Introduction

One of the most frequently occurring tumors in dogs is the mammary gland tumors (Salas et al., 2015; Pastor et al., 2018; Sharma et al., 2018). Histologically and clinically, there are similarities between canine mammary glands and human breast tissue. Hence, dogs can be considered as in vivo models for the human breast cancer (Cassali, 2013; Abdelmegeed and Mohammed, 2018). The architecture of mammary tissue is composed of three cell populations: luminal epithelial cells, basal/myoepithelial cells, and mesenchymal cells (Sorenmo et al., 2011). The wide heterogeneity of these tumors has increasingly made the classification scheme complex (Schlafer, 2016). In 6 out of 34 histological types of neoplasms, myoepithelial cell proliferation was demonstrated; three of them showed malignant transformation of myoepithelial (Goldschmidt et al., 2011).

Myoepithelial cells are well-developed in canine sweat and mammary glands (Beha et al., 2012; Ingthorsson et al., 2015). In mammary tissue, they form a continuous layer around ducts and teat sinuses (spindle-shaped) and a discontinuous layer around alveoli (star-shaped). They are located between luminal epithelial cells and stromal fibroblasts, which makes them suitable to link with these two cell populations and they have characteristics of muscle and epithelial cells (cytokeratins) (Beha et al., 2012).

Different types of epithelial cells synthesize filaments that are products of co-polymerization of at least one type I and one type II cytokeratin. These filaments which are abundant in the cytoplasm of epithelial cells are called soft keratin. They form intra-cytoplasmic cytoskeleton which causes resistance against mechanical stress (Cooper, 2000). Intermediate filaments are the most resistant filaments against mechanical stress among all filaments that form the cytoskeleton. Cytokeratins are important intermediate filaments that are categorized into six types according to the protein structure. Type I and II are acidic (CK-1, CK- 2, CK-3, CK-4, CK-5, CK-6, CK-7, CK-8) and basic (CK-9, CK-10, CK-11, CK-12, CK-13, CK-14, CK-15, CK-16, CK-17, CK-18, CK-19, CK-20) cytokeratins, respectively. Also, according to the molecular weight they are categorized into high molecular weight cytokeratins (squamous keratins) and low molecular weight cytokeratins (simple keratins) (Rekhtman, 2011). They are widely used as epithelial cell markers to assess the tissue origin of cancer (Jacob, 2018). Cytokeratin 7 (CK7) is a type II keratin (basic keratin, low molecular weight) which improves the integrity of cellular structures (Moll et al., 2008). It has been shown that this intermediate filament is expressed in normal canine apocrine glands (Espinosa De Los Monteros et al., 1999). Previously, CK7 expression was subjected to several studies on various canine tissues including cutaneous lesions and mammary glands (Pieper et al., 2015; Eivani and Mortazavi 2016). CK7 is used as a differential diagnosis marker for urothelial tumors, adenocarcinomas, bile duct and lung epithelia (Painter et al., 2010; Pieper et al., 2015; Luo et al., 2017). 

The routine staining method of hematoxylin and eosin (H&E) is incapable of distinguishing various eosinophilic tissue components, though special staining techniques such as Masson’s trichrome have proved to be cost-effective methods to demonstrate the presence and pattern of collagenous tissue. Despite being old-fashioned, this multi-colored method is still a popular stain for the connective tissue assessment in modern histology (Alturkistani et al., 2016). In this study, this method was used to investigate the increase in collagenous tissue, either as a desmoplasia (stromal cell proliferation) or probable changes in myoepithelial cell nature in the neoplastic process.

The current study aimed to investigate the expression of CK7 in canine normal and neoplastic mammary gland epithelial and myoepithelial cells and to evaluate its association with malignancy grade and histologic characteristics of neoplasms. Also, Masson’s trichrome was chosen to differentiate between smooth muscle fibers and collagen fibers.

Materials and Methods

 Samples and Histopathology

This study was based on 17 cases of canine mammary gland neoplasms (n=17) obtained from Department of Pathology, Faculty of Veterinary Medicine, University of Tehran. The samples were fixed in 10% neutral buffered formalin, then cut into 5 µm sections and stained with routine H&E and Masson’s trichrome methods. Histopathological types, subtypes, and grades were evaluated according to the Peña method (Peña, 2013) (Table 1). Histopathological variables included histological type and grade of tumors, intra-tumoral necrosis, and lymphovascular invasion.

Table 1. Method for grading. Modification of Pena method for histologic grading of canine mammary cancer

a * Points

bHPF, high-power field.

Source: Peña, L., De Andres, P.J., et al. (2013) Prognostic value of histological grading in noninflammatory canine mammary carcinomas in a prospective study with two‐year follow‐up: Relationship with clinical and histological characteristics. Vet Pathol 50:94–105. Reproduced with permission of SAGE Publications

Masson’s Trichrome Staining

After deparaffinization and rehydration, the slides were stained with hematoxylin for 10 min, then Biebrich scarlet-acid fuchsin solution for 2 min. After that they were placed in the phosphomolybdic-phosphotungstic acid solution for 10-15 min and aniline blue solution for 5 min. Finally, the slides were placed in 1% acetic acid for 3-5 min, and then dehydration and clearing were performed. 

Antibodies and Immunohistochemistry

Immunohistochemistry for CK7 was performed on formalin-fixed, paraffin-embedded (FFPE) tissue sections at 4 µm diameter. The procedure was performed using ready-to-use mouse monoclonal antibody (clone OV-TL 12/30 Novocastra Leica Biosystems), which recognizes human CK7 protein. A horseradish peroxidase (HRP) polymer detection method was used for the CK7 antibody. After deparaffinization and rehydration, antigen retrieval was carried out by microwave oven at 750 W and 180 W (citrate buffer, 15 min, pH=6, 0.01 M). To neutralize the endogenous peroxidase, all slides were incubated in hydrogen peroxide 3% for 5 min. To reduce the non-specific bindings, all slides were incubated in protein blocker reagents for 5 min. The slides were incubated with the primary antibody CK7, the post-primary antibody, and Novolink polymer (HRP) Each step for 1 h. Next, the diaminobenzidine (DAB) working solution was used as the chromogen for 10 min. Finally, the slides were counterstained in hematoxylin.

Positive and negative controls were set by adjacent normal mammary tissue and replacing the primary antibody by PBS. The semi-quantitative assessment was done based on the Ramalho et al. study (2006) by two observers through randomly chosen 30 fields at high power (×400). Tumors exhibiting brown cytoplasmic staining at >10% neoplastic cells were regarded as positive.

Results

Two out of 17 tumors (11.76%) were benign, and 15 out of 17 were malignant. Among the malignant tumors (n=15), 10 were classified as grade I (66.66%) and 5 were classified as grade II (33.33%). In Masson’s trichrome staining, epithelial cells, myoepithelial cells stained red, and the nucleus of fibroblasts, chondroblasts, and osteoblasts were stained dark blue, and collagen fibers, bone, and cartilage matrices were stained blue. No muscular tissue was observed in the tumor sections. Summarized results of the histopathology and immunohistochemical assessments of the examined sections of neoplastic mammary tissues were presented in Table 2.

Table 2. Classification, grading, and immunohistochemical results of canine mammary gland tumors evaluation

a *Local invasion

b numbers in the brackets are the relative frequencies.

H&E and Connective Tissue Staining Findings

Benign Neoplasms

The proliferation of ducts was observed in a hypocellular stroma in fibroadenoma and desmoplasia in the benign mixed tumor. In Masson’s trichrome staining, the nucleus of spindle and stellate stromal fibroblasts were demonstrated as dark blue, and the thick collagen fibers of fibroadenoma and benign mixed tumor were demonstrated as blue (Figure 1).

Malignant Neoplasms

The malignant neoplasms are divided into malignant epithelial neoplasms and mixed neoplasms. In complex carcinomas (n=8) (malignant neoplasms), the proliferation of both epithelial and myoepithelial cells, areas of myxoid matrix, cholesterol cleft, and lipofuscin laden macrophages were observed. In one ductal carcinoma, areas of squamous differentiation, foci of necrosis, myxoid matrix, cholesterol cleft, and mixed inflammatory infiltration were observed.In carcinoma and malignant myoepithelioma, nests and trabeculae of epithelial cells and malignant proliferation of myoepithelial cells with scant myxoid matrix, necrosis, cholesterol cleft, desmoplasia, and lipofuscin pigments were observed. Periductal, interlobular, and intralobular collagen fibers were demonstrated as blue fibers in Masson’s trichrome stain (Figure 1. A & D). This regular pattern was observed in 8 complex carcinomas, 2 ductal carcinomas, and 1 carcinoma and malignant myoepithelioma. In some tumors, intratumoral collagen fibers were recognized as fine distinct fibers interspersed between proliferative normal and malignant epithelial and myoepithelial cells (1 benign mixed tumor, 8 complex carcinomas, 1 carcinoma and malignant myoepithelioma) (Figure 1).

Figure 1. Canine mammary gland neoplasia. Intratumoral fine (arrows) and thick (pointed arrows) collagen fibers. Masson’s Trichrome. (A & B. Complex carcinoma, 100Î, C. Carcinoma and malignant myoepithelioma, 200Î & D. Fibroadenoma, 100Î).

In carcinoma mixed type (mixed neoplasms), foci of chondroid metaplasia with the proliferation of epithelial cells were observed. In one carcinosarcoma proliferation of epithelial cells, the malignant hemangiopericytoma-like proliferation pattern of stromal fibroblasts and areas of chondroid metaplasia and bone metaplasia were observed. In Masson’s trichrome staining, collagen fibers were recognized as fine distinct fibers in carcinoma mixed type and interlobular and intralobular fibers in carcinosarcomas.

CK7 Immunoreactivity

CK7-labeled epithelial cells were observed in a normal case and four cases of tumors [23.53%] (1 benign mixed tumor, 1 fibroadenoma, 1 complex carcinoma, and 1 carcinoma mixed type) (Figure 2. A & E) and myoepithelial cells in seven cases [41.17%] (1 fibroadenoma, 1 benign mixed tumor, 3 complex carcinomas, 1 ductal carcinoma, and 1 carcinoma mixed type) (Figure 2. C & F). Negative labeling of epithelial (Figure 2. B, D, & F) and myoepithelial (Figure 2. A, B, D, & E) cells were observed in 13 (76.47%) and 10 (58.82%) tumors, respectively (Table 2).

Figure 2. Canine mammary gland carcinoma. Note positive immunolabeling of CK7 in epithelial cells (A, 200Î & E, 100Î) and myoepithelial cells (C, 200Î & F, 400Î) and negative immunolabeling of CK7 in epithelial cells (B, 200Î & F) and myoepithelial cells (A, B & D, 400Î). Proliferative myoepithelial cells stained positive. (Epithelial cells: Pointed arrows, myoepithelial cells: Arrows, A, B & E. Complex carcinoma, C & F. Carcinoma mixed type, D. Carcinoma and malignant myoepithelioma). C & F are from the same samples.

Discussion