Histochemical and immunohistochemical characterization of chordoma in ferrets

Chordomas of the tip of the tail in 6 ferrets were examined using histopathological, histochemical and immunohistochemical procedures. Histopathologically, round neoplastic cells containing numerous cytoplasmic vacuoles of varying sizes, categorized as “physaliphorous cells”, were observed in the amorphous eosinophilic or pale basophilic myxoid stroma. Physaliphorous cells were arranged in lobules and in a “chordoid” or “cobblestone” manner. The neoplasms were diagnosed as benign chordoma without local invasion and metastasis. Histochemically, the cytoplasm of small neoplastic cells was positive for periodic acid-Schiff stain and alcian blue (AB) pH 2.5 and pH 1.0 stains, but negative for hyaluronidase digestion-AB pH 2.5 stain. All neoplastic cells were strongly stained with colloidal ion, negative for high iron diamine AB pH 2.5 and toluidine blue pH 2.5 stains, and positive for Mayer’s mucicarmine stain. Immunohistochemistry using antibodies directed against low-molecular-weight cytokeratins (CK18, CK19 and CK20), vimentin and mucin core protein (MUC5AC) revealed that neoplastic cells had both epithelial and mesenchymal elements. The expression of low-molecular-weight cytokeratins suggests that neoplastic cells acquired the properties of glandular epithelial cells and produced epithelial mucus. Furthermore, the expression of cytokeratins, vimentin, S100 protein, brachyury and epithelial membrane antigen indicates that the neoplasms were equivalent to the classic type of human chordoma. Therefore, immunohistochemistry using these antibodies can be useful for the characterization of ferret chordoma.     

A comparison of six monoclonal antibodies for detection of cytokeratins in normal and neoplastic canine tissues

Twenty normal canine tissue specimens, both fetal and adult; 19 epithelial neoplasms; and 18 nonepithelial neoplasms were examined using 6 commercially available monoclonal antibodies differing in their recognition of various molecular weight cytokeratins in human tissues. Fresh tissue samples were fixed in 100% ethanol and paraffin embedded prior to sectioning. The intermediate filament proteins were identified by an avidin-biotin-immunoperoxidase method. Primary antisera used included AE1/AE3, CAM-5.2, 35BH11, 34BE12, PKK1, MAK-6 cytokeratins, and vimentin. Monoclonal antibodies detected cytokeratins in a wide variety of canine epithelial tissues and neoplasms. Normal mesenchymal tissues and neoplasms, and stromal elements of epithelial tissues, showed no reactivity with anti-cytokeratins, but reacted positively with vimentin. Although PKK1, CAM-5.2, and MAK-6 were the most consistently reactive anti-cytokeratins, the full panel of monoclonals was required to detect cytokeratins in all of the epithelia evaluated.     

Canine synovial sarcoma: a retrospective assessment of described prognostic criteria in 16 cases (1994-1999)

Pertinent patient data and biopsied tissue from 16 cases of canine synovial sarcoma (SS) were reviewed. Histopathological grade, clinical stage, and tissue immunoreactivity to cytokeratin (broad stain, AE1/AE3 and cytokeratin 7) and vimentin were determined and correlated with survival. Effect of treatment on survival was similarly evaluated. Neither clinical stage nor histopathological grade significantly affected survival patterns. Tissues from all cases stained >30% positively with vimentin, whereas no tissue from any case exhibited cytokeratin immunoreactivity. Dogs receiving surgical tumor excision or amputation had a significantly higher survivability than those receiving no treatment (P<0.02). Treatment aggressiveness may be more appropriate than clinical staging or tumor grading in predicting survival. Reliability of diagnosing and prognosticating canine SS with current immunohistochemistry protocols should be questioned.     

DNA measurement and immunohistochemical characterization of epithelial and mesenchymal cells in canine mixed mammary tumours: putative evidence for a common histogenesis.

DNA measurement by image cytometry, and a detailed immunohistochemical study using monoclonal antibodies directed against different human cytokeratin types, muscle-specific actin, vimentin and S100 protein were carried out on normal canine mammary tissue (n =4), benign canine mammary mixed tumours (n =20) and malignant canine mammary mixed tumours (n =13). The results showed that ductal and alveolar luminal cells in normal and neoplastic tissue were immunoreactive with CAM5.2 and AE1/AE3 antibodies recognizing human keratins.Basal/myoepithelial cells were clearly differentiated from ductal and alveolar epithelial cells, since the latter only expressed cytokeratins, whereas the former also expressed vimentin and muscle-specific actin. This immunohistochemical study showed that there is loss of expression of muscle-specific actin and cytokeratins in areas of myoepithelial proliferation, and enhanced expression of vimentin and S100 protein in proliferative areas with osseous and/or chondroid metaplasia. The ploidy studies revealed that 20% (4/20) of benign and 54% (7/13) of malignant mixed tumours of canine mammary gland were aneuploid and that the epithelial and myoepithelial components of the mixed tumours had identical DNA content.Our results reinforce the role of myoepithelial cells in mesenchymal metaplasia in mixed mammary tumours and suggest the possibility of a common origin of both components from a totipotential stem cell with capacity for divergent differentiation.     

Cutaneous plasmacytomas in dogs: a morphologic and immunohistochemical study

Forty-nine cutaneous plasmacytomas in 46 dogs were studied. Tumors occurred at solitary sites in middle-aged to old dogs (mean age, 9.7 years) and most commonly involved the skin of the digits, lips, and ears. Initial diagnosis was made on the basis of light microscopic morphologic findings. Tumors were graded according to the extent of cellular differentiation and immunoreactivity to a panel of immunohistochemical markers (cytokeratins, canine IgG F[ab]2, neurofilament, neuron-specific enolase, S-100 protein, and vimentin). Immunoreactivity was limited to antibodies directed at canine IgG F(ab)2 and vimentin. Vimentin immunoreactivity was usually greater than that of canine IgG F(ab)2, but there was no correlation between immunoreactivity and histologic grade of the tumors. Thirty-six of 39 dogs (92.3%) followed (mean follow-up, 13 months) were cured by surgical excision. The results of this study indicate that canine cutaneous plasmacytomas are benign neoplasms that should be included in the differential diagnosis of cutaneous round cell tumors in dogs.     

Immunocytochemistry of normal pancreatic islets and spontaneous islet cell tumors in dogs

Immunocytochemical studies of the distribution of glucagon, gastrin, insulin, and somatostatin in normal canine pancreatic islets and 20 canine islet cell tumors were done using the peroxidase-anti-peroxidase (PAP) technique. In the normal adult canine pancreas, islets typically consisted of clusters of 20-30 cells, but smaller foci and even individual cells were identified. Alpha cells (glucagon) were often peripherally located, beta cells (insulin) were centrally located and most numerous, and delta cells (somatostatin) were the least numerous and randomly located. Both juvenile and adult canine pancreases did not stain for gastrin. Of the 20 tumors examined, 18 had positive immunoreactivity for insulin, nine for glucagon, 14 for somatostatin, and one for gastrin. Two tumors were uninterpretable due to autolysis. Three tumors were pure insulinomas, but no pure somatostatinomas, glucagonomas, or gastrinomas were identified. Most tumors and metastases had mixed positive immunoreactivity; one neoplastic cell type predominated with lesser numbers of other cell types. Metastatic sites (liver and lymph node) stained for insulin and somatostatin, only. Foci of non-neoplastic islet cell tissue (nesidioblastosis), often located at the pancreatic-mesenteric junction, stained strongly positive for insulin, glucagon, and somatostatin but not for gastrin. The tumor staining pattern did not consistently correlate with tumor function, as determined by blood glucose and serum insulin assays. The PAP technique works well on paraffin-embedded, formalin-fixed tissue using rabbit or guinea pig antisera as the primary antibody. Staining occurred on sections of paraffin blocks stored for up to 7 years.     

Immunohistochemical demonstration of keratin in canine neuroepithelioma

Morphological features and immunoreactivity for cytokeratin (CK), glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) of three canine neuroepitheliomas and three canine ependymomas were investigated. Neuroepitheliomas were in three German shepherds as intradural-extramedullary solitary masses, with spinal cord displacement between T10 and L2. Histologically, they contained tubules and acini, lined by epithelial cells with focal squamous metaplasia, rosette-like structures, and polygonal to spindle-shaped cells between tubules. Acini were empty or filled with a homogeneous, eosinophilic periodic acid-Schiff (PAS)-positive material. Mitotic indices varied from low to moderate. Ependymomas occurred in the third (two cases) and fourth ventricle in adult boxers. Histologically, they were composed of cells with an ill-defined, scant amphophilic cytoplasm, with a central round euchromatic nucleus; cells formed pseudorosettes, with a central fibro-vascular stroma. Neuroepitheliomas stained for CK, but ependymomas did not. Both failed to stain for GFAP, NSE, or phosphotungstic acid hematoxylin (PTAH). Thus, antibodies to cytokeratin are useful to distinguish neuroepitheliomas from ependymomas.     

Immunohistochemical evaluation of intermediate filament expression in canine and feline neoplasms

Specimens of neoplastic tissues from 19 dogs and 4 cats were examined immunohistochemically for intermediate filament expression, using commercially available antibodies. Staining was observed in a wide range of tumor tissues and in normal internal controls by use of antibodies to vimentin, desmin, glial fibrillary acidic protein, and low and high molecular weight cytokeratins. Intermediate filament expression was found to be consistent with light and/or electron microscopic findings, and hence believed to be an accurate indicator of tumor histogenesis in cats and dogs. Three fixatives were evaluated for their relative abilities to preserve antigenicity. Absolute alcohol was superior to B5 fixative and both were superior to formalin. Some tissues that clearly displayed intermediate filament antigens with alcohol and B5 fixative failed to stain when fixed in formalin.     

Immunohistochemical detection of intermediate filament proteins in formalin fixed normal and neoplastic canine tissues.

Normal and well differentiated neoplastic canine tissues were immunohistochemically stained for keratin, vimentin and desmin intermediate filament proteins using commercially available monoclonal antibodies. Keratin was detected in 56 of 57 carcinomas, vimentin in 59 of 62 sarcomas and desmin in three of four muscle cell tumors. Most normal and neoplastic tissues expressed only one type of intermediate filament; exceptions were one hemangiosarcoma and one pulmonary carcinoma in which there was coexpression of vimentin and keratin proteins. Since immunohistochemical detection of intermediate filaments has tissue-specific distribution in the majority of well differentiated canine neoplasms, these stains may be useful in the differential diagnosis of anaplastic canine tumors. However, the monoclonal antibodies to cytokeratin which were tested in this study failed to detect intermediate filaments in liver, pancreas and salivary glands which suggests that these antibodies may also be unable to detect epithelial tumors derived from these tissues. In addition, in nine neoplasms, the normal tissues adjacent to neoplastic cells failed to stain for the intermediate filament normally expressed. When this occurs, evaluation of intermediate filament expression is invalid for the determination of tissue of origin of the neoplastic cells.     

Malignant fibrous histiocytomas in dogs and cats: an immunohistochemical study.

Immunohistochemical staining was performed on seven canine and 10 feline soft tissue tumours histologically diagnosed as malignant fibrous histiocytomas (MFHs) or MFH-like tumours, and eight other histologically specified tumours (non-MFH). This was done to determine if commercially available antibodies that are used routinely in human diagnostic pathology for MFHs would express the same immunohistochemical patterns in canine and feline MFHs and MFH-like tumours. The antibodies were directed against human alpha 1-anti-trypsin (AT), human alpha 1-anti-chymotrypsin (ACT), human lysozyme, bovine S-100 protein and human desmin. AT did not show any immunoreactivity in the tissues investigated. Except for one MFH, all canine MFHs and other soft tissue tumours with a 'histiocytic' character stained for lysozyme and not for S-100. Six out of seven canine MFHs and MFH-like tumours stained positive for desmin as did most non-MFH sarcomas. Most of the canine and feline MFHs and MFH-like tumours were positive for ACT. These findings for ACT staining in canine and feline MFHs and MFH-like tumours are in agreement with the findings in human MFHs. The immunohistochemical results of canine MFHs and MFH-like tumours were different from those in cats. Feline MFHs differed from canine MFHs for both lysozyme and desmin staining.     

The expression of intermediate filaments in canine mammary glands and their tumors

Monoclonal antibodies specific for different types of intermediate filaments (cytokeratin, vimentin, desmin and neurofilaments) were used to study the histogenesis of canine mammary glands and 57 canine mammary tumors by immunocytochemistry. The intra- and interlobular duct epithelium, acinar, and intralobular myoepithelial cells stained positively for cytokeratin. Peripheral ductal and acinar cells, as well as interstitial cells, stained positively for vimentin. A similar staining pattern was seen in adenomas, complex adenomas, benign mixed tumors, ductular carcinomas, and one myoepithelioma-like tumor. Additionally, cytokeratin positive cells were scattered interstitially in one single adenoma, most complex adenomas, some benign mixed tumors, complex carcinomas, and in the malignant mixed tumors. All stromal cells stained positively for vimentin. The fibrosarcomas were positive only for vimentin, while the following expressed both desmin and cytokeratin: epithelial-like cells in one adenoma, three complex adenomas, the myoepithelioma-like tumor, the single comedo carcinoma, two complex carcinomas, the single lobular carcinoma, one malignant mixed tumor, and three osteosarcomas. Epithelial-like cells in one adenoma, six complex adenomas, two benign mixed tumors, two complex carcinomas, the lobular carcinoma, and the malignant schwannoma stained for neurofilaments. Three tumors, one adenoma, one complex adenoma, and the lobular carcinoma expressed both desmin and neurofilaments in addition to cytokeratin and vimentin. The results show the expression of different types of intermediate filaments and indicate that there might be a stem cell origin in most of the canine mammary tumors.     

Immunocytochemical differentiation of canine mesenchymal tumors in cytologic imprint preparations

BACKGROUND: Immunocytochemical techniques are a potentially valuable diagnostic tool to support cytologic diagnosis in dogs. However, detailed studies of staining patterns and intensity in cytologic specimens of mesenchymal tumor types are lacking. OBJECTIVE: The aim of this study was to evaluate commercially available antibodies against human proteins for use in the characterization of canine tumors of mesenchymal origin in cytologic samples. METHODS: Immunocytochemical staining was performed on air-dried imprint specimens of biopsies obtained from 103 mesenchymal neoplasms and 14 metastatic lesions from 98 dogs. All specimens were stained with anti-cytokeratin AE1/AE3 and vimentin. Based on the histologic diagnosis, tumors of muscle, endothelial, histiocytic, and melanocytic origin also were stained with cell-specific antibodies. Staining intensity was subjectively graded and the percentage of positive tumor cells was estimated. RESULTS: All mesenchymal tumors and metastases, with the exception of mesotheliomas, were vimentin-positive and cytokeratin-negative; mesotheliomas (n=6) were positive for both vimentin and cytokeratin. Tumors of muscle (n=5), endothelial (n=15), and histiocytic (n=18) origin stained moderately to strongly positive in a majority of tumor cells with desmin, von Willebrand factor, and lysozyme, respectively. Malignant melanomas (n=15) had variable staining and a variable percentage of positive cells with Melan-A and S100. CONCLUSIONS: Our results indicate that immunocytochemical staining of canine cytologic specimens is a reliable and sensitive technique that may be of benefit for the differentiation of poorly differentiated mesenchymal tumors and metastases. Additional study is needed to assess the specificity of immunocytochemical stains in mesenchymal tumors.     

Two canine Merkel cell tumours: immunoexpression of c‐KIT,E‐cadherin, β‐catenin and S100 protein

Canine Merkel cell tumours are rare neuroendocrine neoplasms that show a relatively benign biological behaviour when compared with their human counterparts. To date, little information is available on their immunohistochemical properties. This report describes the histopathological and immunohistochemical features of two such tumours. The tumours’ immunoreactivity profile was studied with respect to different cellular molecules including chromogranin A (CGA), neurone‐specific enolase (NSE), S100 protein, c‐KIT, the cytokeratins (CKs) detected by pancytokeratin (AE1/AE3) antibodies (i.e. high molecular weight CKs 1, 2, 3, 4, 5, 6, 10, 14, 15 and 16, and low molecular weight CKs 7, 8 and 19) and three markers proposed to correlate with increased malignancy in human tumours: E‐cadherin, β‐catenin and p63 protein. In both lesions, tumour cells were positive for cytokeratins, CGA, NSE, S100 and c‐KIT. No immunostaining was observed for p63 protein, and there was no loss or change in E‐cadherin or β‐catenin immunoexpression. These results suggest that the generally benign behaviour of canine Merkel cell tumours, when compared with their human counterparts, may be partly explained by the conservation of important intercellular adhesion molecules such as E‐cadherin and β‐catenin. Additionally, expression of S100 but not of the p63 protein suggests that these canine tumours present a trend towards neural, rather than basal, epithelial differentiation and do not readily compare with human Merkel cell tumours.     

Immunohistochemistry with keratin,vimentin, desmin,and α‐smooth muscle actin monoclonal antibodies in canine mammary gland: Normal mammary tissue

Summary

Normal canine mammary gland tissue was studied immunohistochemically with monoclonal antibodies (MoAbs) directed against various human keratin types, vimentin, desmin, and α‐smooth muscle actin. Both ductal and alveolar luminal cells were immunoreactive with MoAbs recognizing respectively human keratins no. 7, 8, 18 and 19. In addition, some ductal luminal cells were labelled with a keratin 4 and a keratin 10 MoAb. Basal/myoepithelial cells were immunoreactive only with MoAbs directed against keratin 14, keratins 14 and 17, and a‐smooth muscle actin. The vimentin MoAb merely labelled solitary loose intraluminal cells representing macro‐phages or sloughed epithelial cells. These findings correspond largely to observations made in human breast tissue.     

Distribution and implications of beta-endorphin and ACTH-immunoreactive cells in the intermediate lobe of the hypophysis in healthy equids

The distribution of cells that stain positive for beta-endorphin and ACTH immunoreactivity was studied in the pars intermedia (PI) of the hypophysis in 3 healthy horses and 2 healthy ponies. Serial sections treated with commercial antibodies generated against beta-endorphin or ACTH were processed for immunocytochemical studies, using the avidin biotin immunoperoxidase-complex method. Distribution patterns of cells reacting with antibodies were similar in cells from all equids. Cells immunostained for ACTH were numerous and widely distributed in the PI. Cells immunopositive for ACTH probably contain corticotrophin-like intermediate lobe peptide that cross-reacts with antisera to ACTH. Cells immunopositive for beta-endorphin were fewer in number and had a more limited distribution in the PI. Most beta-endorphin-positive cells were located along the border of the PI adjacent to the lobus nervosus and had abundant eosinophilic cytoplasm when stained with hematoxylin and eosin. Cells with prominent eosinophilic cytoplasm were not common in other areas of the PI. When serial sections were examined, cells that stained positive for beta-endorphin immunoreactivity also appeared positive for ACTH.     

Immunohistochemical and ultrastructural investigation of granular cell tumours in dog, cat, and horse.

Six canine, one feline and one equine granular cell tumours (GCTs) were investigated electron microscopically and immunohistochemically. The tumours were tested for reactivity with monoclonal antibodies against vimentin and desmin and with polyclonal antibodies against cytokeratin, S-100 protein, glial fibrillary acidic protein (GFAP) and neuron specific enolase (NSE). All GCTs were characterized by their PAS positive cytoplasmic granules in light microscopy, which in electron microscopy appeared as lysosome-like granules. In each case two canine GCTs were stained by the antibody against cytokeratin, vimentin and S-100 protein. Cells of the equine GCT showed reactivity with the antiserum against S-100 protein. In the feline GCT no reactivity with any of the antibodies tested was observed. These differences of the immunohistochemical reactions of GCTs suggest a nonuniform histogenesis of GCTs in domestic animals. The reactivity of the tumour cells with the antiserum against NSE is discussed.     

Diagnostic immunohistochemistry of canine round cell tumors

Sixty-five canine skin neoplasms studied using immunocytochemistry, included 22 histiocytomas, 18 amelanotic melanomas, 14 cutaneous lymphosarcomas, six mast cell tumors, and five transmissible venereal tumors. Formalin-fixed, paraffin-embedded sections were stained using the avidin-biotin-peroxidase complex (ABC) immunoperoxidase technique for reactivity with S-100 protein, kappa and lambda immunoglobulin light chains, alpha-1-antitrypsin, alpha-1-antichymotrypsin, leukocyte common antigen (LCA), neuron-specific enolase, keratin, cytokeratin, muramidase, and vimentin. Detection of S-100, kappa and lambda light chains, neuron-specific enolase, and vimentin were most useful for screening these neoplasms. None of the markers examined was consistent in staining histiocytomas. While reactivity of S-100 (ten cases) and neuron-specific enolase (ten cases) was detected in some amelanotic melanomas, lambda light chain immunoglobulin (eight cases) was relatively consistent in cutaneous lymphomas. Mast cell neoplasms reacted with avidin and, therefore, were positive, even on negative control sections. Vimentin reacted strongly on all amelanotic melanomas and transmissible venereal tumors examined. These antibodies are helpful adjuncts in the differential diagnosis of canine skin tumors.     

Expression and distribution of intermediate-filament proteins and laminin during the development of the bovine Müllerian duct

The expression pattern of several intermediate-filament proteins (vimentin, cytokeratin 8, 18 and 19) and the basal lamina component laminin was investigated in the Wolffian and the Müllerian ducts of bovine embryos and fetuses. The material studied comprised sexually undifferentiated stages [crown-rump length (CRL) 0.9 cm/1.0 cm/1.2 cm/1.9 cm/2.5 cm] and female stages (CRL 3.0 cm/4.2 cm/5.1 cm). Laminin could be demonstrated in the basal lamina of the developing Wolffian and Müllerian duct as well as in the stroma surrounding the Müllerian duct. The intermediate-filament protein vimentin was expressed in the mesothelium of the funnel field and in the epithelium of the Müllerian duct in all studied specimens, whereas the epithelial cells of the Wolffian duct only showed vimentin expression from a CRL of 2.2 cm onwards. In the cranial part of the Müllerian ducts only a few cells stained with pan-cytokeratin antibodies, whereas mesothelium and epithelium of the Wolffian duct showed as distinct immunostaining in all investigated stages. Both genital ducts showed no immunostaining with the antibody against cytokeratin 19 at any time of development. We conclude from our immunohistochemical results that the epithelial cells of the Wollfian duct do not contribute cells to the developing Müllerian duct.     

Mixed apocrine sweat gland tumor of the tail in a cow

A 4-year-old native-breed cow had a mass with wide areas of ulceration and hemorrhage at the base of the tail at the same level as the vulva. The tumor was 19 X 13 X 11 cm, appeared red-brown, and was firm to hard, with gritty areas apparent on cut surface. Histologically, the tumor mass was composed of multilayered epithelial cells forming glandular structures with occasional apical blebs and rare solidly packed cells in nests. The stroma included fibrous connective tissue, scattered or periglandular sheets of spindle-shaped cells resembling myoepithelium, several cartilaginous formations, and numerous irregular islands of mineralized osteoid, well-formed bone trabeculae lined by osteoblasts, and many osteoclast-like multinucleated giant cells among or near the neoplastic epithelium. Immunohistochemically, the neoplastic epithelium was positive for pan-cytokeratin (AE1/AE2) and cytokeratin 19 but was negative for cytokeratin 18. Spindle-shaped cells were stained with alpha smooth muscle actin (alphaSMA) and to a lesser extent vimentin antibodies. The cells of osteogenic lineage and spindle cells closely associated with the osteoid showed strong immunostaining for vimentin but not for alphaSMA. Immunostaining for neuron-specific enolase and S100 protein was not observed in any component of the tumor mass. These findings suggested that the origin of bone formation was undifferentiated mesenchymal cells with osteogenic potential.