Immunocytochemical demonstration of intermediate filament cytoskeleton proteins in human endocrine tissues and (neuro-) endocrine tumours

Summary The presence and distribution of intermediate filament proteins, such as cytokeratins, vimentin, neurofilament proteins and glial fibrillary acidic protein were assessed immunohistochemically in pituitary adenomas, medullary thyroid carcinomas, endocrine pancreatic tumours, gastric, intestinal and bronchial carcinoids, parathyroid adenomas, pheochromocytomas, paragangliomas and related non-neoplastic tissues. In some cases, immunohistochemical results were correlated with cytoskeletal proteins as analysed by SDS-polyacrylamide gel electrophoresis. Cytokeratin antibodies with broad range of immunoreactivity (i.e. to murine liver cytokeratin component D) reacted with epithelial cells in all non-neoplastic endocrine tissues and related neuroendocrine tumours studied, except for adrenal medulla, pheochromocytoma and paraganglioma, independently of hormone production and biological behaviour. In contrast, antibodies to epidermis-derived cytokeratins failed to stain endocrine tissues and tumours. Paranuclear cytokeratin accumulations were seen in bronchial, gastric, and intestinal carcinoids and seem to be a common feature of neuroendocrine tumours. One-and two-dimensional SDS-polyacrylamide gel electrophoresis of non-neoplastic endocrine tissues and related tumours revealed two major keratin polypeptides corresponding to cytokeratins No. 8 and 18 of the cytokeratin catalog of human cells (Moll et al. 1982). According to this cytokeratin polypeptide composition, endocrine tissues and related tumours conform to the simple type of epithelia. Vimentin-related immunoreactivity was restricted to stromal cells and to folliculo-stellate cells in normal pituitary gland, Schwann cells in carcinoids and satellite cells in normal adrenal medulla and in pheochromocytomas. Neurofilament protein- (70 kD)-antibodies only stained nerve fibers in normal tissues and at the periphery of carcinoid tumour cell complexes, and, to a variable degree, cells in nontumorous adrenal medulla, pheochromocytomas and paragangliomas. Furthermore, neurofilament reactivity was observed along with cytokeratin expression in two bronchial carcinoids.     

Choroid plexus tumors. An immunocytochemical study with particular reference to the coexpression of intermediate filament proteins.

Sixteen choroid plexus tumors (CPTs) have been investigated for the localization of different immunocytochemical markers of epithelial and nonepithelial nature, namely, simple epithelial-type cytokeratins, vimentin, glial fibrillary acidic protein (GFAP), a panepithelial antigen defined by the lu-5 monoclonal antibody (lu-5 antigen), S-100 protein, and epithelial membrane antigen (EMA). Intermediate filament proteins have been identified in paraffin sections of 14 of 16 cases (87.5%). In all these tumors, cytokeratins and vimentin were constantly coexpressed by the neoplastic cells, in a manner similar to that of the cells lining normal choroid plexus. In 7 of these 14 cases, in addition to cytokeratins and vimentin, the neoplastic cells were shown to coexpress GFAP, which is not synthesized by their normal cell counterpart. The appearance of GFAP immunoreactivity in CPTs might be related to an ependymal differentiation of the neoplastic cells, because normal ependyma and ependymomas constantly coexpress GFAP and vimentin. The simultaneous expression of three distinct intermediate filament proteins by the same neoplastic cells is an exceedingly rare phenomenon, which has never been reported by double labeling technique in neoplasms of the central nervous system. Despite the complex antigenic profile of the CPT, which includes immunoreactivity for lu-5 antigen, S-100 protein, and EMA in most of the cases, positivity for three different epithelial markers indicates that these tumors have an epithelial nature. Moreover, the immunocytochemical typing of CPT with the panel of antibodies used in the current investigation allows differentiation from other primary and metastatic central nervous system tumors.     

Monoclonal antibodies to human intermediate filament proteins. II. Distribution of filament proteins in normal human tissues.

Monoclonal antibodies generated against different human intermediate filament (IF) proteins were assayed on fixed, embedded tissue by the biotin-avidin-immunoperoxidase method for evaluation of the tissue specificity of these antibodies. An antibody (43 beta E8) made to fibroblast IF protein stains mesenchymal tissue such as endothelium, histiocytes, stromal fibroblasts, and Schwann cells but does not stain epithelium, skeletal muscle, lymphocytes, or neurons. Three different anti-cytokeratin antibodies decorate epithelium in three unique patterns. One (35 beta H11) stains all nonsquamous epithelium but fails to recognize squamous epithelium. Antibody 34 beta E12 stains the full thickness of squamous epithelium and ductular epithelium but does not react with hepatocytes, pancreatic acinar cells, proximal renal tubules, or endometrial glands. Antibody 34 beta B4 stains only the suprabasal portion of squamous epithelium. None of these three anti-cytokeratin antibodies reacts with nerve or mesenchymal tissue. Two anti-neurofilament antibodies recognize only neurons, failing to react with epithelial or mesenchymal tissue. We conclude that these anti-intermediate filament antibodies can be used as tissue-specific markers. Neoplasms retain the same intermediate filament patterns as the normal parental tissue; therefore, these antibodies can be used as diagnostic aids in surgical pathology.     

Expression of intermediate filament proteins in adrenal cortex and related tumours

The intermediate filament profile of adrenal cortex and its related tumours has been evaluated. Most adrenocortical cells contained cytokeratin 8 and 18 as demonstrated by monoclonal antibodies CAM 5.2, M20, M9 and RGE53. Cytokeratin immunoreactivity was not confined to a functional zone of the adrenal cortex. Only a small number of the adrenocortical cells showed vimentin immunoreactivity. From normal adrenal cortex through adenomas, to carcinomas, there is a progressive decrease or even loss of cytokeratin immunoreactivity and an increase in vimentin immunoreactivity. Aberrant cytokeratin expression was not found in adrenocortical adenomas and carcinomas with the antibodies used. Awareness of the possible absence of cytokeratin immunoreactivity in adrenocortical carcinomas is important whenever antibodies to cytokeratins and vimentin are used for diagnostic purposes in poorly differentiated neoplasms.     

Expression of intermediate filament proteins in thyroid gland and thyroid tumors

The presence of intermediate filament proteins of cytokeratin/prekeratin type and vimentin type was evaluated in non-neoplastic thyroid glands and in different types of thyroid neoplasms. Follicular epithelium of both normal and goitrous thyroids showed a strong reaction with anticytokeratin antibodies that widely cross-react with various simple epithelia. On the other hand, in normal thyroid, there were only occasionally (in one of 12 cases) solitary cells reacting with antibodies to epidermal prekeratin. In nodular goiters, such cells were often seen (eight of 18), especially among the lining cells of cysts, and in chronic thyroiditis in all (12 of 12) cases. Only the stromal cells and intraluminal macrophages reacted with antibodies to vimentin. Neoplastic cells of papillary carcinomas showed a positive staining reaction both with antibodies to cytokeratins and to epidermal prekeratin. Follicular carcinoma cells, although positive for cytokeratins, could generally not be stained with antibodies to epidermal prekeratin. Medullary carcinoma cells also showed cytokeratin positivity and, only occasionally, positivity for epidermal prekeratin. Anaplastic carcinomas were also reactive with antibodies to cytokeratin but, for the most part, were negative for epidermal prekeratin. Interestingly, some neoplastic cells of all types of thyroid carcinomas also appeared to contain vimentin, as shown with both polyclonal and monoclonal antivimentin antibodies. In contrast to carcinomas, the intermediate filaments of thyroid sarcomas and lymphomas were only of vimentin type. Furthermore, it was found that the papillary structures in benign goiters were only reactive with cytokeratin antibodies and lacked, in contrast to papillary carcinomas, epidermal prekeratin-like immunoreactivity. Hence, the analysis of intermediate filament proteins of thyroid tumors can be utilized to differentiate between papillary and follicular carcinomas and between benign and malignant papillary lesions as well as between anaplastic thyroid carcinomas and sarcomas or lymphomas.     

Antibodies to intermediate filament proteins in the diagnosis and classification of human tumors

Immunohistochemistry of intermediate filaments (IF) is a new and important way to evaluate the epithelial, mesenchymal, muscular, glial, or neural differentiation in tumors. This is based on the stable cell-type-specific expression of IF proteins in normal and neoplastic tissues. Immunohistochemical studies with antibodies to intermediate filaments have also given new perspectives in the histogenesis and biologic nature of many tumors. This article reviews both the recent findings and the authors' experience in the use of intermediate filament antibodies in tumor diagnosis and classification.     

Cytokeratin-specific monoclonal antibodies are reactive with tumours of smooth muscle derivation. An immunocytochemical and biochemical study using antibodies to intermediate filament cytoskeletal proteins

Formalin fixed and paraffin wax embedded tissue from 13 leiomyosarcomas, three leiomyoblastomas, five leiomyomas and fresh tissue from one leiomyosarcoma and one leiomyoma was studied. Tumours were stained by the avidin-biotin-peroxidase technique with antibodies to desmin, vimentin, cytokeratins and S-100 protein. Neoplastic cells in all the cases were positive for desmin and vimentin but were negative for S-100; antibody CAM 5.2 gave strong reactivity with tumour cells of 12 leiomyosarcomas and all leiomyomas, but failed to stain the leiomyoblastomas; LP34 weakly stained two leiomyosarcomas and one leiomyoblastoma. Immunoblot studies using whole cell extracts of one leiomyosarcoma and one leiomyoma respectively, showed 55-58 kd and 35-38 kd bands reactive with monoclonal antibody CAM 5.2. However, no cytokeratin components were demonstrated when cytoskeletal extracts of the same tissue samples were immunoblotted with CAM 5.2. The implications of these findings for the diagnostic histopathologist are discussed.     

Malignant fibrous histiocytoma. Heterogeneous patterns of intermediate filament proteins by immunohistochemistry

Patterns of intermediate filament expression of 10 malignant fibrous histiocytomas (MFHs) were immunohistochemically evaluated using acetone-fixed frozen sections. Seven cases represented the storiform-pleomorphic subtype, 2 were of myxoid type, and 1 was of giant-cell type. All cases had been studied by electron microscopy, and no proof for the diagnoses of liposarcoma, rhabdomyosarcoma, and leiomyosarcoma could be obtained. All tumors showed prominent vimentin immunoreactivity in the tumor cells. Cytokeratin-positive neoplastic cells were found in 2 cases, and in the majority of tumor cells in 1 of these. The 68k neurofilament-positive cells were found in 2 cases. Desmin was not found beyond doubt in the neoplastic cells in any cases, and all cases were negative for glial fibrillary acidic protein. The expression of several types of intermediate filament indicates divergent differentiation properties in MFH and may suggest the heterogeneity of this entity, but more cases should be studied to elaborate any possible consistent patterns of intermediate filament expression in different types of MFH. The expression of multiple types of intermediate filament proteins in MFH can alternatively signify random activation of the corresponding genes in the primitive tumor cells. The complex patterns of intermediate filament proteins in morphologically defined MFHs should be taken into account in the practical immunohistologic analysis of tumors.     

Expression of intermediate filament proteins in normal and diseased thyroid glands.

A total of 67 samples from normal and pathological thyroid glands were stained (as formalin fixed paraffin sections) with a panel of monoclonal antibodies directed against intermediate filament proteins. The study confirmed previous reports of cytokeratin and vimentin coexpression in primary thyroid carcinomas, but coexpression was also detected in normal thyroid and in a range of benign conditions including follicular adenomas, Hashimoto's thyroiditis, and diffuse hyperplasia (thyrotoxicosis). Prekeratin expression was found (using antibodies recognising higher molecular weight cytokeratins) predominantly in areas of squamous change, independent of the underlying thyroid pathology. This study does not therefore support previous findings that prekeratin expression provides a reliable means of distinguishing follicular pattern papillary carcinoma from follicular carcinoma with its poorer prognosis or that it helps distinguish benign from malignant papillary lesions. No evidence of desmin or neurofilament expression was seen, and in particular, neurofilaments could not be detected in any of the cases of medullary carcinoma studied.     

Expression of actin isoforms and intermediate filament proteins in childhood orbital rhabdomyosarcomas

The diagnosis of orbital rhabdomyosarcoma (RMS) in childhood gives rise to several clinical and anatomo-pathological problems. Antibodies recognizing structural proteins and cytoskeletal components have been shown to increase the diagnostic accuracy of different neoplastic lesions. In this study we examined anatomo-clinically and, where possible, by means of immunohistochemistry and electron microscopy, a series of 14 cases of orbital RMS in childhood. In the 12 cases studied by immunohistochemistry, desmin was always present, although showing variable patterns, and alpha-sarcomeric actin was found in 10 cases. alpha-Smooth muscle actin was always absent. The other markers tested (myoglobin, polyclonal actin, vimentin and enolase) proved unreliable for several reasons. We conclude that antibodies against desmin and alpha-sarcomeric actin are useful for the diagnostic definition of RMS. In addition, immunohistochemical analysis supplies data regarding the degree of tumor differentiation and may be applied to monitor radio- and chemotherapy.     

Distribution of intermediate filament proteins in normal and diseased human glomeruli.

The distribution of intermediate filament proteins (vimentin, desmin, and cytokeratin) was studied by means of immunofluorescence in the normal human and rat glomerulus and in pathologic human glomeruli. Antifibronectin antibodies were used as mesangial markers. In normal human glomeruli, vimentin antibodies stained endothelial cells, podocytes, and mesangial cells; desmin antibodies, surprisingly, stained podocytes. In normal rat glomeruli, the pattern of vimentin staining was the same as in humans, but desmin antibodies stained both mesangial cells and podocytes. In human and rat glomeruli cytokeratin staining was confined to segments of Bowman's capsule. In human pathologic glomeruli, vimentin and desmin antibodies stained the structures that were positive in normal glomeruli, giving a characteristic pattern for each pathologic condition examined. These results are compatible with the mesenchymal origin of podocytes and mesangial cells and suggest that both cells have smooth muscle-like phenotypic features. Mesangial cells may have slightly different differentiation paths in humans and rats, leading to a distinct expression of intermediate filament proteins.     

Application of avidin-biotinized peroxidase (ABC) method for immunocytochemical localization of corticotropin-releasing factor (CRF) in rat hypothalamus

Studies on immunocytochemical localization of corticotropin-releasing factor (CRF) were performed in the rat hypothalamus using avidin-biotinized peroxidase (ABC) and PAP techniques. In intact and control animals CRF-immunoreactive nerve fibers were observed within outer layer of median eminence. In the adrenalectomized animals, CRF was also demonstrated in perikarya of neurocytes and in their projections in paraventricular nucleus of the hypothalamus. In both immunocytochemical techniques identical localization of CRF was obtained. However, reaction intensity was greater with the ABC technique than with the PAP one. In bilateral adrenalectomized animals, a greater number of CRF-immunoreactive neural fibers were observed in the median eminence than in control rats and rats subjected to sham operation.     

Rhabdoid tumors of the kidney contain mesenchymal specific and epithelial specific intermediate filament proteins

The intermediate filament proteins of rhabdoid tumors of the kidney were investigated with a panel of monoclonal antibodies to different intermediate filament proteins. Rhabdoid tumor cells are decorated by an antivimentin antibody and by an antibody made against a 54-kilodalton (kd) cytokeratin from human hepatoma cells. The rhabdoid tumor cells fail to react with an antibody generated against keratin from stratum corneum or with an anti-200-kd neurofilament protein antibody. Cytoskeleton preparations of rhabdoid tumor cells grown in vitro demonstrate the presence of vimentin (58 kd) and the 54-kd cytokeratin. Thus, these cells contain two different intermediate filament proteins characteristic of epithelial and mesenchymal cells. We also demonstrate that rhabdoid tumor cells can form tumors in athymic (nude) mice and that the intracytoplasmic globules are present in the nude mouse lesions.     

Expression of intermediate filament proteins in fetal and adult human kidney: modulations of intermediate filament patterns during development and in damaged tissue.

The expression of intermediate filament proteins, particularly individual cytokeratins (CKs), vimentin, and glial filament protein, was immunohistochemically investigated using frozen sections and Carnoy-fixed, paraffin-embedded tissue from normal fetal and adult human kidneys as well as from pathologically altered kidneys. In fetal kidneys, the co-expression of CKs and vimentin was detected in the visceral and parietal epithelium of the glomerulus, the proximal tubules, the thin loops of Henle, and the collecting ducts. In contrast, in the tubules of normal adult kidneys, the presence of vimentin and CKs was nearly always mutually exclusive. While CKs 8 and 18 were present in all tubular epithelia, CKs 19 and 7 each exhibited a distinctive distribution pattern, there being a striking alteration between positive and negative segments and, not infrequently, intratubular heterogeneities. In certain segments, particular cell types (e.g., "plica cells," intercalated cells) could thus be recognized. In tubular epithelia altered by various injurious conditions, novel or enhanced expression of vimentin, CK 19 and CK 7, and, less frequently, CK 17 and glial filament protein was noted in certain segments. The increase in intermediate filament protein expression in altered (particularly proximal) tubules appeared to parallel the reduction in the degree of differentiation. Vimentin was never detected in distal tubules. The present results reveal a considerable similarity between the intermediate filament patterns in non-neoplastic proximal tubules of fetal and damaged kidney tissue and those in clear-cell and chromophilic renal cell carcinomas. They also serve to illustrate that the analysis of both fetal development and reactive cell changes may significantly contribute to our understanding of differentiation phenomena in malignant tumors.     

Mutation analysis of patients with neuronal intermediate filament inclusion disease (NIFID)

Abnormal neuronal aggregates of alpha-internexin and the three neurofilament (NF) subunits, NFL, NFM, and NFH have recently been identified as the signature lesions of neuronal intermediate filament (IF) inclusion disease (NIFID), a novel neurological disease of early onset with a variable clinical phenotype including frontotemporal dementia, pyramidal and extrapyramidal signs. In other neurodegenerative diseases in which protein aggregates contribute to disease pathogenesis, mutations in the encoding protein cause the hereditary variant of the disease. To determine the molecular genetic contribution to this disease we performed a mutation analysis of all type IV neuronal IF, SOD1 and NUDEL genes in cases of NIFID and unaffected control cases. We found no pathogenic variants.     

The intermediate filament cytoskeleton of cutaneous neuroendocrine carcinoma (Merkel cell tumour)

Summary The presence and distribution of cytokeratins, neurofilament proteins, vimentin and glial fibrillary acidic protein were studied in 10 cutaneous neuroendocrine carcinomas (CNEC) by immunohistochemical techniques, using specific antibodies. In all cases tumour cells were specifically stained with antibodies to mouse liver cytokeratin component D in paraffin-embedded formalin-fixed and frozen sections. Moreover, one- and two-dimensional SDS-polyacrylamide gel electrophoresis of high salt/detergent resistant cytoskeletal residues from tumour material, isolated by microdissection from frozen sections, revealed the presence of cytokeratin components 8 and 18 which are characteristic constitutents of cytokeratin filaments of simple epithalia. Neurofilament proteins were detected by immunocytochemistry in tumour cells from 2 patients, from whom frozen material was available, and their presence was also positively identified in cytoskeletal residues by immunoblotting using specific antibodies. Glial fibrillary acidic protein and vimentin could not be demonstrated in tumour cells. Our studies did not confirm the suggested origin of CNEC from epidermal Merkel cells.This work was supported in part by Fonds zur Förderung der wissenschaftlichen Forschung, Grant P4708 to H.D.     

Monoclonal antibodies to human intermediate filament proteins. III. Analysis of tumors

A panel of monoclonal antibodies to human intermediate filament proteins was tested on an unselected series of 246 neoplasms. The antibody panel includes two different anti-cytokeratin antibodies, an anti-vimentin antibody, and an anti-neurofilament antibody (Gown and Vogel, Am J Pathol 114:309, 1984). The studies were done on Carnoy's or methacarn-fixed, paraffin-embedded tissue. When used as a panel, they can unequivocally distinguish carcinomas, melanomas, and lymphomas. All carcinomas react with at least one of the anti-cytokeratin antibodies, and carcinomas can be subtyped based upon the pattern of reactivity with the two anti-cytokeratin antibodies. Melanomas react only with the anti-vimentin antibody, and lymphomas react with none of the antibodies. Neural and neuroendocrine tumors can be identified with the anti-neurofilament antibody. A minority of neoplasms, including lymphomas, seminomas, and some sarcomas, do not react with any of the antibodies. These antibodies are reliable diagnostic reagents that are useful in distinguishing different categories of human tumors.     

Immunohistochemical study of human breast tumors using monoclonal antibodies to intermediate filament proteins (nonproliferating epithelial structures in breast dysplasia)

An immunohistochemical analysis of nonproliferating epithelial structures was carried out in 10 samples of human breast dysplasia and in 4 samples of tissue surrounding mammary gland carcinoma. Monoclonal mouse antibodies against individual prekeratins of rat monolayer epithelial antibodies of clone C12 against rat prekeratin with the molecular mass 49 kilodalton and antibodies of clone E3 against rat prekeratin with the molecular mass 40 kilodalton-monoclonal antibodies against vimentin (clone 30), as well as polyclonal antibodies against smooth muscle myosin and against the basement membrane glycoprotein laminin were used. The lining epithelium of all glandular structures reacted only with C12 antibodies. Two variants of myoepithelial cells containing myosin were detected. Variant I contains myosin and vimentin and is localized in intralobular ducts. Variant 2 contains myosin and prekeratin, recognized by E3 antibodies and is found in extralobular ducts.