GFA protein reactivity in nerve sheath tumors: a polyvalent and monoclonal antibody study.

We studied glial fibrillary acidic (GFA) protein immunoreactivity in 30 schwannomas, including two intracerebral examples, 26 neurofibromas and 12 neuromas using the immunoperoxidase method with a polyvalent antiserum (PVAS) and three well-characterized monoclonal antibody (MAb) preparations. Twelve of the schwannomas, including both intracerebral tumors, two of the neurofibromas and none of the neuromas immunostained with PVAS. Except for one schwannoma, all the PVAS-positive tumors were positive with two of the MAb preparations. While both of the intracerebral schwannomas were positive with the third MAb, none of the extracerebral tumors were. Our results suggest that: 1) human nerve sheath tumors contain cells having polypeptides that share epitopes with GFA protein, but 2) these polypeptides differ from astrocytic GFA protein by at least one epitope, and 3) the location of the tumors in relation to the central nervous system may influence GFA protein immunoreactivity.

Band 1 and voltage-sensitive Na+ channels are not codistributed in cultured rodent neuronal cells.

The relationship of the mouse nervous system specific band 1 protein to the putative high molecular weight component of the Na+ channel was investigated using antibody to band 1. Morphologic differentiation of cultured neuroblastoma cells has been reported to increase the quantity of the putative Na+ channel high molecular weight component. Morphologically differentiated clone NB2a neuroblastoma cells have 2-3 times the amount of band 1 and 1.5 times the relative rate of synthesis of band 1 as undifferentiated cells. The anti-band 1 serum reacts with both adult mouse and rat brain but not 3 cultured rat neuronal lines known to have active Na+ channels. Thus either band 1 is not a component of the Na+ channel or individual cultured murine neuronal lines has distinct macromolecular Na+ channels.

Growth-promoting factors in neurofibroma crude extracts.

Crude extracts of neurofibromas from two unrelated neurofibromatosis (NF) patients were prepared by mincing, homogenizing, and ultracentrifugation in the absence of added solvents. Explant cultures of neurofibromas from other NF patients were grown at low density in culture medium with and without neurofibroma extract supplementation. Differences in growth were monitored by comparing monolayer densities, colony counts, or uptake of [3]H-thymidine. A consistent enhancement of growth rate was demonstrated, and titration curves showed an increasing effect with increasing dosage (ranging from 1.5 microliter/ml to 25 microliters/ml). However, the extract could not substitute for fetal bovine serum. As determined by microscopic examination of Giemsa-stained petri dishes, small spindle-shaped cells, distinct in morphology from ordinary fibroblasts, were the overwhelmingly predominant cell type in most extract-treated cultures. While the specific identity of the growth factor(s) involved is unknown, the following may be stated: The presence of one or more growth factors that may act in an autocrine or paracrine manner in neurofibromas in vivo is demonstrated. There is a preferential effect of such a factor on spindle-shaped cells (presumably Schwann cells), allowing for the selective enrichment of these cells in vitro. There is an enhanced yield of clones derived from single cells, allowing further analysis of the cellular heterogeneity of neurofibromas at the biochemical and molecular levels. These considerations should help to distinguish between those models for neurofibroma growth that emphasize secondary somatic mutations (including allelic exclusion) on the one hand, and cellular interaction on the other hand.

Immunohistochemical diagnosis of nervous system neoplasms.

Classic histopathologic study revealed a series of highly treasured criteria for diagnosing central and peripheral nervous system neoplasms. The advent of immunohistochemistry galvanized further improvements in the accuracy of diagnostic neurooncologic pathology, and moreover, enriched our understanding of tumor histogenesis. Now with the tremendous technical advances in immunodetection and the commercial availability of high-quality monoclonal and polyclonal antibodies, the potential for first-rate diagnostic assessment is available to practically every laboratory, regardless of how small or remotely located. Intensive immunohistochemical analysis of nervous system neoplasms has taught us that neuroectodermal cells have a limited repertoire of highly redundant antigenic phenotypes and that neoplastic cells usually mimic their non-neoplastic immature or fully differentiated counterparts. These observations led to the concept that a panel of antibodies ought to be used to identify the "immuno-gestalt" of tumors, because it is often difficult if not impossible to subcategorize the tumors on the basis of a single immunohistochemical determination. The ensuing tour through the immunodiagnostic neurooncology summarizes approximately 10 years of original work in this field. I have attempted to provide consensus opinions, and whereever appropriate, highlight dissenting arguments. The salient immunohistochemical features of most nervous system tumors also are summarized in tabular form to facilitate diagnostic considerations at your own institutions.

Cellular distribution of cathepsin D in childhood tumors.

Cathepsin D is an acidic endopeptidase that has been found in the cytoplasm of neurons and other cells. To test the hypothesis that the cellular content or distribution of cathepsin D may serve as a marker of neuronal differentiation in childhood neoplasia, we performed an immunocytochemical study of the cathepsin D content of tumors that occurred in 49 pediatric patients. We found diffuse, moderate to heavy staining primarily in differentiating neurogenous tumors and minimal staining in one Wilms' tumor, one Ewing's sarcoma, and one rhabdomyosarcoma. Benign reactive histiocytes stained intensely in most tumors. We concluded that although the presence of cathepsin D in malignant cells is not diagnostic of neural tumors, it may serve as a marker for cellular maturation in neuroblastomas and as a marker of macrophage infiltration in human tumors.

Central nervous system lymphomas. Immunohistochemical and clinicopathologic study of 26 autopsy cases.

Twenty-six malignant lymphomas involving the central nervous system were studied. Eleven were primary (P) and 15 were systemic (S). Eight cases (3 P, 5 S) occurred in immunocompromised patients. Age at presentation in immunocompromised patients was typically younger than in the nonimmunocompromised patients. Presenting complaints of central nervous system involvement included headache, seizures, personality changes, memory lapses, ataxia, cranial nerve symptoms, and impaired consciousness. Cerebrospinal fluid involvement was seen only in 3 S cases. In 8 of the P cases, the diagnosis was first established at autopsy; in 6 of the S cases, central nervous system involvement was first documented at autopsy. Survival was longer in treated patients than in those who received no therapy (5 months in P cases and 9.3 months in S cases; 2.3 months without therapy). Regardless of therapy, the average survival of immunocompromised patients was 2.4 months. The majority of cases were multifocal. Of the P cases, 1 was of low histologic grade, 9 were of intermediate grade, and 1 was of high grade. Of the S cases, 5 were of low grade, 9 were of intermediate grade, and 1 was of high grade. Immunophenotypic studies were performed on formalin-fixed, paraffin-embedded tissue with antisera against common leukocyte antigen (all reactive), B-cell markers (L26, MB2, LN1, and LN2), T-cell markers (UCHL1 and MT1), Leu-M1, Leu-7, and HLA-DR (LN3). Two S cases were of T-cell phenotype; all others were of B-cell derivation. Eleven cases were HLA-DR positive (all of B-cell phenotype). One T-cell lymphoma was reactive for Leu-7. All cases were nonreactive for Leu-M1. All cases in immunosuppressed patients and all P cases were of B-cell phenotype.

Immunohistochemical evaluation of Leu-7, myelin basic-protein, S100-protein, glial-fibrillary acidic-protein, and LN3 immunoreactivity in nerve sheath tumors and sarcomas.

The collective expression of five antigens produced in immature or mature myelin-producing glia was evaluated in nerve sheath tumors and spindle cell sarcomas with histologic features of schwannomas. Myelin-associated glycoprotein (Leu-7), myelin basic-protein (MBP), S100-protein, and, in most cases, glial-fibrillary acidic-protein (GFAP) and HLA-DR/Ia (LN3) immunoreactivity were evaluated immunohistochemically using commercially available antibodies on 53 benign nerve sheath tumors and 12 sarcomas. Leu-7 immunoreactivity was detected by a monoclonal antibody in 12 of 16 schwannomas, 12 of 20 neurofibromas, and 17 of 17 traumatic neuromas. No Leu-7 positivity was seen in the sarcomas. Distinct heavy MBP immunoreactivity, assessed using polyclonal antibodies, was identified only in all 17 traumatic neuromas. Extensive S100-protein positivity was seen in 15 of 16 schwannomas, 17 of 20 neurofibromas, and 17 of 17 traumatic neuromas. Extensive LN3 immunoreactivity was seen in Schwann cells of 50% of the nerve sheath tumors analyzed; however, it was also present in associated interdigitating reticulum cells; GFAP immunoreactivity was not detected. These data suggest that Leu-7 is an important marker of Schwann cell neoplasms, although it is not superior to S100 protein. Moreover, combined immunohistochemical evaluation of potential Schwann cell markers including Leu-7, MBP, GFAP, and LN3 using commercially available antibodies offers no advantage over analysis of S100-protein immunoreactivity alone.

Needle aspiration cytology of a benign and a malignant schwannoma.

The histologic and cytologic features of an uncommon solitary, malignant schwannoma and a benign schwannoma are presented. The benign tumor revealed characteristic interlacing fascicles of spindle-shaped cells on both histology and cytology. The malignant schwannoma produced a variable histologic pattern that was selectively sampled from a recurrence in a scar; only obviously malignant, undifferentiated, spindle-shaped cells not capable of further characterization were seen on cytology. Preliminary experience with immunoperoxidase staining for the neural crest marker S-100 protein has been encouraging and may permit identification of these tumors on aspiration smears.

[Nervous system tumour proteins. G.F.A. and hyaluronectin (author's transl)]. / Les proteéines des tumeurs du système nerveux. G.F.A. et hyaluronectine.

G.F.A. (gliofibrillary acid protein) and hyaluronectin studied here are two antigens defined by the heteroantibodies which appear in the serum of the immunised animal. G.F.A. is associated with gliofilaments and is considered as an astrocyte marker, either normal or tumoral. Hyaluronectin, is found in the Ranvier nodes and at the periphery of neurones. It is not specific to the nervous system. It is found in all tissues and tumors, and in particular marks the intercellular spaces of gliomas, where it is associated with hyaluronic acid for which it has a great affinity. Serum estimation of these Proteins is being undertaken with the view to a clinical application.

[Detection of glial fibrillary acidic protein in central nervous tumors using an immunohistochemical method. Preliminary study of 33 cases (author's transl)]. / Le diagnostic des tumeurs nerveuses centrales par un marqueur de la protéine gliofibrillaire acide. Etude préliminaire d'une série de 33 cas.

Thirty-two cases of human central nervous tumor and one experimental glioma were studied in fixed paraffin or epon embedded tissues using the peroxidase-antiperoxidase method. The present study confirms the usefulness of immunohistochemical methods for the diagnostic evaluation of neuro-epithelial neoplasms. The authors also include some prognostic and histogenetic comments about glial tumors.

Synaptophysin expression in neuroendocrine neoplasms as determined by immunocytochemistry.

Synaptophysin is an integral membrane glycoprotein originally isolated from presynaptic vesicles of bovine neurons. The authors have studied a wide spectrum of neuroendocrine (NE) neoplasms by immunofluorescence microscopy on cryostat sections of freshly frozen tissues using a monoclonal antibody to this protein (SY 38). Without exception, they found the identical--or a very similar--protein expressed in all neuroblastomas, ganglioneuroblastomas, ganglioneuromas, pheochromocytomas, and paragangliomas studied. In these "neural" type NE neoplasms, synaptophysin was coexpressed with neurofilament proteins. Synaptophysin was also demonstrated in NE neoplasms of "epithelial" type in which it was predominantly coexpressed with cytokeratins and desmoplakin. It was invariably found in all variants of islet cell neoplasms and in all medullary thyroid carcinomas. Synaptophysin was also demonstrated in several adenomas of the hypophysis and parathyroids, in the majority of carcinoids of the bronchopulmonary and gastrointestinal tracts, and in many, though not all, NE carcinomas of the same sites, and of the skin. Conversely, SY 38 did not immunostain any of a large number of benign and malignant non-NE epithelial neoplasms; nor was any immunostaining obtained in a group of mesenchymal tumors. It is remarkable that SY 38 did not immunostain a number of malignant melanomas, including several that were immunostained for neuron-specific enolase (NSE) and several neuropeptides. Parallel studies conducted on conventionally fixed, paraffin-embedded tissue sections immunostained by the use of the avidin-biotin complex technique yielded very similar results. The findings indicate that synaptophysin is expressed in the whole range of NE neoplasms without detectable relation to the expression of other NE markers such as NSE, serotonin, and neuropeptides. Nor could the expression of synaptophysin by these tumors be correlated with their epithelial and/or neural cytoskeletal characteristics, their clinical aggressiveness, or the presence or absence of endocrinologic abnormalities. While the consistent expression of synaptophysin by the "neural" type of NE neoplasms would seem predictable its presence in diverse benign and malignant NE tumors of "epithelial" type is remarkable. It is concluded that synaptophysin is a significant as well as novel NE marker, and the use of antibody SY 38 as a broad range marker for the study and diagnosis of NE neoplasms is proposed.

Transferrin receptor expression in tumours of the human nervous system: relation to tumour type, grading and tumour growth fraction.

The expression of transferrin receptor (Tr) was investigated by means of immunohistochemistry in 101 tumours of the human central and peripheral nervous system. The results were compared with the proliferative activity of the tumours, determined by immunostaining for the proliferation-associated antigen Ki-67. In addition to immunostaining of normal and proliferated blood vessel endothelium and of a fraction of tumour infiltrating lymphocytes, we observed staining for Tr in a variable fraction of neoplastic cells of all histological tumour types. Immunoreactivity in the majority of tumour cells was found only in anaplastic tumours such as glioblastomas. Furthermore, a positive correlation between Tr expression and the Ki-67 growth fraction was established for gliomas. Non-glial tumours strongly expressing Tr included one metastatic rhabdomyosarcoma, one intracerebral malignant lymphoma, two of four plasmocytomas and seven of nine metastatic carcinomas. Our results indicate that immunohistochemistry for Tr and Ki-67 can provide additional information about the biological behaviour of nervous system tumours, thus complementing conventional histopathological criteria for anaplasia.

Immunohistopathologic spectrum of the glial fibrillary acidic protein in neurooncology.

To conventional histologic and special staining techniques as well as electron microscopy, immunohistological methods have recently been added to characterize neuroectodermal and non-neuroectodermal tumors of both the central and the peripheral nervous system. An ever increasing panel of these morphologic techniques enables us today, to characterize precisely such tumors in or around the central and peripheral nervous system, sometimes only hampered by preservation of tissue inadequate for the respective methods.

Value of S-100 protein in the diagnosis of soft tissue tumors with particular reference to benign and malignant Schwann cell tumors.

Two hundred and two benign and malignant soft tissue lesions were studied for the presence of S-100 protein by means of the peroxidase-antiperoxidase technique on formalin-fixed, paraffin-embedded tissue. Virtually all benign nerve sheath tumors (neurofibroma, neurilemoma, and granular cell tumor) contained numerous immunoreactive S-100-positive cells. Only one-half (18 of 36) of malignant schwannomas contained the protein, suggesting that its presence is an expression of differentiation in Schwann cell tumors. S-100 protein was not identified within pure neuroblastic tumors (neuroblastoma, neuroepithelioma) but could be identified within rare cells of the ganglioneuroblastoma and within the Schwann cell component of ganglioneuroma. It was also identified within most melanocytic tumors (cellular blue nevus, clear cell sarcoma, and melanoma). In fact, its constant presence in melanoma indicates that it may prove to be an independently reliable method for diagnosing amelanotic forms. It is also sporadically present within a variety of mesenchymal lesions including lipoma, liposarcoma, synovial chondromatosis, chondrosarcoma, fibromatosis, histiocytosis X, and chordoma. Although S-100 protein is highly characteristic of neural crest-derived tumors, it is not restricted to them and, consequently, must be interpreted cautiously. It may prove helpful in select situations such as the distinction of (a) benign nerve sheath tumors from other benign mesenchymal tumors such as fibrous histiocytomas, (b) cellular neurilemomas from malignant schwannomas, (c) malignant schwannomas from conventional fibrosarcoma (d) malignant melanomas from many carcinomas, and, possibly (e) juvenile xanthogranulomas from histiocytosis X.

S-100 protein: is it useful as a tumour marker in diagnostic immunocytochemistry?

A heterogeneous group of 159 tumours was studied for the presence of S-100 protein by the immunoperoxidase technique in order to determine whether this marker may be of value in facilitating immunocytochemical diagnosis. Among cases of melanocytic and pigmented lesions, S-100 was widely distributed and demonstrated the strongest degrees of reactivity. S-100 protein was identified in virtually all nerve sheath tumours such as schwannomas, neurofibromas, myxoid sheath nerve tumour and also in some tumours of controversial histogenesis such as granular cell tumours. The great majority of carcinomas did not express S-100, with only two cases of breast carcinoma displaying focal S-100 staining. In a miscellaneous group of tumours S-100 was demonstrated in chordomas, myoepitheliomas and Wilms' tumour with Schwann cell differentiation. Despite its presence in a wide array of cell types, S-100 protein continues to be an extremely useful marker especially for soft tissue and peripheral nervous system tumours.

Cytokeratin and laminin immunostaining in the diagnosis of cutaneous neuro-endocrine (Merkel cell) tumours.

Nine cutaneous neuro-endocrine tumours have been immunostained with monoclonal antibodies to low molecular weight cytokeratin (CAM 5.2) and neurofilament. Polyclonal antisera to neurone-specific enolase, calcitonin and laminin were also used. All nine cases showed paranuclear, dot-like positive staining with CAM 5.2 and diffuse cytoplasmic staining for neurone-specific enolase. Neurofilament and calcitonin immunoreactivity could not be demonstrated. All tumours were negative for laminin immunoreactivity. The limitations of staining for neurone-specific enolase are discussed and the value of CAM 5.2 in the differential diagnosis of cutaneous neuro-endocrine tumours is emphasized. The histogenetic implications of the absence of laminin staining are considered.

An immunocytochemical demonstration of calcineurin in human nerve cell tumors. A comparison with neuron-specific enolase and glial fibrillary acidic protein.

Human central and peripheral nerve cell tumors were examined in detail using antibodies to calcineurin, glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE). Forty-eight formalin-fixed and paraffin-embedded specimens of human neuronal tumors, including 27 medulloblastomas, were examined. Calcineurin-positive cells were found in all peripheral nerve cell tumors and the two gangliogliomas, whereas 20 of the 27 medulloblastomas and one of the two cerebral neuroblastomas did not contain calcineurin-positive cells. Differentiation of cells along the neuronal lines was positively correlated with calcineurin immunoreactivity. NSE-positive cells were found in all of the tumors with the exception of the one cerebral neuroblastoma. NSE immunoreactivity was not invariably consistent with calcineurin immunoreactivity and non-neuronal cells were often positive. Calcineurin-positive cells were all devoid of GFAP, but NSE-positive cells expressed GFAP in some tumors. GFAP-immunoreactive cells were found only in central nerve cell tumors, and not in peripheral tumors. In addition, GFAP-positive cells in some tumors such as retinoblastoma and medulloblastoma morphologically revealed not only neoplastic but also reactive astrocytic features.

Distribution of endothelial and basement membrane markers in angiogenic tumors of the nervous system.

The distribution of two endothelial cell markers Factor-VIII-related antigen and Ulex europaeus agglutinin was examined by immunoperoxidase and immunofluorescence techniques in paraffin-embedded specimens representing the three main types of angiogenic neoplasms of the nervous system, hemangioblastoma, hemangioendothelioma and hemangiopericytoma. In addition, the distribution of the basement membrane (BM) marker, laminin, was studied in the same tumors. It was found that Ulex europaeus agglutinin was a more sensitive marker of neoplastic endothelial cells than Factor-VIII-related antigen. Both markers only stained endothelial cells, while the tumor cells of hemangiopericytomas and the stromal cells of hemangioblastomas remained unstained. These findings do not support the view that the stromal cells of hemangioblastomas are derived from endothelial cells. With antiserum to laminin a typical staining pattern could be noticed in each tumor, showing the architectural relationships of the cells very clearly. In all three tumor types laminin was only found in the BM of the vessels, not in the interstices of the neoplastic cells outside vessel lumina. Therefore, the reticulin network previously found between the individual cells of hemangiopericytomas does not correspond to BM. It is concluded that both Ulex europaeus agglutinin and laminin antisera could be valuable new aids for the diagnosis of the three tumor types.