Gliosis and glioma distinguished by acridine orange

Acridine orange fluorochrome of nucleic acids was applied to sections of cerebral tissue from 20 patients showing acute or chronic reactive gliosis. The results were compared with the findings in 39 well differentiated and malignant astrocytomas. The orange cytoplasmic fluorescence of ribonucleic acid is lacking in reactive astrocytes of all ages including gemistocytes, but is uniformly present in astrocytoma cells. Acridine orange is a useful supplementary stain for distinguishing between astrocytosis and astrocytoma, particularly for small cerebral biopsies showing scattered or diffusely infiltrating pleomorphic glial cells.     

Fluorescence histochemistry of RNA in human pituitary adenomas

Summary Acridine orange (AO)-RNA fluorescence was studied histochemically in 9 normal human pituitary glands, in 26 secretory and nonsecretory pituitary adenomas, and in a dysgerminoma. Six adenomas showing immunoreactivity for prolactin showed intense orange-red cytoplasmic fluorescence; 6 other tumours exhibiting immunoperoxidase activity of growth hormone showed less intense AO-RNA fluorescence, and 5 adrenocorticotropic hormone-containing adenomas showed still weaker orange fluorescence. Among the chromophobe adenomas without immunoreactivity for secretory products, 5 had uniformly very weak AO-RNA fluorescence, while in 4 other a few scattered cells with strong AO-RNA fluorescence were detected among the majority of weakly fluorescent cells. The pituitary dysgerminoma contained many cells with strong AO-RNA fluorescence. Electron microscopy of these cases showed good correlation of cytoplasmic concentration of ribosomes with AO-RNA fluorescence of the adenomas. AO is useful as an inexpensive, simple supplementary stain for frozen or paraffin sections of pituitary tumours to infer secretory activity as a correlate of RNA concentration and to demonstrate a secretory potential in some cells of hormonally inactive tumours.Supported by a research grant from the Alberta Children's Hospital Foundation, Calgary (HBS)     

Immunolocalization of cathepsin D in the human central nervous system and central nervous system neoplasms

The cellular distribution of the lysosomal proteinase cathepsin D was studied in a series of 76 neoplasms and 18 non-neoplastic tissues from the human central nervous system, using a well-characterized polyclonal antibody in a peroxidase-antiperoxidase technique. In the normal and developing brain, cathepsin D is confined to neurons and choroid plexus epithelium. Strong granular cytoplasmic staining was present in neuronal and choroid plexus neoplasms, and in reactive macrophages. A large variety of other neoplasms also exhibited positive cytoplasmic staining, albeit usually of a weaker diffuse type. Cathepsin D cannot be considered a specific marker for neuronal or choroid plexus neoplasms, but the antiserum used in this study may be of value in antibody panels for the investigation of these tumours. Its localization may also be of value in embryological studies, particularly in the cerebellum, and in investigations of steroid hormone receptor-associated proteins in meningiomas and Schwannomas.     

Use of the silver nucleolar organizer region (AgNOR) technique in the differential diagnosis of central nervous system neoplasia.

Because the distinction between gliosis and low-grade astrocytoma may prove difficult by routine light microscopy, we evaluated the silver nucleolar organizer region (AgNOR) technique in making this distinction. The AgNOR impregnation was performed on formalin-fixed, paraffin-embedded tissue from 49 central nervous system (CNS) biopsies: eight normal brain, 14 gliosis, 14 grade 2 astrocytoma (Daumas-Duport scale), two grade 4 astrocytoma, nine medulloblastoma, one metastatic carcinoma, and one choroid plexus papilloma. Quantitative and qualitative differences were found between gliosis and low-grade astrocytomas. In gliosis, AgNOR counts averaged 1.18 +/- 0.11 (SD) AgNOR/nucleus, while in low-grade astrocytomas AgNOR counts averaged 2.22 +/- 0.39 (p less than 0.001). Compound AgNOR were frequent in 9/14 grade 2 astrocytomas and in both grade 4 astrocytomas, whereas compound AgNOR were extremely rare in cases of gliosis. Quantitative and qualitative differences were also found between normal cerebellar internal granular cells and medulloblastoma cells. Cerebellar granular cells averaged 0.90 +/- 0.10 AgNOR/nucleus whereas medulloblastoma cells had an average of 4.52 +/- 0.95 (p less than 0.001). Compound AgNOR were seen in all medulloblastomas but not in internal granular cells. These findings suggest that the AgNOR technique may be a useful adjunct in the diagnosis of CNS neoplasia.     

Choroid plexus hemangioma with glial nodules in a premature stillborn study

An unruptured hemangioma (vascular malformation) of the choroid plexus of the lateral ventricle in a premature stillborn baby is described. Along the flattened choroid plexus epithelium covering the hemangioma there were small, discrete glial nodules which stained strongly and diffusely for glial fibrillary acidic protein (GFAP). The glial nodules were devoid of surface epithelium and confined within the basement membrane region of the choroid plexus epithelium. GFAP positive cells were not present elsewhere in the choroid plexus. The histogenesis of the glial nodules, whether they have resulted from glial differentiation of reactive choroid plexus epithelium or represented glial heterotopia, remains uncertain.     

Choroid plexus tumours: epidemiologic comparative study of 24 cases

Tumours derived from choroid plexus are rare central nervous system neoplasms affecting mainly children. This study presents a series of 38 patients with neuroectodermal tumours. Twenty four of them had the histological and/or immunohistochemical diagnosis of choroid plexus carcinoma (CPC). Sixteen of these patients were male. The left lateral ventricle was affected in most cases. Main clinical features were: hydrocephalus, intracranial hypertension and convulsion. All the patients were treated by surgery. There were 2 deaths due to surgical complications. Nine patients had recurrence, dying in a intermediate time of 12.3 months and 7 patients remain alive. The other cases did not present available data. CPC is very prevalent in Curitiba when compared to other choroid plexus tumours and it might be related to some pathogenic agent.     

Co-expression of glial fibrillary acidic protein- and vimentin-type intermediate filaments in human astrocytomas

Summary The expression of intermediate filament (IF) proteins was studied in 71 cases of malignant human astrocytoma and in 17 cases of reactive gliosis, using immunocytochemical techniques with polyclonal and monoclonal antibodies to glial fibrillary acidic protein (GFAP) and vimentin. In all cases of astrocytoma, varying in degree of malignancy from grade I to grade IV, co-expression of GFAP and vimentin was found. No change in vimentin- or GFAP-IF expression with increasing anaplasia was seen. In addition astrocytic cells in reactive gliosis showed simultaneous expression of GFAP and vimentin. The intracellular distribution of these IF proteins differed. Vimentin was found to be located in a more juxta-nuclear position, whereas GFAP immunoreactivity showed a more intense staining of the cellular processes. Astrocytes in reactive gliosis behaved more or less like neoplastic cells. However, thin cell processes of reactive astrocytes in the cortex and superficial white matter only contained GFAP immunoreactivity. Simultaneous expression of GFAP and vimentin and their proportion in malignant and reactive glial cells are discussed in the light of earlier reports on the IF content of glial cells during development and maturation, in which vimentin precedes GFAP-expression. The existence of two separate (functional) IF systems in astroglia is suggested.     

Brain involvement in generalized argyria

Cutaneous argyria was diagnosed in a 59-year-old woman. Manic depressive psychosis developed at about the same or a short time thereafter. The patient died 6 years later from a ruptured aortic aneurysm. At autopsy silver deposits were seen in skin, mucous membranes, heart, kidney, and liver. In the central nervous system the leptomeninges and choroid plexuses contained silver granules. In addition, silver granules were visualized in the walls of many intraparenchymal vessels, particularly of the basal ganglia, hypothalamus, substantia nigra, and cerebellum. Progressive glial changes and cellular gliosis were evident in many areas of the brain. With the electron microscope the deposition of silver granules in basal membrane structures of the choroid plexus and intracerebral vasculature was amply confirmed. Furthermore, silver deposition was seen in brain parenchymal cells inside bodies of apparently lysosomal nature. The silver content of various brain regions was determined by absorption spectrophotometry.     

ELECTRON MICROSCOPIC OBSERVATIONS ON MALIGNANT CHOROID PLEXUS PAPILLOMA

Biopsy material from two cases of histologically proven malignant choroid plexus papilloma was studied by electron microscopy. Both tumours differed from benign papillomas in showing poorly differentiated areas with numerous mitoses. Even in such areas, however, a number of ultrastructural features characteristic of normal choroid plexus tissue were observed in both the cases, including cilia, microvilli, a continuous basement membrane and capillary endothelial fenestration. Such findings may be helpful in establishing the primary origin of malignant tumours in the choroid plexus, especially where light microscopy of poorly differentiated biopsy samples makes diagnosis uncertain.     

Antigenic expression in human choroid plexus carcinoma: report of 2 cases

Primary neoplasms of choroid plexus are rare. Six morphological variants have been described: papillary, cystic, acinar, mucus-secreting, oncocytic, and anaplastic. The anaplastic variant, the so-called choroid plexus carcinoma, is the rarest of all and can metastasize. The differential diagnosis of the anaplastic variant of choroid plexus neoplasms with adenocarcinomas, melanomas and undifferentiated neoplasms can be troublesome chiefly in adults. The now large use of immunocytochemical techniques in tissue section has become a powerful tool in the analysis of cell lineages, tumoral and non-tumoral. Nevertheless, the choroid plexus neoplasms have shown a complex and a somewhat confusing pattern of antigenic expression. In two choroid plexus carcinomas (one localized in the right lateral ventricle from a boy of 1 year and 9 months old, and the other localized in the left lateral ventricle from a girl of 3 years old) the following antigens were searched (using the avidin-biotin-peroxidase complex): glial fibrillary acidic protein (GFAP) with monoclonal and polyclonal antibodies; cytokeratins of 40-50kDa, cytokeratins of 60-70kDA (callus cytokeratin), neuronal specific enolase (NSE) and S-100 protein with monoclonal antibodies. The two neoplasms showed immunoreactivity against NSE, S-100 protein and cytokeratin of 40-50kDA. The neoplasm of the boy exhibited glial differentiation having immunoreactivity against GFAP with monoclonal and polyclonal antibodies.     

CXCR3 expression in human central nervous system diseases

The CXCR3 chemokine receptor, expressed on activated T lymphocytes, is seen within the central nervous system (CNS) in inflammatory conditions where a T-cell response is prominent. However, the distribution of CXCR3 in parenchymal CNS cells is unknown. Using a monoclonal antibody against CXCR3 and post-mortem tissue of patients with and without CNS pathology, we have determined its expression pattern. CXCR3 was found in subpopulations of cells morphologically consistent with astrocytes, particularly reactive astrocytes, and in cerebellar Purkinje cells. It was also detected in arterial endothelial and smooth muscle cells, particularly in areas associated with atherosclerotic plaques. CXCR3-positive astrocytes were particularly prominent in the CNS of HIV-positive patients, in patients with Multiple Sclerosis (MS), in ischaemic infarcts and in astrocytic neoplasms. Immunofluorescence studies of mixed adult primary glial cultures and fetal glial cultures also showed expression of CXCR3 in astrocytes. CXCR3 mRNA was detected in Purkinje cells by in situ hybridization with a CXCR3-specific probe. Thus, the predominant expression of CXCR3 in reactive astrocytes may indicate that it plays a role in the development of reactive gliosis in a variety of infectious, inflammatory, vascular and neoplastic processes in the CNS. The relationship between CXCR3 expression in astrocytes to its expression in Purkinje cells, endothelial cells and smooth muscle cells is yet to be determined.     

Immunolocalization of laminin in neoplasms of the central and peripheral nervous systems

Laminin is a basement membrane glycoprotein that is expressed in vitro by immature and neoplastic astrocytes. The expression of laminin in vivo was examined immunohistochemically in normal adult brain and 90 neoplasms of the central and peripheral nervous systems. In normal adult brain, laminin was detected in the vasculature, arachnoid, pial-glial membrane, and choroid plexus. The vasculature in all 90 tumors demonstrated intense laminin immunoreactivity. Deposits of laminin were observed at the glioma-mesenchymal junction in several neoplasms, but never between or within neuroepithelial cells. The glial basement membrane often remained intact although surrounded on both sides by invasive glioma or medulloblastoma. However, there was always fragmentation and disruption of the glial membrane in adjacent fields. Laminin expression by tumor cells was observed in 10/10 schwannomas, 9/10 fibroblastic meningiomas, 3/19 nonfibroblastic meningiomas, and 3/6 mixed glioma-sarcomas. Laminin expression in the normal nervous system and in neuroepithelial neoplasms corresponds to regions of recognized basal lamina formation, including the junction between glial and mesenchymal elements. Although invasive gliomas are able to break down the pial-glial basement membrane and gain access to the perivascular or subarachnoid space, this membrane often remains intact late in the invasive process and may represent a partial barrier to tumor invasion. Laminin may be a useful marker for schwannomas, fibroblastic meningiomas, and vascular neoplasms of the nervous system.     

Anti-neuronal nuclear autoantibodies, types 1 and 2: their utility in the study of tumors of the nervous system

This is a comprehensive immunohistochemical study of selected archival tumors of the nervous system applying human anti-neuronal nuclear autoantibodies of types 1 and 2 (ANNA-1 and -2), serum markers of paraneoplastic syndromes associated primarily with small cell lung cancer (SCLC). Neither ANNA-1 nor ANNA-2 bound to glial tumors regardless of histological grade and subtype; instead they labeled neurons in overrun normal parenchyma. Central neurocytomas and the neuronal components of mixed glioneuronal tumors were also immunoreactive for both. In addition, varying proportions of tumor cells were stained in dysembryoplastic neuroepithelial tumor, subependymal giant cell astrocytoma (SEGA), tuber and neuroblastoma. All other tumors were nonreactive, namely choroid plexus papilloma, pituitary adenoma, pineocytoma, pheochromocytoma, thymic and pulmonary carcinoid, chordoma, meningioma, schwannoma and metastatic melanoma. SCLC was immunonegative for ANNA-1 and ANNA-2 in paraffin preparations, but displayed strong immunoreactivity for both in frozen sections: this discrepancy was not observed in other tumors studied. In conclusion, the human IgG autoantibodies ANNA-1 and ANNA-2 provide novel tools for studying the cytogenesis of tumors of the nervous system in that they permit the identification of both normal and neoplastic, poorly differentiated and small neuronal cells that may escape detection using commercially available anti-neuronal antibodies. Received: 3 February 1998 / Revised, accepted: 12 March 1998     

Differentiation between reactive gliosis and diffuse astrocytoma by in situ hybridization

The authors examined the use of chromosomal analysis by in situ hybridization to differentiate between nonneoplastic reactive gliosis and astrocytomas in cases in which routine histology was inconclusive. Numerical chromosomal aberrations were found in 80% of low-grade astrocytoma specimens and in none of the reactive gliosis specimens. Aneusomic tumor cells were detected in four of 13 stereotactic samples with an initially inconclusive tissue diagnosis, three of which were later diagnosed as astrocytoma. The in situ hybridization procedure may have additional value in the differential diagnosis of reactive gliosis versus low-grade astrocytoma.     

Growth fraction in human brain tumors defined by the monoclonal antibody Ki-67

Summary The monoclonal antibody Ki-67, which reacts with cells in the active part of the cell cycle, was used to evaluate immunocytochemically the growth fraction in 22 primary brain neoplasms. The percentage of labelled cells reflected the histological grade of malignancy of each neoplasms. High percentage of Ki-67-positive cells were observed in one choroid plexus carcinoma (60%), one primary melanoma of meninges (40%), three medulloblastomas (40%–50%), one anaplastic astrocytoma and six glioblastomas (10%–40%). One ependymoma had 7% positive cells. Rare positive cells (1%) were present in one pilocytic astrocytoma and one ganglioglioma. Except one negative case, the meningiomas (five cases) had values of positivity ranging from 1% to 6%. Two acoustic schwannomas were negative. These results suggest that immunocytochemical staining with the Ki-67 may be a useful method for measuring the growth fraction in brain neoplasms.Supported in part by Associazione Italiana Ricerca sul Cancro and Ministero Italiano della Pubblica Istruzione     

A case of cerebellar hamartoma suggesting abnormal cell migration

Summary A rare case of hamartoma of the left cerebellar hemisphere was recognized in an 11-monthold male infant whose mother had a history of unspecified medication in the early gestational period and had a difficult delivery. A notably large head and marked developmental disorders, like hypotonic cerebral palsy, were observed soon after birth. A computed tomogram revealed an iso-minimally enhanced large mass in the left cerebellar hemisphere, which deformed the fourth ventricle and compressed the right cerebellum, as well as moderate cerebral atrophy. Histologically, the border between the cerebellar cortex and this tumor was not apparent. The main tumor, located in the cerebellar white matter, was composed of numerous scattered Purkinje cell-like neurons and glial cells surrounded by abundant GFAP-positive matrix. The small part of the tumor, located near the choroid plexus, was composed of intensely proliferated capillaries such as in capillary hemangioma, and numerous fibrocytes, which were intermingled with several large Purkinje cell-like neurons and some GFAP-positive glial cells. The cerebellar cortex showed a thin molecular layer with some residual external granular cells, a marked decrease of Purkinje cells and a moderate decrease in the internal granular layer, in which large Purkinje cell-like neurons were scattered. Purkinje cells and large Purkinje cell-like neurons scattered in the internal granular layer, cerebellar white matter and choroid plexus showed positive immunoreactivity for anti-Leu-4 monoclonal antibody, which is known to be a marker for Purkinje cells. These findings suggest that this case had the background of abnormal cell migration caused by some kind of disorder during pregnancy.     

Rarity of JC virus DNA sequences and early proteins in human gliomas and medulloblastomas: the controversial role of JC virus in human neurooncogenesis

JC virus (JCV), the agent of progressive multifocal leucoencephalopathy (PML), exerts an oncogenic effect in several laboratory animal models. Moreover, JCV genomic DNA and early viral protein T-antigen have been detected in various types of human central nervous system (CNS) neoplasms. To further explore this association we have studied paraffin-embedded brain biopsy tissue from 60 neoplasms (55 gliomas and five medulloblastomas) and 15 reactive gliosis cases for the presence of JCV DNA sequences and proteins. Four post mortem cases of HIV-associated PML were used as positive controls. Samples were assessed by polymerase chain reaction (PCR) amplification of early (large T antigen) and late (virion protein 3) sequences and immunohistochemistry (IHC) with both PAb 2024 and anti-SV40 large T antigen monoclonal antibodies. Five cases (three neoplasms and two reactive gliosis instances) showed low viral DNA levels when PCR-tested for VP3 or large T, while no case was immunoreactive for any of the two antibodies used. The four PML cases yielded positive results with both PCR and IHC. Additionally, IHC with both antibodies was applied to a tissue micro-array including 109 CNS tumours and 21 reactive gliosis samples. No immunoreactivity was detected in any of these tissue micro-array samples. The rarity of JCV DNA sequences and early proteins in our brain tumours enriches the controversy over the role of JCV in human neurooncogenesis, whose clarification is in need of further molecular and epidemiologic studies.     

Immunohistochemical profile and chromosomal imbalances in papillary tumours of the pineal region

The histopathology of papillary tumours of the pineal region (PTPR) closely resembles that of ependymomas and choroid plexus tumours. Therefore, immunohistochemical staining profiles were investigated in a series of 15 PTPR. In addition to cytokeratin, synaptophysin and glial fibrillary acidic protein expression, PTPR were examined for the presence of dot- or ring-like epithelial membrane antigen (EMA) immunoreactivity typically encountered in ependymoma, staining for inwardly rectifying potassium channel Kir7.1 and stanniocalcin-1 (specifically expressed in choroid plexus tumours) as well as microtubule-associated protein-2 (MAP-2). Furthermore, comparative genomic hybridization was performed in five PTPR. Cytokeratin was expressed in all PTPR examined, whereas glial fibrillary acidic protein and synaptophysin staining were absent. Dot- or ring-like EMA immunoreactivity was only observed in 1 out of 15 PTPR. Membranous Kir7.1 and cytoplasmic stanniocalcin-1 staining were present in the minority of PTPR (3/15 and 4/15, respectively). In contrast, MAP-2 immunoreactivity was encountered in 13 out of 15 PTPR, but was significantly less frequently observed in a series of choroid plexus tumours (7/37). PTPR mainly presented with chromosomal losses affecting chromosomes 10 (4/5 cases) and 22q (3/5 cases) as well as gains on chromosomes 4 (4/5 cases), 8 (3/5 cases), 9 (3/5 cases) and 12 (3/5 cases). To conclude, the majority of PTPR can be distinguished from ependymomas and choroid plexus tumours by absent staining for epithelial membrane antigen, Kir7.1 and staniocalcin-1 as well as the presence of distinct MAP-2 immunoreactivity. Antibodies directed against these antigens are thus expected to be valuable markers in the diagnosis of papillary tumours located in the vicinity of the third ventricle.