Merkel cell tumor coexpressing cytokeratin and neurofilament proteins

Whorled filaments 10 nm in width were identified by anti-intermediate filaments antibodies in a Merkel cell tumor from a 52-year-old man. Immunohistochemical tests revealed that the tumor was stained with anti-keratin antibody and antibodies against the 68-kd and 200-kd subunits of neurofilament proteins but not antibody against the 150-kd subunit. This is the first reported case of Merkel cell tumor expressing a 200-kd subunit of neurofilament proteins.     

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 neurofilament triplet proteins in human neural tumors. An immunohistochemical study of paraganglioma, ganglioneuroma, ganglioneuroblastoma, and neuroblastoma.

Intermediate filaments which are specific to neural cells, ie, neurofilaments, consist of three subcomponents--68, 150, and 200 kd. Thirty human neural tumors were examined for the presence of these three subcomponents by means of their monospecific antisera. All 8 paragangliomas contained cells that were positive for the 68-kd component, but only 5 of them had cells positive for the 150-kd and 200-kd components. All 4 ganglioneuromas and 11 ganglioneuroblastomas contained cells that reacted with antibodies to all three components. All 7 neuroblastomas had cells reacting with antibody to 68 kd, but only 3 of them had cells that reacted with antibodies to 150 kd and 200 kd. In each case, the number of positive cells depended on the antibody used. The largest number reacting with antibody to 68 kd and the smallest with antibody to 200 kd. Furthermore, it was possible to detect tumor cells in which the 68-kd subcomponent existed by itself, but no tumor cells in which the 150-kd or 200-kd subcomponent existed alone could be detected. These results seem to indicate that antibody to the 68-kd component is sufficiently discriminating to be applied diagnostically.     

Synaptophysin and neurofilament proteins as markers for neuroendocrine tumors

Synaptophysin, a membrane glycoprotein of presynaptic vesicles, and neurofilament (NF) proteins were tested as immunohistochemical markers for neuroendocrine tumors. Synaptophysin was consistently present in the tumor cells of pheochromocytomas (10/10), thyroid medullary carcinomas (8/8), and pancreatic islet cell tumors (6/6). Most gastrointestinal and thoracic carcinoid tumors (12/13) were positive, as were neuroendocrine carcinomas (7/9), of which two Merkel cell carcinomas were negative. The NF proteins were present in all pheochromocytomas, in three thoracic and one gastric carcinoid tumors, in four of eight thyroid medullary carcinomas, and in five of six pancreatic islet cell tumors. All intestinal carcinoids were negative for NF proteins, as were neuroendocrine carcinomas, except for two Merkel cell carcinomas that were positive. The 68-kilodalton (kd) NF subunit protein was the most prevalent in all NF-positive neuroendocrine tumors, and the 160-kd subunit was relatively often present, although in a smaller number of cells. The 200-kd NF subunit protein was regularly found in pheochromocytomas and only occasionally found in other neuroendocrine tumors. A series of nonneuroendocrine tumors, such as adenocarcinomas, sarcomas, lymphomas, and melanomas, were negative for both synaptophysin and NF proteins. Thus, synaptophysin is a specific and fairly sensitive marker for neuroendocrine tumors of both low and high grades of malignancy. The NF proteins are good markers for pheochromocytoma, and their presence is of basic tumor biologic interest and of potential diagnostic value in other neuroendocrine neoplasms.     

Intermediate filaments in Merkel cell tumors

A series of ten Merkel cell tumors is described, with special emphasis on intermediate filament expression. The presence of cytoskeletal proteins was studied with a polyclonal antiserum directed against cytokeratin and with monoclonal antibodies against cytokeratin, neurofilament, and vimentin by the immunoperoxidase technique. Cytokeratin was demonstrated in nine of ten tumors. Neurofilament was observed in the two snap-frozen tissues tested and in three of the eight formalin-fixed, paraffin-embedded tissues. No reactivity for vimentin was found. By electron microscopy desmosomes were found to be present in all cases, while tonofilaments were found in only a few cases. neurosecretory granules, although seen in all tumors, were generally present in low numbers. The results of this study indicate that the Merkel cell tumor is a poorly differentiated small cell carcinoma that has the ability to express some neuroendocrine features.     

Cytokeratin and neurofilament in lung carcinomas.

Three monoclonal antibodies, one directed against cytokeratin (clone 80) and two directed against neurofilament (clones 2F11 and 3G6), were used in the study of a series of 77 lung carcinomas by immunohistochemical staining. The anti-cytokeratin antibody, a very broadly reacting antibody directed against an antigenic determinant common to a great number of cytokeratins, was applicable on frozen sections. The two anti-neurofilament antibodies, directed against the 70 kD protein (clone 2F11) and the 160 kD and 200 kD proteins (clone 3G6) of neurofilament, were applicable on both frozen sections and paraffin sections. The staining results on the lung carcinomas indicate that all types of tumors studied, including small-cell anaplastic carcinoma, are markedly positive for cytokeratin. Frozen sections of five and formalin-fixed and paraffin-embedded sections of six other small-cell anaplastic carcinomas were negative with both anti-neurofilament monoclonal antibodies. One poorly differentiated squamous cell carcinoma positive with anti-neurofilament clone 2F11 but negative with clone 3G6. This distribution of cytoskeletal proteins demonstrates the epithelial differentiation of all types of lung carcinomas. Neuroendocrine differentiation of lung carcinomas as found in the small-cell anaplastic types does not result in expression of neurofilament proteins.     

Intermediate filaments and their interaction with membranes. The desmosome-cytokeratin filament complex and epithelial differentiation

Intermediate-sized filaments represent a class of morphologically similar but biochemically and immunologically distinguishable cytoplasmic protein polymer structures. Five major filament types have been identified (cytokeratin, vimentin, desmin, neurofilament protein, glia filament protein) and antibodies to these proteins have been used for distinguishing different cell types and tumors derived therefrom. Epithelial and carcinoma cells are characterized by the presence of cytokeratin filaments and desmosomal elements identified by antibodies to certain high molecular weight proteins of desmosomal plaques. However, the specific pattern of cytokeratin polypeptides is different in different epithelia. The potential value of cell type identification by immunological reactions with antibodies to cytoskeletal proteins in tumor diagnosis is discussed.     

Immunoreactivity of neurofilament proteins in neuroendocrine neoplasms.

We immunostained histologic sections of formalin-paraffin-processed specimens from 112 neuroendocrine tumors to evaluate the expression of intermediate filaments, especially neurofilament (NF) protein. The commercially available anti-human NF protein monoclonal antibody was raised against the Mr 200,000 and 70,000 components of neurofilaments. None of our tumors exhibited unequivocal immunoreactivity for Mr 200,000 or 70,000 neurofilament proteins. The failure of a tumor to express the Mr 200,000 or 70,000 component of NF protein does not question its neuroendocrine nature. However, the unequivocal positivity for Mr 200,000 or 70,000 components of NF in an undifferentiated tumor with a differential diagnosis between neuroendocrine small-cell carcinoma and a neurogenic tumor should support the latter consideration.     

Antineurofilament and antiretinal antibodies in AIDS patients with cytomegalovirus retinitis.

Sera obtained from AIDS patients with cytomegalovirus (CMV) retinitis before and after treatment with foscarnet, AIDS patients with human immunodeficiency virus (HIV) retinopathy, AIDS patients without retinal disease, and normal healthy controls with and without positive CMV serologies were assayed for the presence of antibodies against the 200-kDa outer, 160-kDa middle, and 68-kDa core subunits of the neurofilament triplet. Additional studies were performed to determine the presence of antibodies reactive with proteins extracted from crude human retinal antigen preparations. Antibodies against the 200-, 260-, and 68-kDa proteins of the neurofilament triplet were detected in 15 of 15 AIDS patients with CMV retinitis. The expression of these antibodies was unaffected, qualitatively, by successful treatment with foscarnet. In contrast, only 30% of patients with HIV retinopathy unrelated to CMV, fewer than 35% of AIDS patients with positive CMV titers but without evident retinitis, and fewer than 25% of healthy controls with positive or negative CMV titers possessed antibodies against any of the triplet proteins (P < 0.001). Antibodies against several clusters of retinal antigens were also identified in the sera of patients with CMV retinitis. In summary, the data indicate that retinal elements damaged by CMV infection induce an antibody response against the 200-, 160-, and 68kDa components of the neurofilament triplet as well as other, as yet undefined retinal antigens.     

Comparative immunohistochemical and lectin histochemical studies of ependymal cells and the epithelium of the choroid plexus

In a previous investigation we reported on the distribution of intermediate filaments in normal epithelium of choroid plexus and ependyma (Kasper et al. 1986). The present paper describes the results obtained with an enlarged panel of monoclonal antibodies against intermediate filaments and additionally with lectins. Ependymal cells contain GFAP and vimentin filaments, whereas epithelial plexus cells express cytokeratin, vimentin and neurofilament 200 KD. Using Concanavalin A, we found a strong cytoplasmatic staining of plexus epithelia and a reduced or failed reaction in ependymal cells. The differences in both cell types may find an explanation in their function or are determined during the ontogenic development.     

Immunohistochemical localization of five classes of intermediate filament in a benign pelvic soft tissue tumor of rhabdoid appearance.

A benign pelvic soft tissue tumor from a 50-year-old woman was examined by immunohistochemistry and electron microscopy. The tumor cells had abundant eosinophilic cytoplasm with a hyaline appearance, which was filled with large aggregates of intermediate-sized filaments (IF). The cells were positively immunostained by antibodies against cytokeratin, vimentin, desmin, glial fibrillary acidic protein, and neurofilament proteins. This case represents an extreme example of the simultaneous expression of IF by neoplastic cells, and exemplifies the limited applicability of immunohistochemical detection of IF antigens for pathological diagnosis of neoplasms.     

Immunologic characterization of Ewing''s sarcoma using mesenchymal and neural markers.

The two most recent hypotheses about the histogenesis of Ewing's Sarcoma (ES) are that it has a mesenchymal or neuroectodermal origin. Immunologic markers specific to these two tissue origins were tested on cryostat sections from three primary tumors carrying the chromosomal translocation t(11;22)(q24;q12). Cell lines established in vitro from two of these three primary tumors were also analyzed. Using antibodies directed against neural components (neurone-specific-enolase [NSE], HNK-1, and neurofilament triplet proteins [NFTP]), positive reactions were observed in cells from two primary tumors and their corresponding cell lines. Results of electron microscopic examination of the primary tumors were compatible with the diagnosis of ES. When using antibodies directed against mesenchymal cell surface antigens (common leucocytes, Leu M1, Leu M2, and Leu M3), the weak positive reactions observed in the three primary tumors were attributed to lymphoid infiltrates within tumor cells. Six additional ES cell lines carrying the translocation t(11;22) were also analyzed by immunocytochemical and flow cytometry methods using antibodies directed against mesenchymal and neural components. Positive reactions were observed in all seven cell lines tested using antibodies directed against NSE, HNK-1, and 200 KD subunit of the NFTP, whereas negative reactions were obtained with Leu M2 antibody. These results are consistent with a neuroectodermal origin of ES cells.     

Lewy bodies of Parkinson's disease. Immune electron microscopic demonstration of neurofilament antigens in constituent filaments

Recently it has been shown by light microscopic immunochemistry that Lewy bodies (LBs) react with antibodies raised against neurofilament proteins (NFPs). Because of the ubiquity of the NFPs within neurons, the heterogeneous makeup of the inclusions, and the varying patterns of immunolabeling, we undertook to determine whether the labeled elements are indeed constituent filaments. Employing a preembedding technique, we investigated sections of the same LBs by light and electron immunochemistry. Decoration of the filaments was obtained with a monoclonal anti-NFP antibody. Whereas cores of mature LBs were unreactive by light microscopy, these same cores yielded a positive reaction at the ultrastructural level. Early LBs were intensely labeled in both the core and periphery. These results demonstrate that the filamentous profiles that form the LBs are antigenically identical to neurofilaments and suggest a posttranslational modification of the filaments as they "age" within the inclusion.     

Immunohistochemical Localization of Five Classes of Intermediate Filament in a Benign Pelvic Soft Tissue Tumor of Rhabdoid Appearance

A benign pelvic soft tissue tumor from a 50 year old woman was examined by immunohistochemistry and electron microscopy. The tumor cells had abundant eosinophilic cytoplasm with a hyaline appearance, which was filled with large aggregates of intermediate-sized filaments (IF). The cells were positively immunostained by antibodies against cytokeratin, vimentin, desmin, glial fibrillary acidic protein, and neurofilament proteins. This case represents an extreme example of the simultaneous expression of IF by neoplastic cells, and exemplifies the limited applicability of immunohistochemical detection of IF antigens for pathological diagnosis of neoplasms. Acta Pathol Jpn 41: 65–72, 1991.     

Microtubule-associated protein-2 and neurofilament immunoreactivity in neurons and small, intensely fluorescent cells of an amphibian cardiac ganglion.

The localization of two cytoskeletal proteins was analysed in the cell bodies and processes of ganglionic neurons and small, intensely fluorescent cells of the parasympathetic cardiac ganglion of Necturus maculosus (mudpuppy). Antibodies against microtubule-associated protein-2 and against the highly phosphorylated isoforms of high and middle molecular weight neurofilament subunits were used as somatodendritic and axonal markers, respectively. The ganglionic neurons, which usually have only one major process, and small, intensely fluorescent cells, which have several processes, showed distinctly different staining patterns with the two antibodies. In control and denervated ganglia, the ganglionic cell bodies and several hundred micrometers of the proximal processes were labeled with the antibody against microtubule-associated protein-2, whereas small, intensely fluorescent cells and processes showed a paucity of immunoreactivity. The neurofilament antibody labeled numerous axons in the ganglion but did not label the proximal part of the postganglionic process or small, intensely fluorescent cell processes. Denervation resulted in the presence of phosphorylated neurofilament subunit immunoreactivity in the soma and proximal process of the ganglionic neuron. These data suggest that (i) small, intensely fluorescent cells and ganglionic neurons in the mudpuppy cardiac ganglion contain distinctly different cytoskeletal proteins, (ii) the proximal part of postganglionic "axons" contains dendrite-like and not axon-like cytoskeletal proteins, and (iii) deafferentation promotes the localization of phosphorylated forms of neurofilament subunits in the soma and proximal process of parasympathetic ganglionic neurons.     

Antibodies against mesangial cells and their secretory products in chronic renal allograft rejection in the rat.

Antibodies or cell-mediated immunity can cause chronic rejection of vascularized organ grafts, but the nature and specificity of the antigen(s) involved has remained elusive. We have previously demonstrated the presence of antibodies against cryptic glomerular basement membrane antigens and undefined antigens in the mesangial area in rats with chronic renal allograft rejection. Current experiments were designed to study the post-transplant antibody response against cultured mesangial and endothelial cells in rats with chronic rejection using flow cytometry, indirect immunofluorescent staining, immunoelectron microscopy, confocal microscopy, and Western blots. The results were compared with those obtained with alloantisera raised by immunization with cultured mesangial cells. Post-transplant and post-immunization sera contained IgG antibodies against trypsinized mesangial cells detected by flow cytometry. Indirect immunofluorescent studies using mesangial cells grown on coverslips showed autoantibody binding to cytoplasmic granules in cultures early after plating whereas staining of later cultures showed antibody binding in an interrupted, web-like pattern on the outside of the cells. Immunoelectron microscopy showed autoantibody binding to intracellular secretory granules and to cell surface focal adhesion plaques. The latter finding was confirmed in double-labeling experiments with an antiserum against vinculin. Western blots with mesangial cell culture supernatants demonstrated autoantibody reactivity with antigens in the 40-kd and 60- to 70-kd range, and immunoprecipitation identified these molecules as biglycan and decorin. Absorption of the sera with mesangial cell culture supernatant removed most of the antibodies except those that gave a punctate staining with the mesangial cell surface. However, not all immunostaining of mesangial cells could be explained by antibodies against biglycan and decorin. Post-transplant sera, furthermore, contained low-titered antibodies against endothelial cells. We conclude that rats with chronic renal transplant rejection produce a strong autoantibody response against mesangial cell focal adhesion plaques and proteins secreted by these cells in culture. Such antibodies may cause local damage and interfere in the tissue repair process after injury.     

Neurofilament protein-triplet immunoreactivity in distinct subpopulations of peptide-containing neurons in the guinea-pig coeliac ganglion

A battery of polyclonal and monoclonal antibodies raised against the triplet of identified neurofilament protein subunits was used to investigate neurofilament protein immunoreactivity in neurons of the guinea-pig coeliac ganglion. Using optimal conditions of fixation and tissue processing for each antibody we found that only 20% of the postganglionic sympathetic neurons in the guinea-pig coeliac ganglion contain neurofilament protein-triplet immunoreactivity. Double labelling with neurofilament protein-triplet antibodies raised in different species demonstrated that all of these antibodies labelled the same population of neurons. Double labelling using mouse monoclonal antibodies against neurofilament proteins in combination with rabbit polyclonals to neuronal markers showed that neurofilament protein-triplet immunoreactivity is restricted to specific chemically coded subpopulations of noradrenergic neurons. Approximately 52% of neurons in the ganglion contain neuropeptide Y and are presumed vasomotor neurons projecting to blood vessels in the submucosa of the small intestine. Virtually none of the neuropeptide Y-containing neurons were labelled with neurofilament protein-triplet antibodies. Neurons that contain somatostatin (21%) project to the submucous ganglia of the small intestine. Approximately two-thirds of neurons containing somatostatin are immunoreactive for the neurofilament protein-triplet. The other postganglionic neurons in the ganglion (27%) project to the myenteric plexus of the small intestine and do not contain either neuropeptide Y or somatostatin. Approximately a quarter of these neurons were labelled with neurofilament protein-triplet antibodies. These results suggest that the neurofilament protein-triplet may not be an intrinsic component of the cytoskeleton of all neurons. Furthermore the idea of a chemical coding of neurons should be extended to cytoskeletal proteins. The finding that these neurofilament proteins are confined to specific neuronal subpopulations has important implications for the search for a role of the neurofilament protein-triplet in neurons, for the interpretation of classical neurohistological silver impregnation techniques which appear to stain only neurofilament protein-triplet-containing neurons, as well as for neuropathological conditions that may involve these proteins in disease processes.     

Immunohistochemistry of neuronal inclusions in the cerebral cortex and brain-stem in Lewy body disease

Three cases of Lewy body disease were investigated in order to compare the morphological and immunohistochemical characteristics of the neuronal inclusions in the cerebral cortex (CC) and brain-stem (BS). Ultrastructurally, the CC contained intermediate-sized filaments with variable amounts of granular material and other organelles, whereas the BS consisted of an electron-dense core and an outer area with radially oriented filaments. The cerebral cortex was immuno-reactive with antibodies against tyrosine hydroxylase (TH) and tau protein, and differed from BS. In addition, although the CC were antigenically similar to BS in their neurofilament (70, 160 and 200 kDa) and ubiquitin contents, the localization of neurofilament immunoreactivity differed between them, being confined positively to the core of CC, but to the periphery of the BS. Although Lewy bodies (LB) in idiopathic Parkinson's disease are morphologically similar to BS, they have been reported to differ in their immunoreactivity with antibodies against tau. It has been reported that CC differ from LB with regard to immunoreactivity with antibodies against TH and tropomyosin. It is inferred that these inclusions (CC, BS and LB) differ in morphogenesis.     

Detection of a Cdc2-related kinase associated with Alzheimer paired helical filaments.

By immunocytochemical staining and Western blotting, we detected a Cdc2-related kinase in human brains. The kinase is recognized by antibodies against the carboxyl and the amino termini of p34Cdc2 but is not recognized by antibodies against the PSTAIRE motif. It is slightly smaller than p34Cdc2 in molecular mass (approximately 33 kd). This 33-kd Cdc2-related kinase is present in intracellular neurofibrillary tangles in neurons of elderly humans and in Alzheimer's disease, and it is associated with paired helical filaments (PHF) from Alzheimer's disease brains. Unlike the antibodies to the carboxyl and amino termini of p34Cdc2, antibodies to an abundant brain Cdc2-related kinase PSSLARE/Cdk5 did not immunolabel Alzheimer's disease neurofibrillary lesions. PHF preparations were demonstrated to contain kinases capable of phosphorylating histone H1, PHF-Tau, and a synthetic peptide (VAVVRTPPKSPSSAK). By virtue of its physical association with PHF, the 33-kd Cdc2-related kinase may play a role in transforming normal Tau proteins to PHF-Tau characteristic of Alzheimer's disease.     

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.