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Immunohistochemical expression of p63 protein and calponin in canine mammary tumours
Research in Veterinary Science 123 (2019) 232–238
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Immunohistochemical expression of p63 protein and calponin in canine mammary tumours
T
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Wojciech Łopuszyńskia, , Marek Szczubiałb, Yolanda Millánc, Silvia Guil-Lunac, Raquel Sánchez-Céspedesc, Juana Martin de las Mulasc, Anna Śmiecha, Kamila Bulaka a
Sub-Department of Pathomorphology and Forensic Veterinary Medicine, Department and Clinic of Animal Internal Diseases, University of Life Sciences in Lublin, Poland Department and Clinic of Animal Reproduction, University of Life Sciences in Lublin, Poland c Department of Anatomy and Comparative Pathology, Córdoba University, Spain b
A R T I C LE I N FO
A B S T R A C T
Keywords: p63 protein Calponin Mammary tumours Female dogs Immunohistochemistry
The aim of this study was to compare the expression of p63 protein and calponin in terms of their affinity and specificity for myoepithelial cells in canine mammary tumours. The studied material included 10 benign and 32 malignant mammary tumours from female dogs treated with mastectomy. Primary mouse monoclonal antibodies directed against p63 protein clone 4A4 and calponin clone CALP were used in single- and doublestain system of immunohistochemical reaction. The investigations have shown that majority of myoepithelial cells in benign tumours and carcinomas in situ exhibited strong positive labelling for both markers. In other malignant tumours strong immunoreactivity was observed in resting myoepithelial cells (MECs) and hypertrophic myoepithelial cells (HMECs), while the immunoreactivity in spindle-stellate myoepithelial cells (SMECs) and rounded myoepithelial cells (RMECs) was moderate. The granular-diffuse nuclear expression of p63 protein was observed only in myoepithelial cells. In terms of calponin, diffuse cytoplasmic expression was noted not only in myoepithelial cell but also in some stromal fibroblasts and vascular smooth muscle cells. The epithelial cells did not exhibit specific expression of the investigated markers. The obtained results indicate that p63 is a sensitive and more specific marker of myoepithelial cells in canine mammary tumours compared with calponin. These findings suggest that the immunohistochemical analysis peformed with the use of p63 can be a valuable complement of routine histological examinations of canine mammary tumours facilitating identification of tumours with myoepithelial component.
1. Introduction Mammary tumours are one of the most common neoplasms in female dogs. They are malignant in approximately 50% of cases and lead to substantial reduction in life expectancy in affected animals (Arnesen et al., 2001; Sleeckx et al., 2011). A characteristic feature of canine mammary tumours (CMTs) is the high proportion of myoepithelial cells in the neoplastic proliferation. Their distinction is essential for the histogenetic classification and prognosis. Malignant mammary tumours with myoepithelial component (complex carcinomas) have a better prognosis than simple carcinomas with only epithelial component (Goldschmidt et al., 2011; Misdorp et al., 1999). In a normal mammary gland, myoepithelial cells are located between the epithelial lining and the basement membrane of the secretory portion. The cytoplasm of myoepithelial cells contains fibrils similar to myofibrils present in smooth muscle cells, in addition to the cytokeratin protein
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characteristic for epithelial cells. Furthermore, these cells are similar to smooth muscle cells in terms of the distribution of cell organelles, cell membrane differentiation, and presence of the actin-myosin complex (Pulley, 1973; Vos et al., 1993). Under neoplastic transformation, myoepithelial cells change their normal microscopical appearance and assume unusual shape and arrangement, which has been demonstrated in both histological tissue sections and in cell cultures (SánchezCéspedes et al., 2013b; Zhang et al., 2003). This fact additionally impedes proper identification of myoepithelial cells in routine histopathological examination (Bertagnolli et al., 2009; Zhang et al., 2003). Due to the difficulties in identification of myoepithelial cells in routine histopathological examination of CMTs, various immunohistochemical markers, e.g. cytokeratin (Griffey et al., 1993; Vos et al., 1993), α-smooth muscle actin (Destexhe et al., 1993), calponin (Espinosa de los Monteros et al., 2002), protein 14-3-3-ơ (SuarezBonnet et al., 2011), and antigen CD-10 (Sánchez-Céspedes et al.,
Corresponding author. E-mail address: [email protected] (W. Łopuszyński).
https://doi.org/10.1016/j.rvsc.2019.01.017 Received 26 June 2018; Received in revised form 11 January 2019; Accepted 15 January 2019 0034-5288/ © 2019 Published by Elsevier Ltd.
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temperature of 56 °C for 12 h. After removal from the incubator, tissue sections were deparaffinised in xylene and passed through decreasing concentrations of alcoholic solutions to distilled water. Next, the tissue sections were incubated in a 3% solution of hydrogen peroxide in methanol for 30 min to block endogenous peroxidase. Next step of the procedure involved heat-induced antigen retrieval (water bath at 95–98 °C with 10 mM citrate buffer, pH = 6.0, 8 min). After cooling to room temperature, tissue sections were rinsed in distilled water and TBS buffer, outlined with a paraffin pen, and placed in a humidified chamber. Dilutions of appropriate primary antibodies that were prepared earlier were applied to the tissue sections. The incubation was carried out at a temperature of 37 °C in a humidified chamber for 60 min. Primary mouse monoclonal antibody directed against p63 protein clone 4A4 (Dako, M7242) at dilution 1:50 was used in the investigations. In the next step, tissue sections were rinsed in TBS buffer and a biotinylated secondary antibody, streptavidin-peroxidase complex, and 3,3′-diaminobenzidine tetrahydrochloride DAB (SK-4100; Vector Laboratories, Peterborough, UK) chromogen were applied. The chromogen was prepared 10 min prior to the use as follows: 10 mg DAB in 10 ml 0.05 M Tris-HCl with pH = 7.4 with addition of 100 μl 3% H2O2. Each step was terminated by rinsing in the buffer. Finally, tissue sections were stained with Mayer's haematoxylin for approximately 2–3 min, rinsed with water, dehydrated in alcohol, placed in xylene and sealed in mounting medium. To confirm the myoepithelial phenotype of cells labelled with the anti-p63 antibody, reactions with the primary mouse monoclonal antibody directed against calponin clone CALP (Dako, M3556) at dilution 1: 400 were carried out. In order to reveal coexpression of p63 protein and calponin simultaneously, selected tissue sections were stained with two separate chromogens, i.e. DAB for visualisation of the nuclear reaction after incubation with anti-p63 antibody and Fast Red for visualisation of the cytoplasmic reaction after incubation with anti-calponin antibody. A double control system was used in all reactions, i.e. a negative control, where incubation with the primary antibody was replaced by incubation with relevant non-immunised commercial IgG sera, and a positive control, which was a normal mammary tissue adjacent to neoplastic mass (internal control).
2013a), have been used in the diagnostics of tumours with a myoepithelial component. Despite their high sensitivity, these markers proved to be insufficiently specific for myoepithelial cells in CMTs. Therefore, identification of new diagnostically useful immunohistochemical markers of myoepithelial cells in canine mammary tumours is indispensable. The p63 protein is a product of the p63 gene translation, i.e. a homologue of the p53 suppressor gene. The p63 gene functions as a nuclear transcription factor for proliferation, apoptosis, and differentiation of cells (Maisse et al., 2003). The p63 protein plays an important role in the proliferation, differentiation, aging, and adhesion of epithelial cells and has a number of functions in the origination of tumours (Bertagnolli et al., 2009; Carroll et al., 2006). The p63 protein is a sensitive and specific marker of myoepithelial cells in human breast tumours (Batistatou et al., 2003; Kim et al., 2014; Stefanou et al., 2004). However, there are few reports of the suitability of p63 protein for identification of myoepithelial cells in CMTs (Gama et al., 2003; Ramalho et al., 2006; Sánchez-Céspedes et al., 2016). Calponin is a specific 34-kDa protein involved in regulation of smooth muscle contraction. It is a highly sensitive marker of myoepithelial cells, often used for immunohistochemical analysis of human and animal tissues (Espinosa de los Monteros et al., 2002; Moritoni et al., 2002; Sánchez-Céspedes et al., 2011). However, its value as a marker of myoepithelial cells is limited by the fact that it labels also myofibroblasts and smooth muscle cells as well (Espinosa de los Monteros et al., 2002; Moritoni et al., 2002; Sánchez-Céspedes et al., 2013a). The aim of this study was to compare the expression of p63 protein and calponin in terms of their affinity and specificity for myoepithelial cells in order to assess their usefulness in routine histopathological diagnostics of canine mammary tumours. 2. Material and methods 2.1. Material and histological examinations The studied material included 42 mammary tumours (10 benign and 32 malignant) sampled from 4 to 15-year-old female dogs of different breeds (pedigree and mixed) treated with mastectomy. Depending on the size, up to 6 slices were sampled from each resected tumour from the peripheral zone, macroscopically visible growth zone, and surgical margin. Tissue samples were fixed for 24 h in 10% formalin, pH = 7.2, and passed for 24 h through increasing concentrations of alcoholic solutions, acetone, and xylene to paraffin in a tissue processor (Leica TP1020, Leica Biosystems, Nussloch, Germany). After fixation, hard in palpation tumours with chondrification and/or ossification foci were cut into 5-mm thick sections, decalcified in Osteodec reagent (BioOptica, Milano, Italy) for 24 h, and embedded in paraffin blocks as described above. Tissue samples were sectioned on 4-μm-thick slices on a sledge microtome (Leica SR-200, Leica Biosystems, Nussloch, Germany) and placed on glass slides. Histopathological evaluation of haematoxylin and eosin-stained tissue sections was carried out under a light microscope (Nikon Eclipse E-600, Nikon Instruments, Inc. New York). The microscopic assessment of the tissue sections was based on the histological classification of canine mammary tumours proposed by AFIP/WHO (Misdorp et al., 1999).
2.3. Evaluation of immunohistochemical data Tissue section fields with a distinct histopathological pattern of a given tumour type were selected in each case. Selected fields were analysed for the visualisation of markers, taking into account the nuclear staining in the case of p63 protein expression and the cytoplasmic staining in the case of calponin expression. Based on the morphological appearance and localisation, the analysed cells were subdivided into luminal epithelial cells (LCs), resting myoepithelial cells (MECs), hypertrophic myoepithelial cells (HMECs), spindle-stellate myoepithelial cells (SMECs), and rounded myoepithelial cells (RMECs). Expression of p63 protein was evaluated based on the percentage of cells with a positive reaction and the cell staining intensity. A semiquantity scoring system (SQS) of evaluation of the number of stained cells was used, where + = less than 10% of positive cells, ++ = 11–59% of positive cells, and +++ = more than 60% of positive cells. The staining intensity was assessed in a 3-grade intensity scoring system (IS), where 1i – weak staining, 2i – moderate staining, and 3i – strong staining.
2.2. Immunohistochemical analyses
3. Results
The immunohistochemical examinations were performed in systems of detection of antigen-antibody complexes based on biotinylated secondary antibodies directed against monoclonal mouse primary antibodies in a single staining LSAB plus HRP kit (Dako, K0690) and a double staining EnVision System (Dako, K1395). Tissue sections for immunohistochemical evaluation were deposited on silane-coated Super Frost glass slides (Menzel-Glaser) and kept in an incubator at a
3.1. Results of histological examinations Based on the histopathological examination, 10 (23.8%) benign tumours and 32 (76.2%) malignant tumours were distinguished in the analysed group. Detailed histopathological results are shown in Table 1. 233
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Table 1 Histopathological classification of the 42 canine mammary gland tumours. Tumour types
No. of cases (%)
Benign tumours Duct papilloma Fibroadenoma Complex adenoma Benign mixed tumour Malignant tumours Carcinoma in benign tumour In situ carcinoma Complex carcinoma Simple carcinoma Total
10 (23.80) 2 (4.76) 1(2.38) 4 (9.52) 3 (7.14) 32 (76.20) 3 (7.14) 2 (4.76) 11 (26.20) 16 (38.10) 42 (100.00)
In 16 cases, normal mammary gland tissue, which was the positive internal control in the study, remained attached to the adjacent tumour mass.
Fig. 1. Complex adenoma. In myofibroblasts (short arrow) p63 protein expression is not evident and vascular endothelial cells (long arrow) expressing calponin are seen. Double staining system, immunohistochemistry, Harris's haematoxylin counterstain. Magn. 200×.
3.2. Results of immunohistochemical examinations Granular-diffuse nuclear labelling for p63 protein and diffuse cytoplasmic labelling for calponin was noted in myoepithelial cells. Intensity of the expression, percentage of positive stained cells and number of tumours positive for specific cell type differ depending on the histological tumour type. The results of immunohistochemical reactions are summarised in Table 2. The luminal epithelial cells in the
normal and neoplastic mammary gland did not exhibit expression of p63 protein and calponin. A positive cytoplasmic labelling for calponin was noted in some stromal fibroblasts and vascular smooth muscle cells (Fig. 1).
Table 2 Characterization of cell types with expression of p63 protein and calponin in canine normal and neoplastic mammary gland. Type of tumour
n
Cell morphology*
Immunoreactivity for p63
Immunoreactivity for calponin
n1 (%)
SQS
IS
n1 (%)
SQS
IS
Normal mammary gland
16
LCs MECs
0 16 (100)
– +++
– 3i
0 16 (100)
– +++
– 3i
Benign tumours Duct papilloma
2
LCs MECs HMECs LCs MECs HMECs LCs MECs HMECs SMECs LCs MECs HMECs SMECs RMEC
0 2 2 0 1 1 0 4 3 3 0 3 2 2 2
(100) (66.6) (66.6) (66.6)
– +++ +++ – +++ +++ – +++ +++ + – +++ ++ + +
– 3i 3i – 3i 3i – 3i 2i 2i – 3i 2i 1i 1i
0 2 2 0 1 1 0 4 4 3 0 3 3 2 2
(100) (100) (66.6) (66.6)
– +++ +++ – +++ +++ – +++ +++ ++ – +++ ++ + +
– 3i 3i – 3i 3i – 3i 3i 2i – 3i 3i 2i 2i
LCs MECs HMECs SMECs RMECs LCs MECs HMECs LCs MECs HMECs SMECs LCs MECs HMECs
0 3 (100) 2 (66.6) 1 (33.3) 1 (33.3) 0 2 (100) 2(100) 0 11 (100) 8 (72.7) 5 (45.4) 0 16 (100) 11(68.7)
– ++ + + + – +++ +++ – +++ + + – +++ ++
– 2i 2i 1i 1i – 3i 3i – 3i 2i 2i – 3i 2i
0 3 (100) 3 (100) 2 (66.6) 2 (66.6) 0 2 (100) 2(100) 0 11 (100) 10 (90.9) 7 (63.6) 0 16 (100) 14 (87.5)
– ++ ++ + + – +++ +++ – ++ ++ + – +++ ++
– 3i 2i 2i 1i – 3i 3i – 3i 2i 2i – 3i 2i
Fibroadenoma
Complex adenoma
Benign mixed tumour
Malignant tumours Carcinoma in benign tumour
1
4
3
3
In situ carcinoma
2
Complex carcinoma
11
Simple carcinoma
16
(100) (100) (100) (100) (100) (75) (75)
(100) (100) (100) (100) (100) (100) (75)
*LCs – luminal epithelial cells, MECs – myoepithelial cells, HMECs – hypertrophic myoepitheliel cells, SMECs – spindle-stellate myoepithelial cells, RMECs – rounded myoepitheliel cells, n1 – number of tumours positive for specific cell type, *SQS: – = no stained cells, + = less than 20% positive cells, ++ = 20–59% positive cells, +++ = more than 60% positive cells, IS: 1i – week, 2i – moderate, 3i – strong. 234
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Fig. 2. Coexpression of p63 protein (brown nuclear staining) and calponin (red cytoplasmic staining) in myoepithelial cells of the normal mammary gland. Double staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 200×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Duct papilloma. Multiple papillary projections protruding into the lumen of the pre-existing duct. Prevalent hypertrophic myoepithelial cells and minor in number resting myoepithelial cells with nuclear overexpression of p63 protein. Single staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 100×.
The resting myoepithelial cells (MECs) with elongated shape and flattened nuclei were present in all examined tumour samples and normal mammary gland tissue sections. However, depending on the type of the tumour, MECs formed a single continuous or discontinuous layer around normal secretory acini and ducts as well as around neoplastic structures build up by proliferating luminal epithelial cells. In the normal canine mammary gland, a strong (3i-score) granular-diffuse pattern of nuclear p63 protein expression was evenly noted in MECs along the ductal and acinar structures. Labelling for calponin was uniformly visualized in the cytoplasm of MECs by forming a continuous layer around the ducts and secretory acini. The double staining system revealed marked coexpression of the nuclear p63 protein and cytoplasmic calponin simultaneously in the same resting myoepithelial cells (Fig. 2). In all examined benign and malignant tumours, MECs exhibited strong nuclear expression of p63 protein (SQS = +++, IS = 3i) and a similar cytoplasmic expression of calponin. Hypertrophic myoepithelial cells (HMECs) with a polygonal shape and an ovoid nucleus formed a single continuous or discontinuous layer around the multiform neoplastic structures composed of luminal epithelial cells. There were no such cells in the normal mammary tissue. In two examined duct papillomas presenting as papillary projections protruding into the expanded lumina of pre-existing secretory ducts, HMECs, likewise MECs, formed a single continuous layer under the luminal epithelial lining and tended to build up at the boundary with the connective tissue stroma (Fig. 3). In both examined tumour's samples a vast majority of HMECs exhibited intense positive immunohistochemical reaction (SQS = +++, IS = 3i) for both markers. In the single case of fibroadenoma composed of epithelial elements scattered among a rich connective tissue stroma, the nuclear expression of p63 protein was confined to a thin layer of slightly dispersed HMECs and MECs surrounding the negative luminal epithelial component. The nature of the immunohistochemical reactions was identical as in the case of the duct papilloma (SQS = +++, IS = 3i). The nuclear expression of p63 protein was evident in the HMECs in the case of 3/4 (75%) complex adenomas and 2/3 (66.6%) benign mixed tumours (BMTs). HMECs, similarly to MECs, created a single continuous layer around luminal epithelium deformed by proliferating spindle-stellate myoepithelial cells embedded in an amorphous, faintly eosinophilic matrix, and, in the case of BMTs, by areas of cartilage and/or bony structures with varied degrees of maturation. A majority of HMECs in complex adenomas and BMTs exhibited
Fig. 4. Immunoreactivity of hypertrophic myoepithelial cells. Coexpression of p63 protein and calponin in hypertrophic myoepithelial cells in complex adenoma. Double staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 200× (a). Discontinuous layer of hypertrophic myoepithelial cells showing moderate nuclear expression of p63 protein in simple tubulopapillary carcinoma. Single staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 200× (b).
positive reaction (SQS = +++) with moderate staining intensity (IS = 2i) (Fig. 4a). In carcinomas in situ, HMECs were located in the immediate proximity of MECs and, as in the benign tumours and normal glandular tissue, both types of cells formed a continuous layer serving as a hallmark of non-invasive character of the tumour's growth. In the group of malignant tumours, HMECs formed an incomplete and discontinuous layer around the glandular structures (Fig. 4b). The immunohistochemical staining of HMECs in carcinomas in situ revealed the highest parameters of immunoreactivity (SQS = +++, IS = 3i). The moderate (IS = 2i) nuclear expression of p63 protein was observed in 2/3 (66.6%) cases of carcinomas arising in complex adenomas, in 8/ 11 (72.7%) cases of the complex carcinomas, and in 11/16 (68.7%) cases of the simple carcinomas. In carcinomas in benign tumours and in complex carcinomas, the expression of p63 protein was observed in less than 10% of HMECs (SQS = +), whereas in the group of simple carcinomas positive nuclear reaction was observed in 10–60% of HMECs. The strong cytoplasmic reaction due to the presence of calponin antigen
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Fig. 5. Significant reduction of expression (in number and intensity) of p63 protein in spindle-shaped (a) and stellate (b) myoepithelial cells. Single staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 200×.
Fig. 6. An area of early chondroid differentiation in benign mixed tumour. Weak nuclear expression of p63 protein (arrows) and cytoplasmic positive reaction in the presence of calponin antigen in some rounded myoepithelial cells. Double staining system, immunohistochemistry, Harris's haematoxylin counterstain. Magn. 400×.
in HMECs was visible in all examined benign neoplasms (IS = 3i). Strong staining intensity was observed in two non-infiltrating carcinomas. In other malignant tumours, the expression of calponin in HMECs was moderate (IS = 2i), and the positive reaction was visible in almost all cases (87.5%–100%). Nuclear expression of p63 protein in SMECs forming intertwining branches between deformed structures made up of luminal epithelial cells, was observed in 3/4 (75%) cases of complex adenomas, in 5/11 (45.4%) cases of complex carcinomas, in 2/3 (66.6%) cases of benign mixed tumours, and in 1/3 (33.33%) case of the carcinoma in benign tumour. Weak to moderate staining intensity (IS = 1i–2i) was noted in these types of tumours (Fig. 5). The expression of calponin by SMEC in all analysed tumours was moderate (IS = 2i), and the percentage of positive reactions ranged from 63.6% in the case of the complex carcinomas to 75% in the complex adenomas. Rounded myoepithelial cells (RMECs) exhibiting p63 protein were only found in the areas with immature cartilage formation in 2/3 (66.6%) of BMT and in 1/3 (33.3%) of carcinoma in benign tumour. In these cases the nuclear staining was weak (IS = 1i). The coexpression of calponin and p63 protein in RMECs was demonstrated by doublestain system (Fig. 6), and was characterised by weak to moderate intensity of staining (IS = 1i–2i).
with various antibodies is believed to be the most suitable method for in vivo examination and identification of myoepithelial cells (SánchezCéspedes et al., 2016). This study presents investigations of the expression of p63 protein in benign and malignant CMTs and comparison thereof with the expression of calponin, which is a well-known highly sensitive marker of myoepithelial cells in human breast tumours as well as canine and feline mammary tumours. The results of the investigation indicate that p63 protein is a highly specific and sensitive marker of myoepithelial cells in canine mammary tumours. In both benign and malignant tumours, nuclear expression of p63 protein was noted in the myoepithelial cell layer surrounding the layer of luminal epithelial cells. Epithelial cells, connective tissue cells, and vascular smooth muscle cells were non-reactive with anti-p63 antibody. These results are in agreement with those reported in earlier studies, which described the usage of p63 as an immunohistochemical marker of myoepithelial cells in human breast tumours (Koker and Kleer, 2004; Stefanou et al., 2004; Werling et al., 2003) and canine mammary tumours (Gama et al., 2003; Ramalho et al., 2006; Rasotto et al., 2014). The cytoplasmic expression of calponin in the myoepithelial cells of the analysed benign and malignant canine mammary tumours was comparable to p63 protein nuclear expression. Epithelial cells exhibited no expression of calponin, similarly to p63, whereas positive reaction was found in myofibroblasts and vascular smooth muscle cells. The high sensitivity of calponin as a marker of myoepithelial cells of the canine mammary gland as well as its low specificity were demonstrated in many previous studies (Espinosa de los Monteros et al., 2002; Martin de las Mulas et al., 2004; Rasotto et al., 2014). The results and observations reported by other authors (Ramalho et al., 2006; Sánchez-Céspedes et al., 2016) indicate that nuclear p63 immunolabelling can be used simultaneously with cytoplasmic markers of myoepithelial cells. This greatly facilitates immunohistochemical examinations, as anti-p63 antibodies and antibodies directed against other markers of myoepithelial cells can be placed on the same slide with the analysed tumour section. Our studies with the use of double staining system with two separate chromogens enabled assessment of expression of p63 and calponin in individual myoepithelial cells. Coexpression of p63 and calponin was confirmed in all distinguished subtypes of myoepithelial cells indicating at the same time the difference in their sensitivity. The nuclear expression of p63 turned out to be qualitatively different from cytoplasmic expression of calponin. The
4. Discussion In the normal mammary gland of female dogs, the ductal and lobular portion is lined with two types of cells – an inner luminal epithelial and an outer myoepithelial cell layer (Misdorp, 2002; SánchezCéspedes et al., 2016). Myoepithelial cells are involved in milk ejection and regulation of the growth, differentiation, and morphogenesis of neighbouring cells (Adriance et al., 2005). Unlike in humans and female cats, myoepithelial cells in female dogs are often involved in the neoplastic transformation of the mammary gland tissue. The presence of myoepithelial cells is crucial for the histogenetic classification of canine mammary tumours, and has an impact on the prognosis and evaluation of tumour progression (Misdorp, 2002; Misdorp et al., 1999; Yoshimura et al., 2014). Attention is also paid to the role of myoepithelial cells in identification of carcinomas in situ (Sánchez-Céspedes et al., 2011). However, the identification of myoepithelial cells in routine histopathological examinations of canine mammary tumours is not obvious (Zhang et al., 2003). Many simple carcinomas in haematoxylin and eosin staining turned out to be complex when assessed using immunohistochemical techniques (Martin de las Mulas et al., 2004; Yoshimura et al., 2014). Therefore, immunohistochemical marking 236
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lesions and carcinomas in situ. In the case of canine mammary tumours, the usage of immunohistochemical markers for examination of integrity of the myoepithelial cell layer in diagnostics of carcinoma in situ has not yet been thoroughly investigated. There is only one study with calponin as a suitable marker to analyse the integrity of the myoepithelial cell layer in canine mammary carcinomas in situ (Sánchez-Céspedes et al., 2011). Our research has shown that the p63 and calponin-positive MECs and HMECs formed a continuous layer around the epithelial structures in benign mammary tumours and carcinomas in situ, whereas in malignant mammary tumours, MECs and HMECs formed a discontinuous layer. Furthermore, the expression of p63 protein and calponin in MECs and HMECs in carcinomas in situ was characterised by the highest immunoreactivity parameters (SQS = +++; IS = 3i). This results may suggest that p63 protein and calponin can be used simultaneously as significant markers in diagnostics of carcinoma in situ of the canine mammary gland.
nuclear pattern of p63 expression gave the advantage of accurate distinction of variably stained nuclei of all subtypes of myoepithelial cells from the unstained nuclei of the adjacent cells enabling a more objective assessment. The cytoplasmic expression of calponin was not easily identifiable in attenuated, compressed or misshapen cells. This difference seems to be also related to the function of both p63 proteins and calponin in the cell. Nuclear p63 protein acts as a sequence specific DNA binding transcriptional activator or repressor of proliferation, apoptosis, and differentiation which makes it less dependent on the influence of internal and external interactions. Phosphorylation and changes in cytoskeleton tension can modify the function and structure of calponin leading to its gradual degradation (Liu and Jin, 2016). The double staining system proved to be helpful in distinguishing between calponin-positive myofibroblasts and calponin-positive myoepithelial cells. However, double immunohistochemical technique is more laborintensive and difficult to implement in routine diagnostic laboratory, it proved to be extremely useful in the comparative assessment of p63 and calponin expression. As shown by Reis-Filho et al. (Reis-Filho et al., 2003), p63 protein can also be used as a marker of myoepithelial cells in cytology. Previous studies suggest transformation of myoepithelial cells from MECs to FMECs and SMECs in CMTs (Espinosa de los Monteros et al., 2002). Investigations conducted on mammary tumour cell lines collected from female dogs have shown that the transformed myoepithelial cells were characterised by varied morphology, different growth pattern, and diverse marker labelling (Sánchez-Céspedes et al., 2013b), which was also confirmed in the present study by immunohistochemistry. In examined benign tumours, in MECs in all cases and in HMECs in most cases strong labelling of both markers was noted. In turn, moderate or low immunoreactivity was noted in SMECs and RMECs. In the group of malignant tumours, high expression of p63 protein and calponin was detected in both MECs and HMECs in all cases of carcinomas in situ and only in MECs of all the other malignant neoplasms. HMECs, SMECs, and RMECs exhibited moderate or weak immunoreactivity in the group of malignant tumours with a slightly higher percentage of tumours positive for calponin. Bertagnolli et al. (Bertagnolli et al., 2009) analysed the expression of p63 protein in different subtypes of myoepithelial cells in benign mixed tumours and in carcinomas in benign tumours and found the highest immunoreactivity in resting myoepithelial cells (MECs) of benign mixed tumours. Similar results were reported by other authors (Espinosa de los Monteros et al., 2002). Sánchez-Céspedes et al. (Sánchez-Céspedes et al., 2011) found that the expression of calponin depended on the type of canine mammary tumour and was the highest in dysplasia and low grade malignant tumours. Calponin expression in the group of malignant tumours was decreasing with the increasing histological malignancy grade. This indicates that p63 protein and calponin expression in myoepithelial cells including microscopical presentation of immunohistochemical marking can change with time during tumour expansion and may suggest the benign or malignant nature of the tumour. The mechanism of the loss of immunohistochemical markers in myoepithelial cells during progression and differentiation of CMTs remains unknown, although it is undoubtedly related to gradual transformation from resting myoepithelial cells to cartilage and the loss of epithelial features which are replaced by mesenchymal properties. It could also result from dynamic interaction between epithelial and myoepithelial cells as well as extracellular matrix. It has been suggested that some proteolytic enzymes produced by malignant epithelial cells could affect structure and function of myoepithelial cells (Zhang et al., 2003). In humans, the integrity of the myoepithelial cell layer is an important hallmark of carcinomas in situ. Invasive carcinomas are characterised by the absence of integrity of the myoepithelial cell layer, what can be visualized using immunohistochemical markers (Moriya et al., 2009; Yaziji et al., 2000). Batistatou et al. (Batistatou et al., 2003) reported no expression of p63 protein in invasive breast tumours, whereas positive reaction persisted in myoepithelial cells in benign
5. Conclusion The investigations indicate that p63 protein is a more specific and sensitive marker of myoepithelial cells in canine mammary tumours in comparison with calponin. The immunohistochemical reaction with the use of anti-p63 antibody can be a complementary examination in the routine histological analysis of canine mammary tumours, facilitating identification of tumours with a myoepithelial component. References Adriance, M.C., Inman, J.L., Petersen, O.W., Bissll, M.J., 2005. Myoepithelial cells: good fences make good neighbors. Breast Cancer Res. 7, 190–197. Arnesen, K., Gamlem, H., Glattre, E., Grondalen, J., Moe, L., Nordstoga, K., 2001. The Norwegian Canine Cancer Register 1990–1998. Report from the project “cancer in the dog”. Eur. J. Comp. Anim. Pract. 11, 159–169. Batistatou, A., Stefanou, D., Arkoumani, E., Agnantis, N.J., 2003. The Usefulness of p63 as a Marker of Breast Myoepithelial Cells. In Vivo. 17. pp. 573–576. Bertagnolli, A.C., Cassali, G.D., Genelhu, M.C., Costa, F.A., Oliveira, J.F., Goncalves, P.B., 2009. Immunohistochemical expression of p63 and ΔNp63 in mixed tumors of canine mammary gland and its relation with p53 expression. Vet. Pathol. 46, 407–415. Carroll, D.K., Carroll, J.S., Leong, C.O., Cheng, F., Brown, M., Mills, A.A., Brugge, J.S., Ellisen, L.W., 2006. p63 regulates an adhesion programme and cell survival in epithelial cells. Nat. Cell Biol. 8, 551–561. Destexhe, E., Lespagnard, L., Degeyter, M., Heymann, R., Coignoul, F., 1993. Immunohistochemical identification of myoepithelial, epithelial, and connective tissue cells in canine mammary tumors. Vet. Pathol. 30, 146–154. Espinosa de los Monteros, A., Millan, M.Y., Ordas, J., Carrasco, L., Reymundo, C., Martin de las Mulas, J., 2002. Immunolocalization of the smooth muscle-specific protein calponin in complex and mixed tumors of the mammary gland of the dog: assessment of the morphogenetic role of the myoepithelium. Vet. Pathol. 39, 247–256. Gama, A., Alves, A., Gartner, F., Schmitt, F., 2003. P63: a novel myoepithelial cell marker in canine mammary tissues. Vet. Pathol. 40, 412–420. Goldschmidt, M., Pena, L., Rasotto, R., Zappulli, V., 2011. Classification and grading of canine mammary tumours. Vet. Pathol. 48, 117–131. Griffey, S.M., Madewell, B.R., Dairkee, S.H., Hunt, J.E., Naydan, D.K., Higgins, R.J., 1993. Immunohistochemical reactivity of basal and luminal epithelium-specific cytokeratin antibodies within normal and neoplastic canine mammary glands. Vet. Pathol. 30, 155–161. Kim, S.K., Jung, W.H., Koo, J.S., 2014. p40 (ΔNp63) expression in breast disease and its correlation with p63 immunohistochemistry. Int. J. Clin. Exp. Pathol. 7, 1032–1041. Koker, M.M., Kleer, C.G., 2004. p63 expression in breast cancer: a highly sensitive and specific marker of metaplastic carcinoma. Am. J. Surg. Pathol. 28, 1506–1512. Liu, R., Jin, J.P., 2016. Calponin isoforms CNN1, CNN2 and CNN3: regulators for actin cytoskeleton functions in smooth muscle and non-muscle cells. Gene 585, 143–153. Maisse, C., Guerrieri, P., Melino, G., 2003. P73 and p63 protein stability: the way to regulate function? Biochem. Pharmacol. 66, 1555–1561. Martin de las Mulas, J., Reymundo, C., Espinosa de los Monteros, A., Millan, Y., Ordas, J., 2004. Calponin expression and myoepithelial cell differentiation in canine, feline and human mammary simple carcinomas. Vet. Comp. Oncol. 2, 24–35. Misdorp, W., 2002. Tumors of the mammary gland. In: Meuten, D.J. (Ed.), Tumors in Domestic Animals, fourth ed. Iowa State Press, Blackwell Publishing Company, USA, pp. 575–606. Misdorp, W., Else, R.W., Hellmen, E., Lipscom, T.P., 1999. Histological classification of the mammary tumors of the dog and the cat. 2nd series In: World Health Organization International Histologic Classification of Tumors of Domestic Animals. vol. VII. Armed Forces Institute of Pathology, Washington, D.C., pp. 16–29. Moritoni, S., Kushima, R., Sugihara, H., Bamba, M., Kobayashi, T.K., Hattori, T., 2002. Availability of CD10 immunohistochemistry as a marker of breast myoepithelial cells on paraffin sections. Mod. Pathol. 15, 397–405.
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de los Monteros, A., Martin de las Mulas, J., 2016. Myoepithelial cells in canine mammary tumours. Vet. J. 207, 45–52. Sleeckx, N., de Rooster, H., Veldhuis Kroeze, E.J., Van Ginneken, C., Van Brantegem, L., 2011. Canine mammary tumours, an overview. Reprod. Domest. Anim. 46, 1112–1131. Stefanou, D., Batistatou, A., Nonni, A., Arkoumani, E., Agnantis, N.J., 2004. p63 expression in benign and malignant breast lesions. Histol. Histopathol. 19, 465–471. Suarez-Bonnet, A., Herraez, P., Martin de las Mulas, J., Rodriguez, F., Deniz, J.M., Espinosa de los Monteros, A., 2011. Expression of 14-3-3 ơ protein in normal and neoplastic canine mammary gland. Vet. J. 190, 345–351. Vos, J.H., Van den Ingh, T.S., Misdorp, W., Molenbeek, R.F., Van Mil, F.N., Rutterman, G.R., Ivanyi, D., Ramaekers, F.C., 1993. Immunohistochemistry with keratin, vimentin, desmin, and α-smooth muscle actin monoclonal antibodies in canine mammary gland: Benign mammary tumors and duct ectasis. Vet. Q. 14, 89–95. Werling, R.W., Hwang, H., Yaziji, H., Gown, A.M., 2003. Immunohistochemical distinction of invasive from noninvasive breast lesions: a comparative study of p63 versus calponin and smooth muscle myosin heavy chain. Am. J. Surg. Pathol. 27, 82–90. Yaziji, H., Gown, A.M., Sneige, N., 2000. Detection of stromal invasion in breast cancer: the myoepithelial markers. Adv. Anat. Pathol. 7, 100–109. Yoshimura, H., Nakahira, R., Kishimoto, T.E., Michishita, M., Ohkusu-Tsukada, K., Takahashi, K., 2014. Differences in indicators of malignancy between luminal epithelia cell type and myoepithelial cell type of simple solid carcinoma in the canine mammary gland. Vet. Pathol. 51, 1090–1095. Zhang, R.R., Man, Y.G., Vang, R., Saenger, J.S., Barner, R., Wheeler, D.T., Liang, C.Y., Vinh, T.N., Bratthauer, G.L., 2003. A subset of morphologically distinct mammary myoepithelial cells lacks corresponding immunophenotypic markers. Breast Cancer Res. 5, 151–156.
Moriya, T., Kozuka, Y., Kanomata, N., Tse, G.M., Tan, P.H., 2009. The role of immunohistochemistry in the differential diagnosis of breast lesions. Pathology 41, 68–76. Pulley, L.T., 1973. Ultrastructural and histochemical demonstration of myoepithelium in mixed tumors of canine mammary gland. Am. J. Vet. Res. 5, 55–67. Ramalho, L.N., Ribeiro-Silva, A., Cassali, G.D., Zucoloto, S., 2006. The expression of p63 and cytokeratin 5 in mixed tumors of the canine mammary gland provides new insights into the histogenesis of these neoplasms. Vet. Pathol. 43, 424–429. Rasotto, R., Goldschmidt, M.H., Castagnaro, M., Carnier, P., Caliari, D., Zappulli, V., 2014. The dog as a natural animals model for study of the mammary myoepithelial basal cell lineage and its role in mammary carcinogenesis. J. Comp. Pathol. 151, 166–180. Reis-Filho, J.S., Milanezi, F., Amendoeira, I., Albergaria, A., Schmitt, F.C., 2003. Distribution of p63, a novel myoepithelial marker, in fine-needle aspiration biopsies of the breast: an analysis of 82 samples. Cancer 99, 172. Sánchez-Céspedes, R., Millan, Y., Guil-Luna, S., Garcia-Monterde, J., Reymundo, C., Espinosa de los Monteros, A., Martin de las Mulas, J., 2011. Myoepithelial cell layer integrity in canine mammary carcinoma. J. Comp. Pathol. 145, 25–30. Sánchez-Céspedes, R., Suárez-Bonnet, A., Millán, Y., Guil-Luna, S., Reymundo, C., Espinosa de los Monteros, A., Martin de las Mulas, J., 2013a. Use of CD10 as a marker of canine mammary myoepithelial cells. Vet. J. 195, 192–199. Sánchez-Céspedes, R., Maniscalco, L., Iussich, S., Martignani, E., Guil-Luna, S., De Maria, R., Martin de las Mulas, J., Millan, Y., 2013b. Isolation, purification, culture and characterization of myoepithelial cells from normal and neoplastic canine mammary glands using a magnetic-activated cell sorting separation system. Vet. J. 197, 474–482. Sánchez-Céspedes, R., Millan, Y., Maniscalco, L., Guil-Luna, S., Reymundo, C., Espinosa
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Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Immunohistochemical expression of p63 protein and calponin in canine mammary tumours
T
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Wojciech Łopuszyńskia, , Marek Szczubiałb, Yolanda Millánc, Silvia Guil-Lunac, Raquel Sánchez-Céspedesc, Juana Martin de las Mulasc, Anna Śmiecha, Kamila Bulaka a
Sub-Department of Pathomorphology and Forensic Veterinary Medicine, Department and Clinic of Animal Internal Diseases, University of Life Sciences in Lublin, Poland Department and Clinic of Animal Reproduction, University of Life Sciences in Lublin, Poland c Department of Anatomy and Comparative Pathology, Córdoba University, Spain b
A R T I C LE I N FO
A B S T R A C T
Keywords: p63 protein Calponin Mammary tumours Female dogs Immunohistochemistry
The aim of this study was to compare the expression of p63 protein and calponin in terms of their affinity and specificity for myoepithelial cells in canine mammary tumours. The studied material included 10 benign and 32 malignant mammary tumours from female dogs treated with mastectomy. Primary mouse monoclonal antibodies directed against p63 protein clone 4A4 and calponin clone CALP were used in single- and doublestain system of immunohistochemical reaction. The investigations have shown that majority of myoepithelial cells in benign tumours and carcinomas in situ exhibited strong positive labelling for both markers. In other malignant tumours strong immunoreactivity was observed in resting myoepithelial cells (MECs) and hypertrophic myoepithelial cells (HMECs), while the immunoreactivity in spindle-stellate myoepithelial cells (SMECs) and rounded myoepithelial cells (RMECs) was moderate. The granular-diffuse nuclear expression of p63 protein was observed only in myoepithelial cells. In terms of calponin, diffuse cytoplasmic expression was noted not only in myoepithelial cell but also in some stromal fibroblasts and vascular smooth muscle cells. The epithelial cells did not exhibit specific expression of the investigated markers. The obtained results indicate that p63 is a sensitive and more specific marker of myoepithelial cells in canine mammary tumours compared with calponin. These findings suggest that the immunohistochemical analysis peformed with the use of p63 can be a valuable complement of routine histological examinations of canine mammary tumours facilitating identification of tumours with myoepithelial component.
1. Introduction Mammary tumours are one of the most common neoplasms in female dogs. They are malignant in approximately 50% of cases and lead to substantial reduction in life expectancy in affected animals (Arnesen et al., 2001; Sleeckx et al., 2011). A characteristic feature of canine mammary tumours (CMTs) is the high proportion of myoepithelial cells in the neoplastic proliferation. Their distinction is essential for the histogenetic classification and prognosis. Malignant mammary tumours with myoepithelial component (complex carcinomas) have a better prognosis than simple carcinomas with only epithelial component (Goldschmidt et al., 2011; Misdorp et al., 1999). In a normal mammary gland, myoepithelial cells are located between the epithelial lining and the basement membrane of the secretory portion. The cytoplasm of myoepithelial cells contains fibrils similar to myofibrils present in smooth muscle cells, in addition to the cytokeratin protein
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characteristic for epithelial cells. Furthermore, these cells are similar to smooth muscle cells in terms of the distribution of cell organelles, cell membrane differentiation, and presence of the actin-myosin complex (Pulley, 1973; Vos et al., 1993). Under neoplastic transformation, myoepithelial cells change their normal microscopical appearance and assume unusual shape and arrangement, which has been demonstrated in both histological tissue sections and in cell cultures (SánchezCéspedes et al., 2013b; Zhang et al., 2003). This fact additionally impedes proper identification of myoepithelial cells in routine histopathological examination (Bertagnolli et al., 2009; Zhang et al., 2003). Due to the difficulties in identification of myoepithelial cells in routine histopathological examination of CMTs, various immunohistochemical markers, e.g. cytokeratin (Griffey et al., 1993; Vos et al., 1993), α-smooth muscle actin (Destexhe et al., 1993), calponin (Espinosa de los Monteros et al., 2002), protein 14-3-3-ơ (SuarezBonnet et al., 2011), and antigen CD-10 (Sánchez-Céspedes et al.,
Corresponding author. E-mail address: [email protected] (W. Łopuszyński).
https://doi.org/10.1016/j.rvsc.2019.01.017 Received 26 June 2018; Received in revised form 11 January 2019; Accepted 15 January 2019 0034-5288/ © 2019 Published by Elsevier Ltd.
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temperature of 56 °C for 12 h. After removal from the incubator, tissue sections were deparaffinised in xylene and passed through decreasing concentrations of alcoholic solutions to distilled water. Next, the tissue sections were incubated in a 3% solution of hydrogen peroxide in methanol for 30 min to block endogenous peroxidase. Next step of the procedure involved heat-induced antigen retrieval (water bath at 95–98 °C with 10 mM citrate buffer, pH = 6.0, 8 min). After cooling to room temperature, tissue sections were rinsed in distilled water and TBS buffer, outlined with a paraffin pen, and placed in a humidified chamber. Dilutions of appropriate primary antibodies that were prepared earlier were applied to the tissue sections. The incubation was carried out at a temperature of 37 °C in a humidified chamber for 60 min. Primary mouse monoclonal antibody directed against p63 protein clone 4A4 (Dako, M7242) at dilution 1:50 was used in the investigations. In the next step, tissue sections were rinsed in TBS buffer and a biotinylated secondary antibody, streptavidin-peroxidase complex, and 3,3′-diaminobenzidine tetrahydrochloride DAB (SK-4100; Vector Laboratories, Peterborough, UK) chromogen were applied. The chromogen was prepared 10 min prior to the use as follows: 10 mg DAB in 10 ml 0.05 M Tris-HCl with pH = 7.4 with addition of 100 μl 3% H2O2. Each step was terminated by rinsing in the buffer. Finally, tissue sections were stained with Mayer's haematoxylin for approximately 2–3 min, rinsed with water, dehydrated in alcohol, placed in xylene and sealed in mounting medium. To confirm the myoepithelial phenotype of cells labelled with the anti-p63 antibody, reactions with the primary mouse monoclonal antibody directed against calponin clone CALP (Dako, M3556) at dilution 1: 400 were carried out. In order to reveal coexpression of p63 protein and calponin simultaneously, selected tissue sections were stained with two separate chromogens, i.e. DAB for visualisation of the nuclear reaction after incubation with anti-p63 antibody and Fast Red for visualisation of the cytoplasmic reaction after incubation with anti-calponin antibody. A double control system was used in all reactions, i.e. a negative control, where incubation with the primary antibody was replaced by incubation with relevant non-immunised commercial IgG sera, and a positive control, which was a normal mammary tissue adjacent to neoplastic mass (internal control).
2013a), have been used in the diagnostics of tumours with a myoepithelial component. Despite their high sensitivity, these markers proved to be insufficiently specific for myoepithelial cells in CMTs. Therefore, identification of new diagnostically useful immunohistochemical markers of myoepithelial cells in canine mammary tumours is indispensable. The p63 protein is a product of the p63 gene translation, i.e. a homologue of the p53 suppressor gene. The p63 gene functions as a nuclear transcription factor for proliferation, apoptosis, and differentiation of cells (Maisse et al., 2003). The p63 protein plays an important role in the proliferation, differentiation, aging, and adhesion of epithelial cells and has a number of functions in the origination of tumours (Bertagnolli et al., 2009; Carroll et al., 2006). The p63 protein is a sensitive and specific marker of myoepithelial cells in human breast tumours (Batistatou et al., 2003; Kim et al., 2014; Stefanou et al., 2004). However, there are few reports of the suitability of p63 protein for identification of myoepithelial cells in CMTs (Gama et al., 2003; Ramalho et al., 2006; Sánchez-Céspedes et al., 2016). Calponin is a specific 34-kDa protein involved in regulation of smooth muscle contraction. It is a highly sensitive marker of myoepithelial cells, often used for immunohistochemical analysis of human and animal tissues (Espinosa de los Monteros et al., 2002; Moritoni et al., 2002; Sánchez-Céspedes et al., 2011). However, its value as a marker of myoepithelial cells is limited by the fact that it labels also myofibroblasts and smooth muscle cells as well (Espinosa de los Monteros et al., 2002; Moritoni et al., 2002; Sánchez-Céspedes et al., 2013a). The aim of this study was to compare the expression of p63 protein and calponin in terms of their affinity and specificity for myoepithelial cells in order to assess their usefulness in routine histopathological diagnostics of canine mammary tumours. 2. Material and methods 2.1. Material and histological examinations The studied material included 42 mammary tumours (10 benign and 32 malignant) sampled from 4 to 15-year-old female dogs of different breeds (pedigree and mixed) treated with mastectomy. Depending on the size, up to 6 slices were sampled from each resected tumour from the peripheral zone, macroscopically visible growth zone, and surgical margin. Tissue samples were fixed for 24 h in 10% formalin, pH = 7.2, and passed for 24 h through increasing concentrations of alcoholic solutions, acetone, and xylene to paraffin in a tissue processor (Leica TP1020, Leica Biosystems, Nussloch, Germany). After fixation, hard in palpation tumours with chondrification and/or ossification foci were cut into 5-mm thick sections, decalcified in Osteodec reagent (BioOptica, Milano, Italy) for 24 h, and embedded in paraffin blocks as described above. Tissue samples were sectioned on 4-μm-thick slices on a sledge microtome (Leica SR-200, Leica Biosystems, Nussloch, Germany) and placed on glass slides. Histopathological evaluation of haematoxylin and eosin-stained tissue sections was carried out under a light microscope (Nikon Eclipse E-600, Nikon Instruments, Inc. New York). The microscopic assessment of the tissue sections was based on the histological classification of canine mammary tumours proposed by AFIP/WHO (Misdorp et al., 1999).
2.3. Evaluation of immunohistochemical data Tissue section fields with a distinct histopathological pattern of a given tumour type were selected in each case. Selected fields were analysed for the visualisation of markers, taking into account the nuclear staining in the case of p63 protein expression and the cytoplasmic staining in the case of calponin expression. Based on the morphological appearance and localisation, the analysed cells were subdivided into luminal epithelial cells (LCs), resting myoepithelial cells (MECs), hypertrophic myoepithelial cells (HMECs), spindle-stellate myoepithelial cells (SMECs), and rounded myoepithelial cells (RMECs). Expression of p63 protein was evaluated based on the percentage of cells with a positive reaction and the cell staining intensity. A semiquantity scoring system (SQS) of evaluation of the number of stained cells was used, where + = less than 10% of positive cells, ++ = 11–59% of positive cells, and +++ = more than 60% of positive cells. The staining intensity was assessed in a 3-grade intensity scoring system (IS), where 1i – weak staining, 2i – moderate staining, and 3i – strong staining.
2.2. Immunohistochemical analyses
3. Results
The immunohistochemical examinations were performed in systems of detection of antigen-antibody complexes based on biotinylated secondary antibodies directed against monoclonal mouse primary antibodies in a single staining LSAB plus HRP kit (Dako, K0690) and a double staining EnVision System (Dako, K1395). Tissue sections for immunohistochemical evaluation were deposited on silane-coated Super Frost glass slides (Menzel-Glaser) and kept in an incubator at a
3.1. Results of histological examinations Based on the histopathological examination, 10 (23.8%) benign tumours and 32 (76.2%) malignant tumours were distinguished in the analysed group. Detailed histopathological results are shown in Table 1. 233
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Table 1 Histopathological classification of the 42 canine mammary gland tumours. Tumour types
No. of cases (%)
Benign tumours Duct papilloma Fibroadenoma Complex adenoma Benign mixed tumour Malignant tumours Carcinoma in benign tumour In situ carcinoma Complex carcinoma Simple carcinoma Total
10 (23.80) 2 (4.76) 1(2.38) 4 (9.52) 3 (7.14) 32 (76.20) 3 (7.14) 2 (4.76) 11 (26.20) 16 (38.10) 42 (100.00)
In 16 cases, normal mammary gland tissue, which was the positive internal control in the study, remained attached to the adjacent tumour mass.
Fig. 1. Complex adenoma. In myofibroblasts (short arrow) p63 protein expression is not evident and vascular endothelial cells (long arrow) expressing calponin are seen. Double staining system, immunohistochemistry, Harris's haematoxylin counterstain. Magn. 200×.
3.2. Results of immunohistochemical examinations Granular-diffuse nuclear labelling for p63 protein and diffuse cytoplasmic labelling for calponin was noted in myoepithelial cells. Intensity of the expression, percentage of positive stained cells and number of tumours positive for specific cell type differ depending on the histological tumour type. The results of immunohistochemical reactions are summarised in Table 2. The luminal epithelial cells in the
normal and neoplastic mammary gland did not exhibit expression of p63 protein and calponin. A positive cytoplasmic labelling for calponin was noted in some stromal fibroblasts and vascular smooth muscle cells (Fig. 1).
Table 2 Characterization of cell types with expression of p63 protein and calponin in canine normal and neoplastic mammary gland. Type of tumour
n
Cell morphology*
Immunoreactivity for p63
Immunoreactivity for calponin
n1 (%)
SQS
IS
n1 (%)
SQS
IS
Normal mammary gland
16
LCs MECs
0 16 (100)
– +++
– 3i
0 16 (100)
– +++
– 3i
Benign tumours Duct papilloma
2
LCs MECs HMECs LCs MECs HMECs LCs MECs HMECs SMECs LCs MECs HMECs SMECs RMEC
0 2 2 0 1 1 0 4 3 3 0 3 2 2 2
(100) (66.6) (66.6) (66.6)
– +++ +++ – +++ +++ – +++ +++ + – +++ ++ + +
– 3i 3i – 3i 3i – 3i 2i 2i – 3i 2i 1i 1i
0 2 2 0 1 1 0 4 4 3 0 3 3 2 2
(100) (100) (66.6) (66.6)
– +++ +++ – +++ +++ – +++ +++ ++ – +++ ++ + +
– 3i 3i – 3i 3i – 3i 3i 2i – 3i 3i 2i 2i
LCs MECs HMECs SMECs RMECs LCs MECs HMECs LCs MECs HMECs SMECs LCs MECs HMECs
0 3 (100) 2 (66.6) 1 (33.3) 1 (33.3) 0 2 (100) 2(100) 0 11 (100) 8 (72.7) 5 (45.4) 0 16 (100) 11(68.7)
– ++ + + + – +++ +++ – +++ + + – +++ ++
– 2i 2i 1i 1i – 3i 3i – 3i 2i 2i – 3i 2i
0 3 (100) 3 (100) 2 (66.6) 2 (66.6) 0 2 (100) 2(100) 0 11 (100) 10 (90.9) 7 (63.6) 0 16 (100) 14 (87.5)
– ++ ++ + + – +++ +++ – ++ ++ + – +++ ++
– 3i 2i 2i 1i – 3i 3i – 3i 2i 2i – 3i 2i
Fibroadenoma
Complex adenoma
Benign mixed tumour
Malignant tumours Carcinoma in benign tumour
1
4
3
3
In situ carcinoma
2
Complex carcinoma
11
Simple carcinoma
16
(100) (100) (100) (100) (100) (75) (75)
(100) (100) (100) (100) (100) (100) (75)
*LCs – luminal epithelial cells, MECs – myoepithelial cells, HMECs – hypertrophic myoepitheliel cells, SMECs – spindle-stellate myoepithelial cells, RMECs – rounded myoepitheliel cells, n1 – number of tumours positive for specific cell type, *SQS: – = no stained cells, + = less than 20% positive cells, ++ = 20–59% positive cells, +++ = more than 60% positive cells, IS: 1i – week, 2i – moderate, 3i – strong. 234
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Fig. 2. Coexpression of p63 protein (brown nuclear staining) and calponin (red cytoplasmic staining) in myoepithelial cells of the normal mammary gland. Double staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 200×. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Duct papilloma. Multiple papillary projections protruding into the lumen of the pre-existing duct. Prevalent hypertrophic myoepithelial cells and minor in number resting myoepithelial cells with nuclear overexpression of p63 protein. Single staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 100×.
The resting myoepithelial cells (MECs) with elongated shape and flattened nuclei were present in all examined tumour samples and normal mammary gland tissue sections. However, depending on the type of the tumour, MECs formed a single continuous or discontinuous layer around normal secretory acini and ducts as well as around neoplastic structures build up by proliferating luminal epithelial cells. In the normal canine mammary gland, a strong (3i-score) granular-diffuse pattern of nuclear p63 protein expression was evenly noted in MECs along the ductal and acinar structures. Labelling for calponin was uniformly visualized in the cytoplasm of MECs by forming a continuous layer around the ducts and secretory acini. The double staining system revealed marked coexpression of the nuclear p63 protein and cytoplasmic calponin simultaneously in the same resting myoepithelial cells (Fig. 2). In all examined benign and malignant tumours, MECs exhibited strong nuclear expression of p63 protein (SQS = +++, IS = 3i) and a similar cytoplasmic expression of calponin. Hypertrophic myoepithelial cells (HMECs) with a polygonal shape and an ovoid nucleus formed a single continuous or discontinuous layer around the multiform neoplastic structures composed of luminal epithelial cells. There were no such cells in the normal mammary tissue. In two examined duct papillomas presenting as papillary projections protruding into the expanded lumina of pre-existing secretory ducts, HMECs, likewise MECs, formed a single continuous layer under the luminal epithelial lining and tended to build up at the boundary with the connective tissue stroma (Fig. 3). In both examined tumour's samples a vast majority of HMECs exhibited intense positive immunohistochemical reaction (SQS = +++, IS = 3i) for both markers. In the single case of fibroadenoma composed of epithelial elements scattered among a rich connective tissue stroma, the nuclear expression of p63 protein was confined to a thin layer of slightly dispersed HMECs and MECs surrounding the negative luminal epithelial component. The nature of the immunohistochemical reactions was identical as in the case of the duct papilloma (SQS = +++, IS = 3i). The nuclear expression of p63 protein was evident in the HMECs in the case of 3/4 (75%) complex adenomas and 2/3 (66.6%) benign mixed tumours (BMTs). HMECs, similarly to MECs, created a single continuous layer around luminal epithelium deformed by proliferating spindle-stellate myoepithelial cells embedded in an amorphous, faintly eosinophilic matrix, and, in the case of BMTs, by areas of cartilage and/or bony structures with varied degrees of maturation. A majority of HMECs in complex adenomas and BMTs exhibited
Fig. 4. Immunoreactivity of hypertrophic myoepithelial cells. Coexpression of p63 protein and calponin in hypertrophic myoepithelial cells in complex adenoma. Double staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 200× (a). Discontinuous layer of hypertrophic myoepithelial cells showing moderate nuclear expression of p63 protein in simple tubulopapillary carcinoma. Single staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 200× (b).
positive reaction (SQS = +++) with moderate staining intensity (IS = 2i) (Fig. 4a). In carcinomas in situ, HMECs were located in the immediate proximity of MECs and, as in the benign tumours and normal glandular tissue, both types of cells formed a continuous layer serving as a hallmark of non-invasive character of the tumour's growth. In the group of malignant tumours, HMECs formed an incomplete and discontinuous layer around the glandular structures (Fig. 4b). The immunohistochemical staining of HMECs in carcinomas in situ revealed the highest parameters of immunoreactivity (SQS = +++, IS = 3i). The moderate (IS = 2i) nuclear expression of p63 protein was observed in 2/3 (66.6%) cases of carcinomas arising in complex adenomas, in 8/ 11 (72.7%) cases of the complex carcinomas, and in 11/16 (68.7%) cases of the simple carcinomas. In carcinomas in benign tumours and in complex carcinomas, the expression of p63 protein was observed in less than 10% of HMECs (SQS = +), whereas in the group of simple carcinomas positive nuclear reaction was observed in 10–60% of HMECs. The strong cytoplasmic reaction due to the presence of calponin antigen
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Fig. 5. Significant reduction of expression (in number and intensity) of p63 protein in spindle-shaped (a) and stellate (b) myoepithelial cells. Single staining system, immunohistochemistry. Harris's haematoxylin counterstain. Magn. 200×.
Fig. 6. An area of early chondroid differentiation in benign mixed tumour. Weak nuclear expression of p63 protein (arrows) and cytoplasmic positive reaction in the presence of calponin antigen in some rounded myoepithelial cells. Double staining system, immunohistochemistry, Harris's haematoxylin counterstain. Magn. 400×.
in HMECs was visible in all examined benign neoplasms (IS = 3i). Strong staining intensity was observed in two non-infiltrating carcinomas. In other malignant tumours, the expression of calponin in HMECs was moderate (IS = 2i), and the positive reaction was visible in almost all cases (87.5%–100%). Nuclear expression of p63 protein in SMECs forming intertwining branches between deformed structures made up of luminal epithelial cells, was observed in 3/4 (75%) cases of complex adenomas, in 5/11 (45.4%) cases of complex carcinomas, in 2/3 (66.6%) cases of benign mixed tumours, and in 1/3 (33.33%) case of the carcinoma in benign tumour. Weak to moderate staining intensity (IS = 1i–2i) was noted in these types of tumours (Fig. 5). The expression of calponin by SMEC in all analysed tumours was moderate (IS = 2i), and the percentage of positive reactions ranged from 63.6% in the case of the complex carcinomas to 75% in the complex adenomas. Rounded myoepithelial cells (RMECs) exhibiting p63 protein were only found in the areas with immature cartilage formation in 2/3 (66.6%) of BMT and in 1/3 (33.3%) of carcinoma in benign tumour. In these cases the nuclear staining was weak (IS = 1i). The coexpression of calponin and p63 protein in RMECs was demonstrated by doublestain system (Fig. 6), and was characterised by weak to moderate intensity of staining (IS = 1i–2i).
with various antibodies is believed to be the most suitable method for in vivo examination and identification of myoepithelial cells (SánchezCéspedes et al., 2016). This study presents investigations of the expression of p63 protein in benign and malignant CMTs and comparison thereof with the expression of calponin, which is a well-known highly sensitive marker of myoepithelial cells in human breast tumours as well as canine and feline mammary tumours. The results of the investigation indicate that p63 protein is a highly specific and sensitive marker of myoepithelial cells in canine mammary tumours. In both benign and malignant tumours, nuclear expression of p63 protein was noted in the myoepithelial cell layer surrounding the layer of luminal epithelial cells. Epithelial cells, connective tissue cells, and vascular smooth muscle cells were non-reactive with anti-p63 antibody. These results are in agreement with those reported in earlier studies, which described the usage of p63 as an immunohistochemical marker of myoepithelial cells in human breast tumours (Koker and Kleer, 2004; Stefanou et al., 2004; Werling et al., 2003) and canine mammary tumours (Gama et al., 2003; Ramalho et al., 2006; Rasotto et al., 2014). The cytoplasmic expression of calponin in the myoepithelial cells of the analysed benign and malignant canine mammary tumours was comparable to p63 protein nuclear expression. Epithelial cells exhibited no expression of calponin, similarly to p63, whereas positive reaction was found in myofibroblasts and vascular smooth muscle cells. The high sensitivity of calponin as a marker of myoepithelial cells of the canine mammary gland as well as its low specificity were demonstrated in many previous studies (Espinosa de los Monteros et al., 2002; Martin de las Mulas et al., 2004; Rasotto et al., 2014). The results and observations reported by other authors (Ramalho et al., 2006; Sánchez-Céspedes et al., 2016) indicate that nuclear p63 immunolabelling can be used simultaneously with cytoplasmic markers of myoepithelial cells. This greatly facilitates immunohistochemical examinations, as anti-p63 antibodies and antibodies directed against other markers of myoepithelial cells can be placed on the same slide with the analysed tumour section. Our studies with the use of double staining system with two separate chromogens enabled assessment of expression of p63 and calponin in individual myoepithelial cells. Coexpression of p63 and calponin was confirmed in all distinguished subtypes of myoepithelial cells indicating at the same time the difference in their sensitivity. The nuclear expression of p63 turned out to be qualitatively different from cytoplasmic expression of calponin. The
4. Discussion In the normal mammary gland of female dogs, the ductal and lobular portion is lined with two types of cells – an inner luminal epithelial and an outer myoepithelial cell layer (Misdorp, 2002; SánchezCéspedes et al., 2016). Myoepithelial cells are involved in milk ejection and regulation of the growth, differentiation, and morphogenesis of neighbouring cells (Adriance et al., 2005). Unlike in humans and female cats, myoepithelial cells in female dogs are often involved in the neoplastic transformation of the mammary gland tissue. The presence of myoepithelial cells is crucial for the histogenetic classification of canine mammary tumours, and has an impact on the prognosis and evaluation of tumour progression (Misdorp, 2002; Misdorp et al., 1999; Yoshimura et al., 2014). Attention is also paid to the role of myoepithelial cells in identification of carcinomas in situ (Sánchez-Céspedes et al., 2011). However, the identification of myoepithelial cells in routine histopathological examinations of canine mammary tumours is not obvious (Zhang et al., 2003). Many simple carcinomas in haematoxylin and eosin staining turned out to be complex when assessed using immunohistochemical techniques (Martin de las Mulas et al., 2004; Yoshimura et al., 2014). Therefore, immunohistochemical marking 236
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lesions and carcinomas in situ. In the case of canine mammary tumours, the usage of immunohistochemical markers for examination of integrity of the myoepithelial cell layer in diagnostics of carcinoma in situ has not yet been thoroughly investigated. There is only one study with calponin as a suitable marker to analyse the integrity of the myoepithelial cell layer in canine mammary carcinomas in situ (Sánchez-Céspedes et al., 2011). Our research has shown that the p63 and calponin-positive MECs and HMECs formed a continuous layer around the epithelial structures in benign mammary tumours and carcinomas in situ, whereas in malignant mammary tumours, MECs and HMECs formed a discontinuous layer. Furthermore, the expression of p63 protein and calponin in MECs and HMECs in carcinomas in situ was characterised by the highest immunoreactivity parameters (SQS = +++; IS = 3i). This results may suggest that p63 protein and calponin can be used simultaneously as significant markers in diagnostics of carcinoma in situ of the canine mammary gland.
nuclear pattern of p63 expression gave the advantage of accurate distinction of variably stained nuclei of all subtypes of myoepithelial cells from the unstained nuclei of the adjacent cells enabling a more objective assessment. The cytoplasmic expression of calponin was not easily identifiable in attenuated, compressed or misshapen cells. This difference seems to be also related to the function of both p63 proteins and calponin in the cell. Nuclear p63 protein acts as a sequence specific DNA binding transcriptional activator or repressor of proliferation, apoptosis, and differentiation which makes it less dependent on the influence of internal and external interactions. Phosphorylation and changes in cytoskeleton tension can modify the function and structure of calponin leading to its gradual degradation (Liu and Jin, 2016). The double staining system proved to be helpful in distinguishing between calponin-positive myofibroblasts and calponin-positive myoepithelial cells. However, double immunohistochemical technique is more laborintensive and difficult to implement in routine diagnostic laboratory, it proved to be extremely useful in the comparative assessment of p63 and calponin expression. As shown by Reis-Filho et al. (Reis-Filho et al., 2003), p63 protein can also be used as a marker of myoepithelial cells in cytology. Previous studies suggest transformation of myoepithelial cells from MECs to FMECs and SMECs in CMTs (Espinosa de los Monteros et al., 2002). Investigations conducted on mammary tumour cell lines collected from female dogs have shown that the transformed myoepithelial cells were characterised by varied morphology, different growth pattern, and diverse marker labelling (Sánchez-Céspedes et al., 2013b), which was also confirmed in the present study by immunohistochemistry. In examined benign tumours, in MECs in all cases and in HMECs in most cases strong labelling of both markers was noted. In turn, moderate or low immunoreactivity was noted in SMECs and RMECs. In the group of malignant tumours, high expression of p63 protein and calponin was detected in both MECs and HMECs in all cases of carcinomas in situ and only in MECs of all the other malignant neoplasms. HMECs, SMECs, and RMECs exhibited moderate or weak immunoreactivity in the group of malignant tumours with a slightly higher percentage of tumours positive for calponin. Bertagnolli et al. (Bertagnolli et al., 2009) analysed the expression of p63 protein in different subtypes of myoepithelial cells in benign mixed tumours and in carcinomas in benign tumours and found the highest immunoreactivity in resting myoepithelial cells (MECs) of benign mixed tumours. Similar results were reported by other authors (Espinosa de los Monteros et al., 2002). Sánchez-Céspedes et al. (Sánchez-Céspedes et al., 2011) found that the expression of calponin depended on the type of canine mammary tumour and was the highest in dysplasia and low grade malignant tumours. Calponin expression in the group of malignant tumours was decreasing with the increasing histological malignancy grade. This indicates that p63 protein and calponin expression in myoepithelial cells including microscopical presentation of immunohistochemical marking can change with time during tumour expansion and may suggest the benign or malignant nature of the tumour. The mechanism of the loss of immunohistochemical markers in myoepithelial cells during progression and differentiation of CMTs remains unknown, although it is undoubtedly related to gradual transformation from resting myoepithelial cells to cartilage and the loss of epithelial features which are replaced by mesenchymal properties. It could also result from dynamic interaction between epithelial and myoepithelial cells as well as extracellular matrix. It has been suggested that some proteolytic enzymes produced by malignant epithelial cells could affect structure and function of myoepithelial cells (Zhang et al., 2003). In humans, the integrity of the myoepithelial cell layer is an important hallmark of carcinomas in situ. Invasive carcinomas are characterised by the absence of integrity of the myoepithelial cell layer, what can be visualized using immunohistochemical markers (Moriya et al., 2009; Yaziji et al., 2000). Batistatou et al. (Batistatou et al., 2003) reported no expression of p63 protein in invasive breast tumours, whereas positive reaction persisted in myoepithelial cells in benign
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