Immunohistochemical evaluation of canine ovarian tumors

Canine ovarian tumors (epithelial tumor, sex-cord stromal tumor, germ cell tumor) classifying into 9 histological types were examined immunohistochemically using placental alkaline phosphatase (PLAP), cytokeratin7 (CK7), desmin, S100, AE1/AE3, inhibin alpha, vimentin, and alfa feto-protein (AFP). The papillary and tubular types observed in epithelial tumors were immunoreactive for desmin and AE1/AE3. The papillary type was also immunoreactive for PLAP and CK7. The solid type, nest type, cord type, palisade type, cystic type and spindle type, which were observed in sex-cord stromal tumors, showed a positive immunoreaction for S100 but little or no positive immunoreaction for inhibin alpha with an exception of positive result in the palisade type. Most of the sex-cord stromal tumors were AE1/AE3-positive except for the palisade type. In the cobblestone type observed in germ cell tumors, only vimentin and AFP were positive. The present study elucidated the detailed histological and immunohistochemical characteristics of canine ovarian tumors.     

p63: a novel myoepithelial cell marker in canine mammary tissues

Several immunohistochemical markers have been used to demonstrate the presence of myoepithelial cells in order to determine their role in the histogenesis of mammary tumors. p63, a recently characterized p53 homologue, is consistently expressed in myoepithelial cells of the human breast; however, no assessment of its immunoreactivity has been reported so far in canine mammary tissues. We investigated p63 immunohistochemical expression, as a novel myoepithelial cell nuclear marker, in 81 samples of normal (n = 2), hyperplastic (n = 11), and neoplastic (n = 68) canine mammary tissues. Myoepithelial phenotype was confirmed by using complementary monoclonal antibodies: alpha-smooth muscle actin, cytokeratin 14, cytokeratin AE1/AE3, and vimentin. p63 expression was observed in 91.4% (74/81) of the samples evaluated. Normal mammary glands, mammary hyperplasias, and benign tumors showed 100% immunoreactivity, with p63 expression restricted to myoepithelial cell nuclei. In general, benign mixed tumors showed a basal cell compartment immunoreactive to p63, with a gradual decrease of its expression during myoepithelial transformation. p63 expression was found in 72% of malignant tumors, allowing myoepithelial or basal cell identification in spindle-cell carcinomas (2/2), tubulopapillary carcinomas (8/9), solid carcinomas (7/10), and carcinosarcomas (1/3). The osteosarcoma analyzed was p63 negative. In our series, stromal components were consistently nonreactive to p63. In conclusion, the present study reveals p63 as a sensitive and highly specific marker of myoepithelial cells in canine mammary tissues, and the authors suggest p63 as an additional marker for defining myoepithelial histogenesis.     

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.     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

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.     

Comparative immunohistochemical characterization of canine seminomas and Sertoli cell tumors

Primary testicular tumors are the most common causes of cancer in male dogs. Overall, the majority of canine patients should be cured by testicular surgery. However, tumor markers are not well-known in veterinary medicine. We sought to determine using immunohistochemistry whether the combined human testicular tumor markers (placental alkaline phosphatase, OCT3/4, CD30, alpha-fetoprotein, inhibin-alpha, vimentin, c-KIT, and desmin) are expressed in canine seminomas and Sertoli cell tumors (SCTs). We examined 35 canine testicular tumors, 20 seminomas and 15 SCTs. c-KIT was expressed markedly in canine seminomas. Both inhibin-alpha and vimentin were expressed significantly in canine SCTs. The results of this study demonstrate differences and similarities between tumor marker expression of testicular tumors in dogs and humans. All the main markers in current routine use are discussed as well as potential useful markers for benign and malignant tumors, and tumor progression.     

Immunohistochemical features of canine ovarian papillary adenocarcinoma and utility of cell block technique for detecting neoplastic cells in body cavity effusions

Dogs with ovarian papillary adenocarcinoma occasionally present with ascites and/or pleural effusion. These aspirated fluids often contain a large number of cells, and distinction between neoplastic cells and activated mesothelial cells can be difficult. In this study, 7 cases of canine ovarian papillary adenocarcinoma, including 3 with ascites and pleural effusion, were immunohistochemically examined. Ovarian tumor cells were positive for cytokeratin CAM5.2 (CAM5.2), Wilms’ tumor 1 (WT-1) and progesterone receptor (PR) in all 7 cases. A metastatic lesion of the mediastinum in one case was also positive for CAM5.2, WT-1 and PR. Immunohistochemistry on cell blocks obtained from ascites and/or pleural effusion of 2 cases revealed the presence of PR-positive epithelial cells. Whereas, activated mesothelial cells in ascites or pleural effusion collected from dogs without neoplastic lesions were negative for PR. In addition, surface epithelium and subsurface epithelial structures (SES) of normal canine ovaries, that are considered to be the cell of origin for ovarian papillary adenocarcinoma, were also positive for CAM5.2, WT-1 and PR. These results indicate that, together with CAM5.2, WT-1 and PR is a useful diagnostic marker for canine ovarian papillary adenocarcinoma. Expression of PR may be associated with progesterone-dependent nature of canine ovarian papillary adenocarcinoma.     

Metastatic iridociliary adenocarcinoma in a labrador retriever

An enucleated left eye from a 15-year-old female spayed Labrador Retriever was received by the Comparative Ocular Pathology Laboratory of Wisconsin (COPLOW) for histopathologic evaluation. Routine histologic preparation included staining with hematoxylin and eosin, and with alcian blue periodic acid-Schiff (PAS). At necropsy 9 months later, all grossly abnormal tissues (ipsilateral orbit and lung) were submitted to the COPLOW for histopathologic evaluation. Histopathologic evaluation of the globe revealed extensive invasion of the uvea and sclera by a pleomorphic cell population that formed disorganized cords and exhibited PAS-positive basement membrane material. Necropsy revealed a morphologically similar tumor in the ipsilateral orbit and lung. On immunohistochemical examination, the intraocular tumor stained diffusely immunopositive for vimentin, S-100, and neuron-specific enolase and multifocally, sparsely immunopositive for cytokeratin AE1/AE3. The orbital and thoracic tumors stained positively for vimentin but negatively for cytokeratin AE1/AE3. There are few reports of canine metastatic iridociliary adenocarcinoma in the literature; this is the first with immunohistochemical analysis.     

Histological and Immunohistochemical Evaluation of Canine Ovary

In the present study, 15 canine ovaries without morphological lesions were examined histologically and immunohistochemically by using a large number of proteins including AE1/AE3, cytokeratin7 (CK7), CK13, CK20, vimentin, desmin, alpha smooth muscle actin (alphaSMA), calponin, S100, Neurofilaments, Inhibinalpha, placental alkaline phosphatase (PLAP) and neuron-specific enolase. Ovarian structures observed in this study included surface epithelium (SE), cortical tubules (CT), tunica albuginea (TA), stromal cells (SC), internal endocrine cells (IE), rete ovarii (RO) and fallopian tubes (FT). SE, CT, RO and FT were broadly immunoreactive for desmin. Besides AE1/AE3 and vimentin, desmin was also closely linked to these structures. Rete ovarii forming a reticular structure showed a positive reaction to S100. Surface epithelium was immunoreactive for PLAP at a significantly high level. In conclusion, these results indicate a specific segment of immunoreactivity as well as the broad range of immunoreactivity in canine ovary. The distinct patterns of immunoreactive for various kinds of proteins will play an important role in facilitating their identification and discrimination even in a normal canine ovary with a complex structure.     

Coordinate expression of cytokeratins 7 and 20 in feline and canine carcinomas.

Forty-seven feline and 60 canine epithelial tumors were studied to test the coordinate expression of cytokeratin 7 (CK 7) and cytokeratin 20 (CK 20) using commercially available monoclonal antibodies and an avidin-biotin immunoperoxidase staining technique. Previously, the distribution of both cytokeratins was examined in normal tissues from 4 cats and 4 dogs. The pattern of distribution of CK 7 in normal tissues was similar, with minor differences, to that described in humans, whereas the reactivity pattern of CK 20 in cats and dogs was wider than that in humans. The subset of tumors strongly expressing CK 7 and CK 20 included pancreatic adenocarcinomas (100%), transitional cell carcinomas (75%), and endometrial carcinomas (67%) in the cat. None of the canine tumors had this immunophenotype. Feline (50%) and canine (56%) mammary gland carcinomas and canine cholangiocarcinomas (67%) were the only tumors presenting the CK 7 +/CK 20- immunophenotype, whereas the CK 7-/CK 20+ immunophenotype included thyroid carcinomas (100%), intestinal adenocarcinomas (60%), bronchioloalveolar carcinomas (50%), and renal carcinomas (50%) in the cat and intestinal adenocarcinomas (56%), gastric adenocarcinomas (50%), and ovarian carcinomas (50%) in the dog. The CK 7-/CK 20- immunophenotype included the rest of the analyzed tumors. The immunohistochemical evaluation of coordinate expression of both CK 7 and CK 20 in feline and canine carcinomas using monoclonal antibodies provides important information that can help to discriminate among carcinomas from different primary sites and could be particularly helpful in the determination of the primary site of origin of carcinomas presenting as metastatic disease.     

Immunocytochemical study of tissues from clinically normal dogs and of neoplasms, using keratin monoclonal antibodies.

Three commonly used keratin monoclonal antibodies (MAB)--AE1:AE3, CAM 5.2, and MAK-6--were compared with routinely used cytokeratin antibody. The expression of these antibodies was analyzed in several tissues obtained from clinically normal dogs and in a variety of neoplasms from dogs. Using appropriate enzymatic digestion, paraffin-embedded tissues processed in routine manner retained their typical keratin expression. Differentiated and poorly differentiated epithelial neoplasms, lymphomas, and melanomas were studied by use of the avidin-biotin-peroxidase technique. All 4 of the aforementioned antibodies had similar staining profiles. Of 3 anaplastic carcinomas, 2 had positive reaction to all 4 antibodies. All lymphomas, plasma cell tumors, and amelanotic melanomas had negative reaction to MAK-6, CAM 5.2, AE1:AE3, and cytokeratin MAB. Three basal cell epitheliomas had positive reaction to all 4 antibodies, whereas 1 basal cell tumor with a solid pattern had negative staining reaction. Two carcinoids had negative reaction to all markers and 1 of 2 malignant chemodectomas and 1 transitional cell carcinoma had staining reaction to only AE1:AE3 MAB. Comparing the 4 antibodies, use of AE1:AE3 MAB produced the strongest staining intensity followed by cytokeratin, MAK-6, and CAM 5.2 MAB. All 4 antibodies had low background staining. In conclusion, AE1:AE3 and MAK-6 MAB are as useful as cytokeratin MAB for identification of poorly differentiated epithelial neoplasms in dogs and cats.     

Histologic and immunohistochemical characterization of a testicular mixed germ cell sex cord-stromal tumor and a leydig cell tumor in a dog

Mixed germ cell sex cord-stromal tumors (MGSCTs) of the testis are rare in dogs. We describe the histopathology and immunohistochemical characteristics of an MGSCT associated with a Leydig cell tumor in a cryptorchid testis. Histologically, MGSCT consisted of two nodules of seminiferous tubules lined by germ cells and Sertoli cells in variable proportions. Germ cells had variable size and nuclear features, with frequent giant cells. Germ cells were evenly mixed with Sertoli cells or located in the center of tubules. Markers that labeled mainly germ cells and few or no Sertoli or Leydig cells were calretinin, KIT, and PGP 9.5. E-cadherin, GATA-4, inhibin-alpha (INH-alpha), and neuron-specific enolase (NSE) were predominantly detected in Sertoli cells, whereas melan A was particularly expressed in Leydig cells and vimentin in all three cell types. OCT3/4 was not detected in any cell type. Although more cases of canine MGSCT need to be examined, our results suggest that an immunohistochemical panel of E-cadherin, GATA-4, INH-alpha, KIT, NSE, PGP 9.5, and melan A will help distinguish the three main cell types in canine testicular germ cell and sex cord-stromal tumors.     

Cytological features of canine ovarian tumours: a retrospective study of 19 cases

The cytological features of 19 histologically confirmed canine ovarian tumours were retrospectively examined. Seven cases were cytologically classified as papillary adenocarcinoma, eight cases as granulosa cell tumours, two cases as mature ovarian teratomas, one case as a dysgerminoma and one case as a mixed granulosa cell tumour/dysgerminoma. On cytology, papillary adenocarcinoma was characterised by a papillary glandular pattern and tight cohesiveness. Granulosa cell tumours showed monolayered clusters of loosely cohesive granulosa cells. Call-Exner-like bodies were found in five of seven cases. Granulosa cells appeared to be heterogeneous and usually contained several intracytoplasmic vacuoles. Teratoma was characterised cytologically by keratin debris (two cases) and a mixture of epithelial cells with sebaceous, basaloid, columnar/palisading or ciliated appearance (one case). The dysgerminoma contained severely atypical round cells admixed with small lymphocytes. The mixed dysgerminoma/granulosa cell tumour had a mixture of germinal and granulosa cells. Cytological diagnosis was in agreement with histopathology in 18 of 19 (94.7 per cent) cases.     

Immunohistochemical expression of HER - 2 and Ki - 67 in granulosa cell tumor in bitches

Granulosa cell tumour, an ovarian neoplasm of stromal origin, is an important tumour related to oestrogenic dominance syndrome and cystic endometrial hyperplasia–pyometra complex. In order to analyse ovarian tumour´s malignant potential, immunohistochemical markers can be used, such as anti-HER2 and anti-Ki-67. The aim of this study was to evaluate the expression of immunohistochemical markers HER-2 and Ki-67 in granulosa cell tumour from bitches´ ovaries. In HER-2 immunomarker analysis using the HercepTest^® method, most tumours were classified as 2+ (moderate labelling). Concerning Ki-67 immunomarker, only one case was described as having a high proliferative index. An association was found between immunostained cell percentage by anti-HER-2 antibodies and high pleomorphism, represented by the pattern of follicular/trabecular tumour arrangement. There was no correlation between anti-Ki-67 and anti-HER-2 antibody immunostaining intensities, probably due to only one case with a high Ki-67 index. With an effective protocol for HER-2 and Ki-67 immunohistochemical identification in granulosa cell tumours in bitches, it was possible to characterize this neoplasm proliferation profile.     

Medullary sponge kidney in a 10-year-old Shih Tzu dog

Medullary sponge kidney was diagnosed in a 10-year-old male Shih Tzu dog with a long history of hyposthenuria, but with no other findings indicating renal failure or hormonal aberration. At the dog's death from heart failure, an autopsy was performed. On gross morphology, bilateral kidneys were normal size and had many cysts ranging from the corticomedullary junction to renal papillae. Histopathologic findings showed that almost all of the cysts were lined by monolayered or multilayered and columnar or cuboidal epithelium with chilium similar to epididymis. Immunohistochemically, all of these cells were strongly positive for AE1/AE3 and negative for vimentin. Many of these cells were positive for cytokeratin 7 (CK7), and only a few cells were positive for desmin. The results of staining are the same as those for epithelium of the collecting duct of normal canine kidney. This is the first report of this pathologic entity in the canine kidney.     

Epithelial‐to‐mesenchymal transition: immunohistochemical investigation of related molecules in canine cutaneous epithelial tumours

Background –  Epithelial‐to‐mesenchymal transition (EMT) is a multistep process, important in tumour invasion and metastasis, characterized by loss of epithelial markers, redistribution of β‐catenin and gain of mesenchymal markers. Hyposthesis/Objectives –  Our aim was to investigate the immunohistochemical aberrant expression of cytokeratin, vimentin, survivin and heat shock protein 72 (Hsp72) in canine cutaneous epithelial tumours, to understand the association of expression of these molecules with features of malignancy and their role in the EMT phenotype. Methods –  Ten canine squamous cell carcinomas (SCCs; one with lymph node metastasis), 30 canine hair follicle tumours (six pilomatricomas, eight infundibular keratinizing acanthomas, six trichoepitheliomas and 10 trichoblastomas) and five normal skin samples were investigated by immunohistochemistry using specific anti‐vimentin, ‐cytokeratin, ‐survivin and ‐Hsp72 antibodies. A semi‐quantitative method was used to analyse the results, as follows: 0 to <5%; ≥5 to <10%; ≥10 to <25%; and ≥25% of positive cells. Immunofluorescence was performed to investigate survivin–vimentin and survivin–Hsp72 colocalization in selected SCCs. Results –  In malignant hair follicle tumours and SCCs, a reduced intensity of cytokeratin and increased survivin and Hsp72 expression were observed. In SCCs, loss of cytokeratin expression and vimentin immunolabelling, suggestive of the EMT phenotype, were evident in <5% of neoplastic cells in the front of tumour invasion. In the same areas, strong nuclear survivin and cytoplasmic Hsp72 staining was evident, often colocalizing. Only a few neoplastic cells in the front of tumour invasion showed vimentin–survivin colocalization. Conclusions and clinical importance –  A possible simultaneous involvement of survivin and Hsp72 in tumour invasion and the multistep process of EMT of cutaneous epithelial tumours of dogs is suggested.     

Eimeria organisms develop in the epithelial cells of equine small intestine

Histopathologic and immunohistochemical examinations were performed to determine the origin of host cells parasitized by Eimeria in the small intestines collected from five foals. Eimeria organisms at various stages (mainly microgametes and macrogametes) were frequently found in the cytoplasm of hypertrophied host cells in the lamina propria at the tips of villi of the jejunum and ileum. The cytoplasm of the host cell was immunohistochemically positive for cytokeratin AE1/AE3 and cytokeratin 13 and was negative for vimentin, desmin, alpha-smooth muscle actin, chromogranin A, neuron-specific enolase, and factor VIII. The host cells parasitized by Eimeria species had the immunostaining characteristics of epithelial cells but not of mesenchymal cells, endothelial cells of lacteals or capillaries, smooth muscle cells or neuroendocrine cells. These results suggest that the host cell of Eimeria species is possibly derived from intestinal epithelial cells and then displaced into the lamina propria of the small intestine.     

Anti‐Muellerian hormone concentration in bitches with histopathologically diagnosed ovarian tumours and cysts

Increased concentrations of Anti‐Muellerian hormone (AMH) can indicate a granulosa cell tumour as shown in women, mares and cows. To investigate AMH to differentiate canine granulosa cell tumour from other ovarian pathologies, we evaluated the ovaries of 63 bitches. Blood serum samples were collected before surgery for AMH analysis. Ovaries were submitted for histopathological examination. Fourteen bitches showed normal ovaries. These bitches had AMH values between 0.12 and 0.99 ng/ml. In 20 bitches ovarian cysts i.e., follicular cysts (n = 8), corpora lutea cysts (n = 7), subsurface cysts (n = 5) were diagnosed. These dogs had AMH values of 0.11–2.09 ng/ml. Bitches with small luteinized follicular cysts had slightly higher AMH values than those without ovarian alteration. In 29 cases ovarian neoplasms i.e., granulosa cell tumour (n = 9), epithelial tumours (n = 16), dysgerminomas (n = 3) and one sarcoma were identified. Anti‐Muellerian hormone values of bitches with an ovarian neoplasm except granulosa cell tumour ranged from 0.18 to 1.18 ng/ml. The AMH values of bitches with granulosa cell tumour ranged from 1.12 to ≤23 ng/ml and were significantly higher (p < .05) than in all of the other bitches. The cut‐off of 0.99 ng/ml gave a sensitivity of 100% and a specificity of 94.44% to diagnose granulosa cell tumour. In conclusion, markedly elevated AMH concentrations in bitches are indicative for a granulosa cell tumour. However, negative testing does not rule out the existence of small one. Differentiation of GCT from luteinized follicular cysts may especially be difficult.     

The expression of p63 and cytokeratin 5 in mixed tumors of the canine mammary gland provides new insights into the histogenesis of these neoplasms

Cytokeratin 5 and p63 have been described as basal and myoepithelial cell markers in human breast. Mixed tumors of the canine mammary gland have been associated with a myoepithelial origin. Cytokeratin 5 expression has not been evaluated in these tumors. We investigated the relation between cytokeratin 5 and p63 double-immunohistochemical expression in 23 mixed tumors of the canine mammary gland (10 benign mixed tumors and 13 carcinomas arising from benign mixed tumors) and their origin. Cytokeratin 5 and p63 co-expression was observed in myoepithelial cells of benign mixed tumors, as well as in squamous differentiation of carcinoma arising from benign mixed tumors. Though a few interstitial spindle cells of the mesenchymal components expressed both p63 and cytokeratin 5, the basal epithelial cells were labeled only by cytokeratin 5. The co-expression of p63 and cytokeratin 5 in myoepithelial cells and squamous differentiation suggest that, like in human breast, cytokeratin 5 can also be considered a myoepithelial- and squamous-cell differentiating marker in canine tumors. The presence of some interstitial spindle cells stained for p63 and cytokeratin 5 might be associated with a myoepithelial origin of the mesenchymal component of mixed tumors of the canine mammary gland. Moreover, contrary to p63, basal epithelial cells were labeled by cytokeratin 5, indicating that cytokeratin 5 may not represent an exclusive myoepithelial cell marker but also a basal epithelial cell marker in canine mixed tumors. According to these data, basal epithelial cells may be related to the origin of the epithelial component of mixed tumors of the canine mammary gland.