER may indicate that ER lacks clinical significance. High levels of PR found in a small group of histologically aggressive tumors in several series may indicate that hormonal therapy may be especially useful in this difficult subset of patients.

65% of all intracranial meningiomas and 80% of the intraspinal ones are seen in women. Meningiomas often grow larger and present an increased incidence of clinical signs and symptoms during pregnancy and also in the luteal phase of the menstrual cycle. In this study the oestradiol and progesterone receptors were determined in 30 patients for the first time via the double ligand assay. In about 10% of the patients we found slight oestradiol receptor activity, whereas approx. 63% of the meningiomas examined had specific progesterone receptors. So far there has been no evidence of a definite dependence of receptor concentrations on the sex and age of the patients or on the pathohistology and site of the tumours. Results can be compared with the previously published data on oestradiol and progesterone receptors in meningiomas to a limited extent only, since different biochemical techniques have been employed and also since pre-operative glucocorticoid treatment has been different from study to study. However, we can safely assume that a specific progesterone receptor does exist. Since proliferation of meningiomas with high concentrations of circulating gestagens has been established, the occurrence of significant concentrations of progesterone receptors could imply therapeutic consequences (eg anti-gestagens) in a large proportion of tumours. We may neglect the clinical relevance of the oestradiol receptor because it occurs in low concentrations only. The epidemiological and clinical data on patients with meningiomas, as well as the existence of sex hormone receptors, may contribute to the development of a theory on the pathogenesis of this tumour.

Fifty meningiomas of four major histological types have been examined by immunocytochemical methods applied to formalin fixed paraffin embedded material and using readily available commercial antisera. The expression of GFAP, S100, NSE, vimentin, cytokeratin, fibronectin and desmoplakin was investigated. The majority of tumours contained NSE (86%) and S100 (78%), and all irrespective of histological type, contained vimentin and fibronectin. The epithelial marker cytokeratin was found in 56% of cases, but showed only focal expression. The relevance of these findings to histogenesis and metabolism is discussed and it is suggested that intermediate filament typing provides valuable information on meningiomas structure and function. The possible role of NSE and S100 in meningeal neoplasm is discussed.

Cyclic 3-5-adenosine-monophosphate and 3-5-guanosinemonophosphate concentrations were studied in the bioptate of brain tumors, examined during neurosurgical operation and in normal brain tissues. The correlations between cyclic nucleotides in the tumors appeared to be significantly decreased in comparison to the intact tissue, being a characteristic signs for all types of tumors. There were significant fluctuations in the level of cyclic nucleotides both in the tumours and nontumorous brain tissues. There were no correlations found between the level of cyclic adenosine-monophosphate and cyclic guanosine-monophosphate on the one hand and the duration of the disease, localization of the tumor, degree of the brain oedema and the type of narcosis on the other.

Antisera against fibronectin (FN) and glial fibrillary acidic protein (GFAP) were used to combined on frozen sections of surgical biopsies to distinguish between astrocytoma and hemangioblastoma, meningioma, or sarcoma. The nonglial neoplasms contained FN in their parenchyma, whereas the gliomas contained GFAP. The immunofluorescent staining procedure takes 10 min. The immunoperoxidase staining for the permanent files takes 30 min. The stains have been used in this institute for more than 1 year, and 26 tumors have been evaluated.

A meningioma was discovered in a 66 year old man with non-progressive alcoholic cirrhosis and abstinence for the 5 previous years. The development of polycythemia ten months later was related to an inappropriate secretion of erythropoietin. Conventional causes for this phenomenon could not be found. Erythropoietin assays in erythroblast cultures from both the patient's blood and post-mortem, from tumor fragments demonstrated a significant elevation of erythropoietic activity. The diagnosis of polycythemia secondary to meningioma is suggested. Only one similar case has apparently been reported.

Meningiomas are more common in females and their course can be affected by changes in the hormonal milieu of the patient. Recently, a few studies have reported the presence of one or more steroid receptors in a small number of meningiomas. We have studied the estrogen and progesterone receptors (ER and PgR) of 11 surgically excised meningiomas. The receptors were assayed biochemically and localized at the cellular level using fluoroceinated estradiol and rhodamine-conjugated progesterone. Considerable amounts of ER were found in 4/11 cases, and PgR was high in 6/11 tumors. Cytoplasmic localization of the steroid binding sites was seen using both tracers. The presence of 10% of cells with two ++ intensity of the fluorescence correlated with tumors having positive ER values (greater than 10 Fm/mg protein). The presence of nuclear fluorescence and the higher level of PgR seen in some tumors indicate that the ER present is functioning. This study confirms the presence of steroid binding sites in meningiomas and suggests that there may be a role for hormonal manipulation in selected patients.

The results of histoautoradiographic and immunohistochemical studies of biopsy specimens of 15 brain tumours are reported. The specimens were labeled with 3H-thymidine using an in vitro technique. Meningiomas, oligodendrogliomas and well differentiated astrocytomas showed a median S-phase fraction of about 1%. In contrast, the labeling indices of 4 from 7 anaplastic astrocytomas were higher (2.1, 3.0, 3.5, 11.4). With increasing degree of malignancy the proliferative heterogeneity of the tumours increases. In every glioma varying amounts of glial fibrillary acidic protein (GFAP) were detected immunohistochemically (PAP technique). In 3 high-grade gliomas (2 glioblastomas, 1 anaplastic astrocytoma) an inverse relation of the investigated parameters (high S-phase fraction, low GFAP expression) was found. An exact prediction on biological behaviour of an individual tumour by GFAP detection immunohistochemically is not possible, because a high GFAP content can be detected also in some malignant tumours. However, the 3H-thymidine labeling indices of viable parts of the tumours, probably reflecting the growth fraction seem to be clinically important parameters, especially in respect to the prognosis.

Ten cases of intracranial meningiomas in patient who differed by age, sex and histological characteristics of the lesion have been considered for the purpose of evaluating the lesion's receptor capacity and the changes in it with the above variations. Receptors were found and they varied with age and histological features.

Polyamine levels have been studied in brain tumor patients. We focused our study on the relationship between tumor, cerebrospinal fluid (CSF) and red blood cell (RBC) polyamine levels. Our results are the following: Polyamine levels in CSF are consistently increased, whatever the histological type may be. The highest tumoral concentrations are found in medulloblastoma. In glioblastomas, the RBC spermidine levels are higher than in the other types of tumors and there is a highly significant correlation between the spermidine/spermine ratio in tumor and RBC. Therefore, RBC polyamine determination might be of clinical interest in the monitoring of patients with glioblastomas.

The relationship between meningeal hemangiopericytoma (angioblastic meningioma), meningiomas of meningothelial derivation, and peripheral hemangiopericytoma is controversial; and immunohistochemical studies have yielded conflicting results. Likewise, immunohistochemistry has been touted as a reliable means of differentiating fibrous meningioma from acoustic schwannoma. By the immunoperoxidase method, we studied 40 meningiomas (11 meningotheliomatous, four transitional, 11 fibrous, three secretory, four metaplastic, one xanthomatous, one papillary, four atypical, one malignant), five arachnoid granulations, 13 angioblastic meningiomas, nine peripheral hemangiopericytomas, and seven acoustic schwannomas. Antisera to vimentin, epithelial membrane antigen (EMA), keratin, S-100 protein, carcinoembryonic antigen (CEA), desmin, factor VIII, Ulex europeaus, and glial fibrillary acidic protein (GFAP) were utilized. All meningiomas and arachnoid granulations stained for vimentin and EMA; 15% and 12% of meningiomas were S-100 and keratin positive, respectively. The latter was noted primarily in areas of secretory (pseudopsammomatous) differentiation. In contrast, all angioblastic meningiomas stained for only vimentin. This profile of immunoreactivity was also seen in the peripheral hemangiopericytomas, with the exception of single cases that stained focally for EMA and S-100 protein, respectively. Acoustic schwannomas all stained positively for S-100 protein, vimentin, and were variably reactive for EMA, a pattern not distinct from meningioma. We conclude that (a) meningiomas express both epithelial and mesenchymal markers as do arachnoid granulations, (b) that angioblastic meningiomas demonstrate only mesenchymal markers, (c) that angioblastic meningiomas express identical markers to peripheral hemangiopericytoma and should thus be considered a variant thereof, (d) among meningiomas, CEA and keratin appear to be relatively specific markers for the "secretory" variant, and (e) because of overlap in S-100 and EMA reactivity, these markers are unreliable in differentiating meningioma from acoustic schwannoma.

Meningiomas are rich in progestin receptors, whereas estrogen receptors (ER) are virtually undetectable. The present experiments were performed to evaluate whether the absence of ER from the majority of human meningioma cytosols can be attributed to: occurrence of only a small number of ER-positive cells in an otherwise ER-negative tissue; resistance of nuclear receptors to extraction; or impairment of steroid binding. Twenty-one specimens were selected from our total series of 67 meningiomas. Based on cytosol assays, five of these meningiomas were considered to be ER positive (10-42 fmol/mg protein) and five had marginal ER concentrations (4-9 fmol/mg protein), whereas the remaining tissues were ER negative. For comparison, human breast cancer tissues were used. Tissues were sectioned at 6 micron and stained immunocytochemically using a monoclonal antibody against the human ER. The breast cancer specimens showed specific nuclear staining in part of the tumor cells. The sensitivity of the immunocytochemical assay was found to be sufficient to detect staining in breast cancer tissues containing as little as 17 fmol ER/mg cytosol protein. No specific staining was observed in meningioma tissues. It is concluded that the majority of meningiomas are truly devoid of ER and that the estrogen binding agent detected in low concentration in some meningioma cytosols is immunologically different from the human breast cancer ER. The presence of progestin receptors in meningioma apparently does not require the continuous presence of ER.

The content of neurospecific proteins S-100, GFA and D2 was measured in malignant cerebral tumors by electrophoresis with the use of monospecific antisera. Concomitant measurement of proteins S-100 and GFA is a more reliable diagnostic criterion as to the tumor histogenesis than study of each protein alone. D2 protein appeared to be the most stable specific marker.

Tissue samples from 17 intracranial tumors were analysed for the cytosolic estrogen and progestin receptor concentration. Three of the four meningiomas were progestin receptor-positive, but all were estrogen receptor-negative. All the four meningioma patients were postmenopausal women. The intracranial metastasis of a mammary carcinoma contained both estrogen and progestin receptors. Low progestin receptor concentration was found in an astrocytoma of a postmenopausal women. In addition, the presence of estrogen and progestin receptors was studied in four prolactinomas, two glioblastomas, two oligodendrogliomas, two mixed-cell carcinomas and one astrocytoma. All these tumors were estrogen and progestin receptor-negative.

The relationship between malignant vascular meningeal tumours and typical meningiomas remains controversial, despite the need for accurate diagnostic distinction between the two, and some forms of vascular meningioma may be more closely allied to haemangioblastomas or extracranial haemangiopericytomas than to true meningiomas. In order to try to clarify the diagnostic characteristics and origins of the entity known as haemangiopericytic meningioma, 10 histologically typical cases were stained by the immunoperoxidase technique with a panel of seven antibodies. The results were compared with those obtained from typical and angiomatous meningiomas, haemangioblastomas and haemangiopericytomas from extracranial sites. Both the haemangiopericytic meningiomas and the extracranial haemangiopericytomas showed a similar staining pattern, which differed from that of the typical and angiomatous meningiomas in the strikingly focal nature of the vimentin staining and the lack of reactivity with antibodies to epithelial elements. The haemangioblastomas were less consistent in their individual staining characteristics, but had a quite different overall pattern from all the other tumour types. It is, therefore, suggested that so-called haemangiopericytic meningiomas are in fact primary haemangiopericytomas of the meninges, antigenically distinct from true meningiomas and displaying a malignant potential appropriate to haemangiopericytomas arising in any other sites.

During the last decade several strategies have been developed to identify proteins which could serve as markers in tumor biology. One avenue of great promise to detect such proteins seems to be the separation of prefractionated organelles from tumor cells by high resolution two-dimensional electrophoresis. Using detergent-lysed nuclei from several human tumor cell lines, especially from brain tumors, and two-dimensional electrophoresis, we analyzed the nuclear protein pattern obtained after sequential salt extraction of tumor cell nuclei. In addition to proteins occurring in all tumor cell lines, the pattern of different tumor cell lines exhibits considerable differences when proteins were visualized by silver staining, thus emphasizing the specificity of nuclear proteins with respect to the cell type. Even quantitative variations of the nuclear phosphoproteins 23/4 were detectable, indicating a potential correlation between their synthesis/phosphorylation and the proliferation behavior of tumor cells. The data indicate that nuclear proteins with their distinct heterogeneity and tissue specificity may represent a powerful source in determining tumor-specific proteins. The extent of chromosomal protein heterogeneity may be additionally increased by their covalent modification by nuclear kinases; therefore, tumor-specific nuclear proteins may occur as quantitative and qualitative variations.

Peroxidase-antiperoxidase (PAP) technique was selected for visualizing the binding of peanut lectin (PNL) to the most frequent human brain tumors. The randomly selected material included neoplasms of neuroectodermal and mesenchymal origin. We employed 1--5 micrometers of routinely processed and paraffin-embedded tissues. PNL receptors were detected to a variable extent on the cell surface of astrocytoma, glioblastoma multiforme, oligodendroglioma, ependymoma, meningotheliomatous meningeoma, and plexus papilloma. In gliomas as increase in malignancy seems to be associated with a decrease in PNL binding. Except for the plexus papillomas, neuraminidase pretreatment had neither a qualitative nor a quantitative influence on the binding behavior of PNL. Intracellular PNL receptors could be detected in "granular cells" and in the perinuclear region of malignant gliomas.

Benign and malignant brain tumors and normal cerebral cortex were assayed for calmodulin content by enzymatic and radioimmunoassay techniques. Normal cerebral cortex contained more (8.31 +/- 1.27 vs 3.30 +/- 0.42 micrograms/mg protein) calmodulin than the brain tumors. The contents of calmodulin in the malignant glioblastomas were significantly higher than the meningiomas (5.41 +/- 0.31 vs 2.97 +/- 0.16 micrograms/mg protein). These differences were independent of tumor location and persisted when calmodulin content was normalized for DNA rather than protein content. This data supports differences in the tissue calmodulin contents with normal cortex greater than primary malignant tumors greater than benign tumors greater than metastatic tumor tissue.

Polymorphous tumor cells with bizarre giant nuclei are characteristic of the so-called atypical meningioma. Some of these cells are iron-positive. Meningioma cells apparently are able to store iron excessively. It is discussed whether an extremely high intracellular iron uptake is responsible for atypical meningioma growth. Derangement of mitotic spindles may lead to polyploid chromosome sets and thereby to giant nuclei. This seems likely because induction of tumor growth after iron injection has been described. Spodographic examinations showed that even histologically inconspicuous tumor cells contain fine-grained iron. Application of the Prussian-blue reaction to spodograms reveals distinctly higher iron contents than common paraffin sections had proved.

Considering the increasing use of lasers in neurosurgery and the increasing number of wavelengths of laser light becoming available, we evaluated optical properties between 200 and 900 nm of meninges, normal human brain tissue, and brain tumors. We used a two-beam spectral photometer with an integrating sphere as the measuring instrument. The material consisted of 13 brains and 1 specimen each of dura mater, falx, and arachnoid obtained at autopsy and 30 samples of brain tumors removed during operation. In tissue samples more than 5 mm thick, the relative levels of absorption and scattering were estimated from the relative level of reflection measured according to the Kubelka-Munk theory. In thin tissue slices, penetration depth was calculated according to Beer's law from measurements of reflection and transmission. Generally, in all tissues there was an increase of reflection, scattering, and penetration depth and a decrease of absorption from the ultraviolet up to the near infrared spectral range interrupted by the absorption bands of hemoglobin. Within the ultraviolet spectral range, no major differences of optical properties were observed. Within the visible and near infrared spectral range, white matter reflected most of the incident power and showed the lowest level of absorption and the shortest penetration depth. Low grade gliomas revealed optical properties similar to those of gray matter. In comparison with normal brain tissue, meningiomas and glioblastomas showed significantly higher levels of absorption calculated according to the Kubelka-Munk theory from reflection measurements in thick tissue samples, but also deeper penetration obtained from measurements of reflection and transmission in thin slices, especially within the near infrared spectral range.

The presence of progesterone receptors in meningioma tissue is demonstrated by use of highly specific monoclonal antibodies against the rabbit progesterone receptors which cross-react with human progesterone receptors in breast cancer cells, thus giving evidence of the existence of genuine progesterone receptors in human meningiomas.

Normal human brain nuclei and nuclei obtained from glioblastoma, as well as other primary brain and metastatic tumors, were analyzed for their sodium content. The greatest concentration of sodium was found in glial tumor nuclei with lesser amounts in normal glial nuclei and least amounts in nuclei from nonglial tumors. This data supports our hypothesis that glial nuclei sequester brain sodium and also suggests that sodium in the nucleus may be important in glial mitogenesis.

The expression of muscarinic binding sites was examined in a collection of primary brain tumors of different cellular origins and various degrees of dedifferentiation, as compared to control specimens. Eleven gliogenous tumors were examined, all of which contained substantial amounts of muscarinic binding sites. Most of the other tumor types examined did not display detectable binding of [3H]N-methyl-4-piperidyl benzilate ([3H]4NMPB). Scatchard analysis indicated the existence of homogeneous antagonist sites in both normal forebrain and glioblastoma multiforme, with Kd values of 1.2 nM and 0.9 nM, respectively. The density of muscarinic binding sites varied between tumors from different patients, and also between specimens prelevated from different areas of the same tumor. This variability, as well as the average density of binding sites, appeared to be larger in highly malignant tumors than in less malignant ones. In contrast, the density of muscarinic receptors from control specimens was invariably high, but within the same order of magnitude. To test whether the muscarinic binding activity in the brain tumors is correlated to other cholinoceptive properties, cholinesterase activity was also examined. Individual data for density of [3H]4NMPB binding sites were then plotted against corresponding values of cholinesterase activity. The pattern of distribution of these values was clearly different in tumor specimens, when compared to that observed in samples derived from non-malignant brain. Our observations indicate that human brain cells of gliogenous origin are capable of expressing muscarinic binding sites, and that, if a correlation exists between muscarinic receptors and cholinesterase levels in gliogenous tumors, it differs from that of non-malignant brain tissue.

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.

Meningiomas possess features indicative of epithelial differentiation. The present study further documents this finding by a multimodality approach on serial sections from 25 meningiomas of all histologic and clinical categories. Standard light microscopic examination, immunohistochemical staining with a variety of epithelial and nonepithelial markers, and histochemical staining for mucin were performed on each case. Directed electron microscopic examination was performed on selected examples of epithelial feature-positive and -negative cases. All cases were immunoreactive for vimentin and epithelial membrane antigen. Thirty-two percent were reactive for cytokeratin and 39% for S-100. Twenty percent showed mucin positivity by histochemistry. Electron microscopic examination of cytokeratin-positive cases showed either intracellular lumina (secretory areas) or cytoplasmic tonofibrils, whereas cytokeratin-negative tumors lacked lumina and tonofibrils. Previous studies showing epithelial features in meningiomas are reviewed with emphasis on the studies using immunohistochemistry. These histochemical, immunohistochemical, and ultrastructural characteristics are of utility in the examination of tumors of suspected meningeal origin, particularly when dealing with atypical presentations or histomorphologic features.

The availability of [125I]estradiol and [3H]R5020, a synthetic gestagen, have made it possible to determine estradiol and progesterone receptors simultaneously in 70 meningiomas with a double-labeling assay. With this technique, complete Scatchard analysis can be carried out for both receptors, even with small amounts of tumor tissue such as biopsy specimens. Low levels of estradiol receptor reactivity were found in 11%, whereas specific progesterone receptors were demonstrated in 76%, of the meningiomas examined. No correlation of receptor concentration with sex or age of the patients or with location of the tumor has yet been demonstrated. However, indications of a correlation between gestagen binding activity and histological subtypes were found. The presence of progesterone receptors in a large proportion of meningiomas could have implications for tumor therapy.

Bovine papilloma virus (BPV) DNA was labeled in vitro under conditions of repair synthesis and subsequently used as a "probe" in DNA-DNA reassociation studies to detect BPV-specific DNA sequences in a viral-induced calf meningioma and hamster fibroma. In vitro labeled BPV DNA had denaturation characteristics expected for duplex DNA and denatured DNA reassociated with apparent second-order kinetics. Analysis of in vitro labeled BPV DNA reassociation rates in the presence of excess tumor DNA revealed that the calf meningioma contained approximately 700 to 800 BPV genome equivalents per diploid cell whereas the hamster fibroma contained about 150 incomplete BPV genome equivalents per diploid cell. Thermal denaturation of in vitro labeled BPV DNA which reassociated in the presence of the two tumor DNA preparations indicated less than 1.5% base pair mismatching.

Fat within meningiomas is histologically not uncommon. Focal areas of low density on computed tomography scans within meningiomas have been demonstrated and are most often intratumoral cysts. The case presented demonstrates a focal area of low density within a meningioma that is proved histologically to be fat.

Flow cytofluorometric analysis was used to determine the distribution of the DNA content in cells from selected areas of normal human brain and in benign and malignant brain tumors. Propidium iodide was employed as DNA fluorochrome and the analysis was carried out on a suspension of single cells. Normal, nonstimulated human lymphocytes were used as diploid controls. With nonneoplastic tissue an average of 91% of the cells were diploid (presumably in G0 or G1 stage of the cell cycle). The cells of most benign tumors were mainly diploid (77-98%), nine specimens of pituitary adenomas had large numbers of aneuploid cells. In glioblastoma multiforme the proportion of diploid cells was significantly diminished and polyploid cells were frequently seen. Similar results were obtained in other malignant tumors, with metastatic tumors showing the greatest ploidy variation, which included triploid, tetraploid, and hypertetraploid cells. The analytical method used provides valuable information of significant clinical importance on the DNA distribution in brain tumor cells.

Using immunocytochemical methods, we localized several glycoproteins of the extracellular matrix to leptomeningeal cells and meningiomas in vitro. Three cell lines derived from normal human leptomeninges and seven from meningiomas were studied by indirect immunofluorescence to evaluate the cellular production of fibronectin, laminin, collagen type IV, and procollagen type III. All leptomeningeal cell lines stained intensely and uniformly for all matrix proteins; all meningioma cell cultures stained uniformly, but the intensity of staining varied considerably. After removal of the cells in culture adherent to glass with 25 mM ammonium hydroxide, indirect immunofluorescence demonstrated an exuberant residual extracellular residue enriched with fibronectin, laminin, collagen type IV, and procollagen type III. Electron microscopic examination of all leptomeningeal and meningioma cultures revealed desmosomes and dense tonofilament formation; in addition, granular, filamentous basement membrane-like material was abundant in the extracellular spaces of all cultures. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the cell layer of two leptomeningeal and four meningioma cultures showed production of interstitial collagen types I and III; diethylaminoethyl (DEAE)-cellulose chromatography of the medium demonstrated preferential production of procollagen type I. Our findings show conclusively that normal arachnoid cells in vitro synthesize several of the collagen subtypes and may be responsible for the "fibrous response" of the leptomeninges to trauma, infection, or infiltration by tumor. The similarities between leptomeningeal cells and meningiomas demonstrated by electron microscopy and by indirect immunofluorescence support the notion that meningiomas are derived from arachnoid cells. The localization of various mesenchymal glycoproteins within the intra- and extracellular spaces and the ubiquity of specialized intercellular junctions suggest that leptomeningeal cells in culture have the potential to behave like both stromal and epithelial cells.

Tumors were induced in experimental animals in order to investigate early tumor growth with conventional histology and gliofibrillary acid protein (GFAP) demonstration with the peroxidase-antiperoxidase (PAP) method. In one experiment, the animals were killed after transplacental ENU administration, when microtumors were suspected; in the second experiment, the animals were followed up to their natural death and microtumors found at random were used for analysis. Conventional histology revealed 3 types of microtumors: growth restricted to the subventricular matrix, growth in the neighbourhood of the ventricles with obvious or probable connection to the ventricular zones and small tumors observed exclusively in the white matter. The latter tumors by conventional staining were composed of small round cells, considered to be oligodendrocytes. They did not contain GFA-protein positive cells within the tumor. The tumor in presumable and visible connection with the ventricular lining did contain GFAP-positive astrocytes. In the very small subventricular tumors, the small round cells (oligodendrocytes) were in continuous contact with the identical interfascicular glial cells, while GFAP positive astrocytes seemed to stem from the subventricular astrocytes (tanycytes, ependymoglia). The ependyma itself was always preserved. A twofold origin of these experimental tumors with probable development into one common cytological glial type is assumed.

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.

Recently the presence of steroid hormone receptors (SHR) have been reported in a series of nervous system human tumors, mainly in meningiomas. The possibility of characterizing brain tumors deserves attention because of the poor knowledge of endocrine-related behaviour of such tumors. The reliability of biochemical characterization of various receptor types should allow for better understanding of biological profile of primary and metastatic brain tumor, together with new avenues for combined modality treatment. In the future the highest priority might be assigned to the definition of the cut-off value of positivity for single receptors and to the receptor-endocrine modulation complex relationship for individual patients. A clear definition of the role of SHR in human brain tumors might improve our therapeutic potentials.

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.

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.

Cell proliferation potential was assessed by measuring the labeling indices of the monoclonal antibody Ki-67 and of 5-bromodeoxyuridine (BUdR), and the cellular deoxyribonucleic acid (DNA) content in 48 human brain tumors. The diagnostic and prognostic value of flow-cytometric DNA analysis was also evaluated using ethanol-fixed paraffin-embedded BUdR-labeled specimens; these were the same specimens as were used for measuring the BUdR and Ki-67 labeling indices. Both the Ki-67 and the BUdR labeling indices correlated with the degree of malignancy estimated from conventional histological preparations. The Ki-67 labeling index was 1.7 times greater than the BUdR labeling index. The relationship of DNA aneuploidy to the labeling indices or to morphology in cases of glioma was examined. All of the tumors with an aneuploid line corresponded to malignant glioma classified by histological criteria, although malignant glioma did not always show DNA aneuploidy. In addition, the cases with aneuploid lines showed high BUdR and Ki-67 labeling indices. The cell kinetic data, which indicate the biological character of tumors, allowed prediction of the prognosis of the patients with gliomas. In contrast, despite the presence of an aneuploid line, three of 13 meningiomas showed a benign histological pattern without an aggressive clinical course, and neither the Ki-67 nor the BUdR labeling index was high. These results indicate an unequivocal relationship between DNA aneuploidy and clinical behavior; in general, both labeling indices may prove to be objective indicators of the outcome of patients with brain tumors.

The occurrence and the distribution of GAGs have been studied histochemically in 224 human cerebral tumors by means of Alcian blue techniques. In the normal peritumoral gray matter the alcianophilia is stronger than in the white matter and demonstrated the presence of HA and CS. In the glioma group the alcianophilia, due to HA and CS, is mainly related to the presence of infiltrated cortex. In the other tumors, GAGs are histochemically disclosed in relation to collagen, reticulin, mesodermic areas, etc. The vessels of every tumor show a positive staining for HA, CS and HS. The histochemical findings are consistent with the biochemical ones as reported in Part I, even though the significance of GAGs in cerebral tumors remains unknown.