Canine mammary tumors: a review and consensus of standard guidelines on epithelial and myoepithelial phenotype markers, HER2, and hormone receptor assessment using immunohistochemistry.

Although there have been several studies on the use of immunohistochemical biomarkers of canine mammary tumors (CMTs), the results are difficult to compare. This article provides guidelines on the most useful immunohistochemical markers to standardize their use and understand how outcomes are measured, thus ensuring reproducibility of results. We have reviewed the biomarkers of canine mammary epithelial and myoepithelial cells and identified those biomarkers that are most useful and those biomarkers for invasion and lymph node micrometastatic disease. A 10% threshold for positive reaction for most of these markers is recommended. Guidelines on immunolabeling for HER2, estrogen receptors (ERs), and progesterone receptors (PRs) are provided along with the specific recommendations for interpretation of the results for each of these biomarkers in CMTs. Only 3+ HER2-positive tumors should be considered positive, as found in human breast cancer. The lack of any known response to adjuvant endocrine therapy of ER- and PR-positive CMTs prevents the use of the biological positive/negative threshold used in human breast cancer. Immunohistochemistry results of ER and PR in CMTs should be reported as the sum of the percentage of positive cells and the intensity of immunolabeling (Allred score). Incorporation of these recommendations in future studies, either prospective or retrospective, will provide a mechanism for the direct comparison of studies and will help to determine whether these biomarkers have prognostic significance. Finally, these biomarkers may ascertain the most appropriate treatment(s) for canine malignant mammary neoplasms.

Recommended guidelines for submission, trimming, margin evaluation, and reporting of tumor biopsy specimens in veterinary surgical pathology.

Neoplastic diseases are typically diagnosed by biopsy and histopathological evaluation. The pathology report is key in determining prognosis, therapeutic decisions, and overall case management and therefore requires diagnostic accuracy, completeness, and clarity. Successful management relies on collaboration between clinical veterinarians, oncologists, and pathologists. To date there has been no standardized approach or guideline for the submission, trimming, margin evaluation, or reporting of neoplastic biopsy specimens in veterinary medicine. To address this issue, a committee consisting of veterinary pathologists and oncologists was established under the auspices of the American College of Veterinary Pathologists Oncology Committee. These consensus guidelines were subsequently reviewed and endorsed by a large international group of veterinary pathologists. These recommended guidelines are not mandated but rather exist to help clinicians and veterinary pathologists optimally handle neoplastic biopsy samples. Many of these guidelines represent the collective experience of the committee members and consensus group when assessing neoplastic lesions from veterinary patients but have not met the rigors of definitive scientific study and investigation. These questions of technique, analysis, and evaluation should be put through formal scrutiny in rigorous clinical studies in the near future so that more definitive guidelines can be derived.

Recommended guidelines for the conduct and evaluation of prognostic studies in veterinary oncology.

There is an increasing need for more accurate prognostic and predictive markers in veterinary oncology because of an increasing number of treatment options, the increased financial costs associated with treatment, and the emotional stress experienced by owners in association with the disease and its treatment. Numerous studies have evaluated potential prognostic and predictive markers for veterinary neoplastic diseases, but there are no established guidelines or standards for the conduct and reporting of prognostic studies in veterinary medicine. This lack of standardization has made the evaluation and comparison of studies difficult. Most important, translating these results to clinical applications is problematic. To address this issue, the American College of Veterinary Pathologists' Oncology Committee organized an initiative to establish guidelines for the conduct and reporting of prognostic studies in veterinary oncology. The goal of this initiative is to increase the quality and standardization of veterinary prognostic studies to facilitate independent evaluation, validation, comparison, and implementation of study results. This article represents a consensus statement on the conduct and reporting of prognostic studies in veterinary oncology from veterinary pathologists and oncologists from around the world. These guidelines should be considered a recommendation based on the current state of knowledge in the field, and they will need to be continually reevaluated and revised as the field of veterinary oncology continues to progress. As mentioned, these guidelines were developed through an initiative of the American College of Veterinary Pathologists' Oncology Committee, and they have been reviewed and endorsed by the World Small Animal Veterinary Association.

Correlation between the histopathological diagnosis by AgNOR count and AgNOR area in canine mammary tumors.

BACKGROUND: Mammary tumors are the most common type of tumor in female dogs. The histopathological diagnosis is usually made by a hematoxylin-eosin (HE) staining of the tumor, which then requires a pathologist's judgment for assessment of malignancy. The purpose of this study was to investigate an alternative silver staining of some argyrophilic nucleolar organizer regions (AgNOR) for improving the diagnostic accuracy with mammary tumors. HYPOTHESIS: There is a correlation between the histopathological diagnosis by AgNOR count and AgNOR area in canine mammary tumors. ANIMALS: Seventy-three canine mammary tumors from 33 female dogs. MATERIALS AND METHODS: The AgNOR staining was evaluated retrospectively in 73 canine mammary tumors with a parallel HE staining as a "Gold Standard." Both a quantitative manual counting method and a qualitative computerized morphometric method were tested. RESULT: The result from both methods indicated a clinically relevant difference in the mean values of the AgNOR in the following 4 categories: malignant, benign, hyperplastic, and normal mammary tissue. The counting method was superior, with 89% of the cases given a correct diagnosis of a malignant or a nonmalignant canine mammary tumor. The 2 methods were then compared to test their ability to classify the tumors correctly. Again, the counting method was the most reliable method, with a sensitivity of 80% and a specificity of 76% when the upper 50% of the AgNOR counts were presumed malignant. CONCLUSION AND CLINICAL IMPORTANCE: The results indicated that an AgNOR test could be an aid to pathologists as a prognostic indicator or to assist them in deciding between a benign or a malignant diagnosis in questionable cases.

Melatonin and IL-25 modulate apoptosis and angiogenesis mediators in metastatic (CF-41) and non-metastatic (CMT-U229) canine mammary tumour cells.

BACKGROUND: Melatonin has oncostatic actions and IL-25 is active in inflammatory processes that induce apoptosis in tumor cells AIM: The aim of this study was to evaluate melatonin and IL-25 in metastatic (CF-41) and non-metastatic (CMT-U229) canine mammary tumor cells cultured as monolayers and tridimensional structures. MATERIALS AND METHODS: The cells were treated with melatonin, IL-25 and IL-17B silencing gene and performed cell viability, gene and protein expression of caspase-3 and VEGFA (Vascular endothelial growth factor A) and an apoptosis membrane protein array. RESULTS: Treatment with 1 mM of melatonin reduced cell viability of both tumor cell lines, all treatments alone and combined significantly increased caspase-3 cleaved and proteins involved in the apoptotic pathway and reduced pro-angiogenic VEGFA, confirming the effectiveness of these potential promising treatments. CONCLUSION: This is the first study evaluating the potential use of these strategies in CF-41 and CMT-U229 cell lines and together encourages subsequent in vitro and in vivo studies for further exploration of clinical applications.

Tumour-associated macrophages influence canine mammary cancer stem-like cells enhancing their pro-angiogenic properties.

Cancer stem-like cells as cells with ability to self-renewal and potential to differentiate into various types of cells are known to be responsible for tumour initiation, recurrence and drug resistance. Hence a comprehensive research is concentrated on discovering cancer stem-like cells biology and interdependence between them and other cells. The aim of our study was to evaluate the impact of macrophages on cancer stem-like cells in canine mammary carcinomas. As recent studies indicated presence of macrophages in cancer environment stimulates cancer cells into more motile and invasive cells by acquisition of macrophage phenotypes. From two canine mammary tumour cell lines, CMT-U27 and P114 cancer stem-like cells were stained with Sca1, CD44 and EpCAM monoclonal antibodies and isolated. Those cells were next co-cultured with macrophages for 5 days and used for further experiments. Canine Gene Expression Microarray revealed 29 different expressed transcripts in cancer stem-like cells co-cultured with macrophages compared to those in mono-culture. Up-regulation of C-C motif chemokine 2 was considered as the most interesting for further investigation. Additionally, those cells showed overexpression of genes involved in non-canonical Wnt pathway. The results of 3D tubule formation in endothelial cells induced by cancer stem-like cells co-cultured with macrophages compared to cancer stem-like cells from mono-cultures and with addition of Recombinant Canine CCL2/MCP-1 revealed the same stimulating effect. Based on those results we can conclude that macrophages have an impact on cancer stem-like cells increasing secretion of pro-angiogenic factors.

Analysis of microRNA expression in canine mammary cancer stem-like cells indicates epigenetic regulation of transforming growth factor-beta signaling.

Cancer stem cells (CSCs) display both unique self-renewal ability as well as the ability to differentiate into many kinds of cancer cells. They are supposed to be responsible for cancer initiation, recurrence and drug resistance. Despite the fact that a variety of methods are currently employed in order to target CSCs, little is known about the regulation of their phenotype and biology by miRNAs. The aim of our study was to assess miRNA expression in canine mammary cancer stem-like cells (expressing stem cell antigen 1, Sca-1; CD44 and EpCAM) sorted from canine mammary tumour cell lines (CMT-U27, CMT-309 and P114). In order to prove their stem-like phenotype, we conducted a colony formation assay that confirmed their ability to form colonies from a single cell. Profiles of miRNA expression were investigated using Agilent custom-designed microarrays. The results were further validated by real-time rt-PCR analysis of expression of randomly selected miRNAs. Target genes were indicated and analysed using Kioto Encyclopedia of Genes and Genomes (KEGG) and BioCarta databases. The results revealed 24 down-regulated and nine up-regulated miRNAs in cancer stem-like cells compared to differentiated tumour cells. According to KEGG and BioCarta databases, target genes (n=240) of significantly down-regulated miRNAs were involved in transforming growth factor-beta signaling, mitogen-activated protein kinases (MAPK) signaling pathway, anaplastic lymphoma receptor tyrosine kinase (ALK) and peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1A) pathways. The analysis of single-gene overlapping with different pathways showed that the most important genes were: TGFBR1, TGFBR2, SOS1, CHUK, PDGFRA, SMAD2, MEF2A, MEF2C and MEF2D. All of them are involved in tumor necrosis factor-beta signaling and may indicate its important role in cancer stem cell biology. Increased expression of TGFBR2, SMAD2, MEF2A and MEF2D in canine mammary cancer stem-like cells was further confirmed by real-time-qPCR. The results of our study point at epigenetic differences between cancer stem-like cells and differentiated tumour cells, which may be important not only for veterinary medicine but also for comparative oncology.

Expression and molecular characterization of the growth hormone receptor in canine mammary tissue and mammary tumors.

GH synthesis has been documented in canine mammary tissue and mammary tumors. In the present report, the characteristics of the GH receptor (GHR) are studied in these tissues. First, using immunohistochemistry, GHR was found to be present throughout normal and tumorous mammary tissues, being localized in epithelial and myoepithelial/spindle cell components and in the activated fibroblasts of desmoplastic tumor stroma. GHR expression seemed to be down-regulated only in terminally differentiated alveoli in normal tissue. GHR immunoreactivity in particular mammary (adeno)carcinomas was heterogenous. Second, the canine GHR was characterized at the molecular level. Northern blot analysis revealed a major GHR transcript of approximately 4.2 kb. The coding sequence of the canine GHR shows extensive homology with the GHR of several species. Seminested RT-PCR (using primers annealing in exons 4-5, exon 6, and exon 9) generated, next to the primary product, four different products in mammary tissues and the canine mammary tumor cell line CMT-U335, which seemed to be alternative GHR transcripts. These alternative GHR transcripts were characterized by exon 8 skipping, exon 7 skipping, and use of alternative splice donor and acceptor sites. Especially, the transcript that is missing exon 8 may encode a GH binding protein. In most malignant mammary samples, only the primary transcript was present; and alternative transcripts could not be detected. The absence of alternative GHR transcripts in mammary carcinomas, and thus putative inhibitors of GH-induced signal transduction, may contribute to enhanced sensitivity of malignant tumors to GH.

The difference in DNA ploidy pattern between some canine and human neoplasms appears to be genuine and a reflection of dissimilarities in DNA aneuploidy evolution.

In a reaction to the article by Deitch et al, (Anticancer Res 13: 2117-2118, 1993) evidence is presented that flow cytometrically detected DNA-hypodiploidy in canine neoplasms is genuine and not an artefact caused by autolysis or chemotherapy. Intervals between removal of tumors and freezing in our studies were much shorter (average 15 min, maximum 30 min) than e.g. for human breast tumors in which the percentage of hypodiploidy is about 2%. Also average CVs for the G0, 1 peaks in our FCM analysis of canine tumors (mammary 2.27 + 0.06, n = 179); thyroid 2.57 + 0.13, n = 88) were equal to or less than those usually found in the comparable human tumors. Biological arguments in favor of the existence of genuine hypodiploid stemlines are the finding of tetraploidized subclones of the original hypodiploid clone, the reappearance of the same hypodiploid stemline in distant metastases during clinical follow up, and the isolation of a cytogenetically and flow cytometrically hypodiploid cell line from a primary canine mammary carcinoma. It is concluded that Deitch et al, incorrectly have invoked autolysis as a source of hypodiploidy in our original studies on canine neoplasms. Our evidence for interspecies differences in the evolution of aneuploidy in tumors of the same organ therefore remains unchallenged.

P53 mutations in mammary tumor cell lines and corresponding tumor tissues in the dog.

Alterations in the p53 gene are frequently observed in a wide variety of human cancers. To elucidate the role of p53 in tumorigenesis of the dog, we analyzed nine mammary tumor cell lines, and the primary or metastatic tumors used for their establishment, for the presence of genomic p53 abnormalities. Possible genomic rearrangements were analyzed by Southern blotting using a canine cDNA probe. More subtle alterations were identified by single strand conformation polymorphism (SSCP) analysis for which we partially characterized the canine p53 gene (codon 109-388 as compared to the human gene). The presence of mutations in SSCP fragments with altered mobility was confirmed by DNA sequencing. Three of the nine cell lines showed a mutated p53 gene. All were missense mutations accompanied by loss of the wild type allele. The point mutations, at codon 176 (TGC * TTC), 236 (TAC * AAC) and 245 (GGC * GCC), were all located in one of the four regions that are frequently affected in human cancers. Analysis of the DNA extracted from the tumors of origin demonstrated the presence of two of these point mutations. These findings indicate the involvement of the p53 gene in the genesis of canine tumors in a way comparable to that of human tumors.

Growth hormone induces tyrosyl phosphorylation of the transcription factors Stat5a and Stat5b in CMT-U335 canine mammary tumor cells.

It has now been well documented that the normal and tumorous canine mammary glands can be extra-pituitary sites of substantial growth hormone (GH) synthesis. Until now, attempts to reproduce the GH synthesis in-vitro using canine mammary explants or mammary tumor cells have not been successful. Therefore, the response of CMT-U335 canine mammary tumor cells to administered porcine GH (pGH) was investigated as an in-vitro model to study the possible effects of GH synthesis on this site. CMT-U335 cells spontaneously express the growth hormone receptor (GHR) as well as the prolactin receptor (PRLR). Twenty five minutes after administration, GH induced, in a dose-dependent manner, phosphorylation of the transcription factors Stat5a and Stat5b. Clear phosphorylation was induced by 10(-7) M and 10(-8) M pGH, with virtually no phosphorylation at 10(-9) M pGH. A similar dose-dependent phosphorylation of Stat5a by ovine prolactin was found in these cells. Although at high concentrations binding of pGH to the canine PRLR can occur (albeit with a low pKa), the similar dose-dependent effect of oPRL on Stat5a phosphorylation indicated that pGH signaled through the GHR. Remarkably, pGH induced a moderately decreased proliferation of CMT-U335 tumor cells, which may indicate that GH induces differentiation in these tumor cells. The GH-induced activation of Stat5a and Stat5b in these cells, as part of the JAK/Stat signal transduction pathway, is consistent with mammary GH playing a role in autocrine and/or paracrine stimulation of (tumorous) mammary cells.

The accuracy of cytology in diagnosis and DNA analysis of canine mammary tumours.

Fine-needle aspirates from 84 spontaneous canine mammary tumours were used to assess the accuracy of this method in flow cytometric DNA analysis and cytological diagnosis. Defined samples from different tumour parts were analysed for histological diagnosis and DNA ploidy as a control. The DNA ploidy in cytology specimens and those obtained from the defined tumour samples, when testing for independence, was highly significant (P = 0.0001). The total accuracy of cytology was 79 per cent, with a sensitivity of 65 per cent and a specificity of 94 per cent. The results show the possibility of combining cytology and DNA analysis of fine-needle aspirates from canine mammary tumours. The method is suitable for preoperative diagnosis of canine mammary tumours as well as for measuring DNA ploidy, which makes it useful if DNA ploidy turns out to be of diagnostic and prognostic value in these tumours.

Progression of canine mammary tumours as reflected by DNA ploidy in primary tumours and their metastases.

Thirty dogs with metastasizing mammary tumours (carcinomas, n = 22; sarcomas, n = 8) were necropsied. Flow cytometric DNA analysis was carried out on frozen primary tumours and on selected metastases from the dogs. Ductular carcinomas (n = 13) had a varying growth pattern, in terms of histology, in both the primary tumours and metastases and between different metastases. The different types of DNA ploidy, including hypodiploidy, in the primary ductular carcinomas were also seen in the tumour metastases. Dogs with primary anaplastic carcinomas (n = 7) had multiple metastases, which were in most cases near-diploid or hyperdiploid. Two dogs had spindle-cell carcinomas, which were hypodiploid in both the primary tumour and the metastases. The DNA ploidy in the metastases was retained in 16 of the 22 dogs with primary carcinomas. Fibrosarcomas (n = 5) showed different types of DNA ploidy. In two of the three dogs with diploid or near-diploid osteosarcomas, the DNA ploidy was retained in the metastases. There was a statistically significant association between mammary tumours and metastases (P = 0.0001) in terms of both histological diagnosis and DNA ploidy. The association was retained when the carcinomas were tested separately (P = 0.0001); in the sarcomas it was retained weakly in terms of histology (P = 0.0183) but not DNA ploidy (P = 0.6659). The retention of the DNA ploidy in most carcinoma metastases indicated that selection of DNA ploidy had taken place prior to metastasis. The differences in patterns of DNA ploidy between ductular and anaplastic carcinomas may reflect different pathogenesis in these types of canine mammary tumour.

S100 protein is not specific for myoepithelial cells in the canine mammary gland.

The distribution of protein S100 in the canine mammary gland was studied, this substance having been described as specific for myoepithelial cells. Two immunohistochemical methods, peroxidase-anti-peroxidase complex (PAP) and alkaline phosphatase-anti-alkaline phosphatase complex (APAAP) were used on frozen sections of normal canine mammary gland. Both the myoepithelial and epithelial cells stained positively with PAP. With APAAP, staining was also seen in the controls, presumably because endogenous alkaline phosphatase was not blocked. As protein S100 was shown to be present both in myoepithelial and epithelial cells of the mammary gland, it was concluded that S100 is not a specific marker for myoepithelial cells.

Transcriptomic profile of two canine mammary cancer cell lines with different proliferative and anti-apoptotic potential.

The aim of the study was to identify the genes responsible for the high growth rate and antiapoptotic potential in selected canine mammary cancer cells. cDNA canine microarrays were used to compare the transcriptome in simple carcinoma CMT-U27 and spindle-cell tumor CMT-U309 cell lines. In CMT-U27 cell line the growth rate (shorter cell cycle), anti-apoptotic potential (higher expression of Bcl-2) was higher and spontaneous and induced apoptosis was lower. Comparison of transcriptomes revealed 333 genes which expression differed similarly. We focused on genes involved in cell proliferation, adhesion and apoptosis, and selected 29 of them. The high growth rate and anti-apoptotic potential in CMT-U27 cells was associated with enhanced expression of genes (at the level of transcripts) involved in Ca(2+) signaling pathway (Calmodulin 1, 2, 3 and SPSB2) and growth hormone cellular pathway. The low-proliferative and pro-apoptotic phenotype of CMTU309 cells was more dependent on TGFbeta, neuregulin 1 pathways and adhesion-related molecules.

Plasticity of cloned canine mammary spindle cell tumor, osteosarcoma and carcinoma cells.

Female dogs are frequently affected by mammary tumors, both carcinomas and sarcomas. The mechanisms behind mammary-tumor formation and the high degree of heterogeneity are not understood. To provide insight into this issue, it is important to determine the properties of the cells forming the different types of tumors. One question is if individual neoplastic cells can give rise to phenotypically distinct tumor types, i.e., show plasticity. We studied 3 different tumors (a spindle-cell tumor, an osteosarcoma, and a carcinoma) and followed the change of lineage marker expression between the primary canine mammary tumors, the clones derived from the corresponding tumors and in tumors generated after inoculation of tumor clones into nude mice (n = 75). Inoculation of clones derived from the spindle-cell tumor gave rise to spindle-cell tumors in nude mice. Several of these contained bone tissue, a sign of plasticity. Clones derived from the osteosarcoma were negative for a panel of lineage markers but, when inoculated into nude mice, they were able to form bone, again a sign of plasticity. In contrast to the primary carcinoma, most of the clones derived thereof lacked keratin expression, but keratin expression was recovered in most of the tumors formed after inoculation of clones into nude mice. Moreover, tumors generated from the carcinoma clones, in contrast to the primary tumor, were positive for smooth-muscle-cell markers. Our results point to plasticity in canine mammary tumors, as shown both by morphologic criteria and by expression patterns for lineage specific markers.

Canine mammary tumour cell lines established in vitro.

Mammary tumours are the most common tumours in the female dog. The tumours have a complex histology and exist in epithelial, mixed and mesenchymal forms. To study the biology of canine mammary tumours, five cell lines have been established and characterized. The results indicate that canine mammary tumours might be derived from mammary stem cells and that the tumour growth is independent of oestrogens. The established canine mammary tumour cell lines will be valuable tools in further studies of the histogenesis and pathogenesis of these tumours.

Characterization of four in vitro established canine mammary carcinoma and one atypical benign mixed tumor cell lines.

Five spontaneous canine mammary tumors were cultured in vitro and cell lines were established. The tumors included three frozen carcinomas, fine-needle aspirate from one fresh carcinoma, and one fresh atypical benign mixed tumor. The cell lines have so far been cultured for about 2 yr and passaged between 45 and 200 times. The cell lines expressed different types of intermediate filaments, including a heterogenous pattern. In some cases no intermediate filaments were expressed. Ultrastructure studies showed epithelial cells and cells intermediate between epithelial and myoepithelial types. Retrovirus associated A-particles were found in two carcinomas. The mixed mammary tumor cell line formed ductlike structures in collagen substrate. The cell lines grew when inoculated s.c. into male nude mice. Two carcinomas caused lymph node metastases in two mice and another carcinoma single lung metastases in one tested mouse. DNA hypodiploidy, studied by flow cytometry, in one of the primary carcinoma was retained in vitro, and this cell line showed polyploidy during later passages. The other cell lines had a more unstable DNA profile, although a tendency for polyploidy was found. These findings were also illustrated in chromosome studies.

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.

Immunohistochemical investigation into the distribution pattern of myoepithelial cells in the bovine mammary gland.

The distribution pattern of myoepithelial cells in the bovine mammary gland was investigated by an immunohistochemical technique, using monoclonal antibodies against cytokeratins 5, 6 and 18 and cytokeratins 8 and 14 and against alpha-smooth-muscle actin filaments. Myoepithelial cells were shown to be present as a continuous basal cell layer in the intralobular ducts, as discontinuous cell rows in the basal cell layer of the interlobular ducts, and as single cells dispersed in the basal cell layer of the quarter cisterns; while they were apparently absent in the teat cisterns. Unlike the case with myoepithelial cells of the human breast, anti-cytokeratin 14 was less specific as a marker of bovine myoepithelial cells than was anti-alpha-smooth-muscle actin.