NOTE Pathology

Atypical Canine Mammary Adenoma Characterized by Cystic Ducts Comprising a Single Layer of Basaloid Cells with Myoepithelial Differentiation

Kyohei YASUNO1), Ryosuke KOBAYASHI1), Takayuki MINESHIGE1), Go SUGAHARA1), Masahiko NAGATA2), Junichi KAMIIE3) and Kinji SHIROTA1,3)*

1)Research Institute of Biosciences, Azabu University, 1–17–71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252–5201, Japan2)Animal Specialist Center, 1–3–2 Jindanjihigashi-cho, Chofu, Tokyo 182–0012, Japan3)Laboratory of Veterinary Pathology, School of Veterinary Medicine, Azabu University, 1–17–71 Fuchinobe, Chuo-ku, Sagamihara,

Kanagawa 252–5201, Japan

(Received 9 January 2013/Accepted 18 March 2013/Published online in J-STAGE 1 April 2013)

ABSTRACT. This report describes an atypical mammary adenoma with a rare histological feature characterized by proliferating single-layered cystic ducts composed of basaloid cells with frequent myoepithelial differentiation. A 9-year-old, intact female Miniature Pinscher dog had mammary tumors on the thorax. Histologically, one of tumors comprised the proliferation of two types of tubular structures; the single-layered cystic ducts lined by flattened cells and double-layered tubules with luminal cells and outer spindle cells. The former ducts were predominant in the tumor and contained pale basophilic mucus, which was Alcian blue (pH 2.5)-positive, but periodic acid Schiff-negative. Immunohistochemical staining indicated that the cells lining single-layered cystic ducts were negative for the luminal epithelial marker, cytokeratin (CK) CAM5.2, but were constantly positive for basal cell markers CK14 and p63 and frequently positive for SMA. Electron microscopy revealed fine, parallel myofilaments within these single-layered neoplastic cells. These histological and immunohistological examinations suggested that the origin of the tumor was bipotential mammary progenitor cells with predominant differentiation into the myoepithelial progenitor linage.KEY WORDS: adenoma, basaloid cells, canine, mammary gland, myoepithelial cells.

doi: 10.1292/jvms.13-0016; J. Vet. Med. Sci. 75(8): 1095–1099, 2013

The histopathological classification of canine mammary tumors was first established in 1974 [7] and modified in 1999 [10]. Benign mammary tumors classified in 1999 included simple adenoma, complex adenoma, basaloid adenoma, fibroadenoma, benign mixed tumor and duct papilloma. Re-cently, Goldschmidt et al. [4] proposed a new classification and grading of canine mammary tumors in 2011. Accord-ing to this classification system, benign mammary tumors were classified as adenoma-simple, intraductal papillary ad-enoma, ductal adenoma (basaloid adenoma), fibroadenoma, myoepithelioma, complex adenoma (adenomyoepithelioma) and benign mixed tumor. The new system suggests that both components of the bilayered epithelium of the mammary gland have the potential to form benign neoplastic lesions independently; inner luminal epithelial cells and outer myo-epithelial cells could form adenomas and myoepitheliomas, respectively [4]. Thus, the new system could be helpful to di-agnose benign canine mammary tumors on the basis of their histology and cell origin. However, a few reported atypical adenomas could not be classified using this system [5, 9, 16]. This paper shows a rare case of canine mammary adenoma with proliferating cystic ducts comprising a single layer of basaloid cells with myoepithelial differentiation.

A 9-year-old, Miniature Pinscher, intact female dog had three tumors on the right (4.4 × 4.0 × 2.5 cm) and left thorax and on the ventral neck. The right thorax tumor that was found 8 months before surgery by the dog’s owner had gradually grown since its discovery. The tumors were surgically excised and examined histopathologically at our laboratory. Cut surface of the two thorax tumors was white to light brown with partial hemorrhage and well-demarcated. Mucoid fluid was exudated from the cut surface of the right thorax tumor. Regional lymph nodes of the mammary gland showed no enlargement. No local recurrence or metastasis was detected for 5 months after surgical excision.

Excised tissues were fixed in 10% neutral buffered for-malin, embedded in paraffin, sectioned and stained with he-matoxylin and eosin (HE) and Alcian blue (pH 2.5)-periodic acid Schiff (PAS) double stain.

The expression of several cell differentiation markers, such as cytokeratins (CKs), p63 and α-smooth muscle actin (SMA), has been described in canine mammary tissues. CK CAM5.2 is considered to be a luminal epithelial cell marker, whereas CK14 and p63 are expressed in basal/myo-epithelial cells [3, 6, 15]. SMA is exclusively expressed in myoepithelial cells [1]. Immunohistochemical staining was performed using an immunoenzyme polymer method with primary antibodies shown in Table 1. Peroxidase-conjugated anti-mouse immunoglobulin (Ig) G (Histofine Simple Stain MAX-PO (M); Nichirei, Tokyo, Japan) was used as secondary antibody. After immunoreactions, sections were colorized with diaminobenzidine (DAB) and counterstained with Mayer’s hematoxylin. Sections stained with normal

*CorrespondenCe to: shirota, K., Laboratory of Veterinary Pathol-ogy, School of Veterinary Medicine, Azabu University, 1–17–71, Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252–5201, Japan.

e-mail: shirota@azabu-u.ac.jp©2013 The Japanese Society of Veterinary Science

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mouse IgG under identical conditions were used as negative controls. For immunofluorescence, sections were incubated with 4% Block-AceTM (Yukijirushi, Sapporo, Japan) for 10 min at room temperature and then incubated overnight with rabbit anti-type IV collagen antibody (LSL, Tokyo, Japan) at 4°C. After washing with PBS, the fluorescent secondary antibody fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (EY Laboratories, San Maeto, CA, U.S.A.) was applied, and cells were incubated for 30 min at room temperature. To determine the co-expression of CK14 and SMA in neoplastic cells, double-label immunofluorescence testing was performed using anti-CK14 and anti-SMA an-tibodies as primary antibodies (Table 1). This assay used FITC-conjugated goat anti-mouse IgG (EY Laboratories) and rhodamine-conjugated goat anti-mouse IgG (Chemicon, Temecula, CA, U.S.A.) to label anti-CK14 and anti-SMA, respectively. Fluorescence was analyzed using a FSX100 fluorescence microscope (Olympus, Tokyo, Japan).

Formalin-fixed tissue specimens from the right thorax tumor were cut into 1-mm3 cubes, re-fixed in 2.5% glutaral-dehyde and post-fixed in 1% OsO4 for 2 hr. These specimens were then dehydrated through ascending grades of alcohol and embedded in epoxy resin. Ultra-thin sections were dou-ble-stained with uranyl acetate and lead citrate and examined using a JEM 1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan) at 80 kV.

Histologically, the right thorax tumor was a nodular, well-circumscribed, and multilobulated mammary lesion separated by small to moderate amount connective tissues (Fig. 1). The tumor was basically composed of two types of tubular structures which were cystic dilated ducts compris-ing a single layer of flattened cells with oval nuclei and small prominent nucleoli (Fig. 2) and double-layered tubules with cuboidal to columnar luminal epithelium and outer spindle-shaped cells (Fig. 3). Both types of tubular structures were irregularly distributed: however, the former type was pre-dominant in the tumor. The single-layered cystic ducts con-tained pale basophilic mucus that was Alcian blue-positive but PAS-negative (Fig. 4) and were occasionally lined by cuboidal cells with surface apocrine snout-like protrusions (Fig. 2, inset). In double-layered tubules, luminal epithelial cells had round to oval nuclei with prominent nucleoli, and surrounding outer cells showed heterochromatic oval or flattened nuclei. This type of neoplastic tubules contained PAS-positive, but Alcian blue-negative fluid in the lumens. Some neoplastic tubules were composed of heterogeneous linings with both characters of the single- and double-

layered tubular structures. In the tumor, mitotic cells and Ki-67-positive cells were extremely rare (data not shown). The left thorax and ventral neck tumors were diagnosed as a complex adenoma and a lipoma, respectively. No particular cystic ducts were observed within the left thorax and ventral neck tumors, and no metastasis was observed in local lymph nodes.

Table 2 and Fig. 5 summarize the immunohistochemical staining results of tumor cells. The lining cells of the single-layered cystic ducts were negative for CK CAM5.2 (Fig. 5a), but positive for the basal cell markers CK14 (Fig. 5b) and p63 (Fig. 5c). Many of these tumor cells were coincidentally positive for SMA (Fig. 5d), which was also comfirmed by double-labeled immunofluorescence microscopy (Fig. 6). These CK14/SMA double-positive cells were directly ex-posed to the luminal surface of the cystic ducts surrounded by a continuous PAS-positive basement membrane compris-ing type IV collagen (Fig. 7). Electron microscopy identi-fied myofilaments within the cytoplasm of cystic ductular cells (Fig. 8a and 8b). These filaments were distinguishable from tonofilaments or intermediate filaments, because they exhibited a parallel arrangement and focal densities along their course. Apocrine snout-like structures observed in the cystic ducts by light microscopy did not exhibit decapitation secretion, but contained several organelles indicating degen-erative process. In the bilayered tubules, luminal cells were positive for CK CAM5.2, and the surrounding outer cells were positive for CK14, p63 and SMA (Table 2).

To our knowledge, this is the first case report of a mam-mary adenoma comprising a single layer of basaloid cells with myoepithelial differentiation. Several tubular structures were lined only by SMA-positive myoepithelial cells and characteristically contained accumulated Alcian blue-posi-tive acid mucin in the lumens.

On the basis of these special and immunohistochemical stainings, the neoplastic cells in the present case are consid-ered to be originated from bipotent progenitor cells of the mammary gland. In human studies, these bipotent progenitor cells are derived from the mammary stem cells and differen-tiate into myoepithelial and luminal progenitor cells [11, 13]. A similar hierarchy of cellular differentiation is proposed in canine mammary glands, because most of the basal cells of the mammary gland, also referred as basal/myoepithelial cells, exhibit the immunophenotypes of both epithelial and smooth muscle cells [14]. Some studies proposed a close relationship between myoepithelial compartments and pro-genitor cells of the mammary gland [2, 12]. In the present

Table 1. Primary antibodies and immunostaining protocola)

Antibody Clone Dilution Antigen retrievalb) SourcePan-CK CAM5.2 Prediluted Proteinase K, 37°C, 10 min Becton Dickinson, Heidelberg, GermanyCK14 LL002 1:100 MW, pH 6.0, 90°C, 10 min Serotec, Wiesbaden, Germanyp63 4A4 1:150 AC, pH 6.0, 121°C, 10 min Thermo Fisher Scientific, Waltham, MA, U.S.A.SMA 1A4 1:100 No treatment Dako A/S, Glostrup, DenmarkKi-67 MIB-1 1:100 MW, pH 6.0, 95°C, 20 min Dako A/S, Glostrup, Denmark

a) CK=cytokeratin; SMA=α-smooth muscle actin. b) MW=microwave/citrate buffer (pH 6.0) or antigen activation solution (pH 9.0; Nichirei, Tokyo, Japan); proteinase K (Wako Pure Chemical Industries, Osaka, Japan), 0.4 mg/ml; AC=autoclave/citrate buffer (pH 6.0).

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case, the neoplastic cells lining the single-layered cystic ducts exhibited the definite immunophenotype of the basal cells, and most of these cells differentiated into myoepithelial cells as confirmed by ultrastructural morphology and SMA

expression. These ductular lumens contained Alcian blue-positive mucin, which might include chondroitin sulfate as seen in the stroma of complex adenoma or benign mixed tumor [8]. Therefore, we propose that the origin of the cells lining the single-layered cystic ducts might be myoepithelial progenitor cells, which differentiate into myoepithelial cells producing chondroitin sulfate. Histopathological and im-munohistological features of the double-layered neoplastic tubules shared common characteristics with those of com-plex adenoma [3].

The differential diagnosis of this tumor was myoepi-thelioma, complex adenoma, ductal adenoma and lobular hyperplasia with duct ectasia. Myoepithelioma is a rare benign neoplasm exclusively composed of spindle-shaped neoplastic cells with immunohistological characteristics

Fig. 1. Photomicrograph of mammary gland tumor at the right thorax from a 9-year-old, Miniature Pinscher, intact female dog. The tumor is a well-circumscribed and composed of nodular proliferation of tubular structures separated by thin connective tissues. Two types of tubular structures were identified within the tumor. Hematoxylin and eosin (HE) stain.

Fig. 2. Single-layered cystic ducts within the canine mammary tumor. The ducts contain light basophilic myxoid material in the lumens. Lining cells are basically flat, but occasionally cuboidal (inset, Bar=20 µm). These cuboidal cells exhibit apocrine snout-like protru-sion on their apical surface (arrowheads, Bar=50 µm). HE stain.

Fig. 3. Another type of tubular structures in the tumor is lined by cuboidal to columnar luminal epithelial cells and spindle outer cells. Bar=50 µm. HE stain.

Fig. 4. The myxoid material in the single-layered cystic ducts is positive for Alcian blue, but negative for PAS. Alcian blue (pH 2.5)–periodic acid Schiff (PAS) double stain. Bar=50 µm.

Table 2. The results of immunostaining

Tumor cell type CK CAM5.2 CK14 p63 SMASingle-layered cystic ducts Flattended luminal cells − + + + Cuboidal cells + + + −Double-layered tubules Luminal epithelial cells + − − − Outer cells − + + +

CK=cytokeratin; SMA=α-smooth muscle actin; − = negative; + = positive.

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Fig. 6. Double-label immunofluorescence staining of single-layered cystic ducts. CK14 and SMA were labeled using fluorescein isothiocyanate (FITC, green) and rhodamin (red), respectively. A number of CK14/SMA double-positive cells were directly exposed to the luminal surface of the ducts. Some of these were lined by only CK14/SMA double-positive cells (*). Nuclei are colored purple with 4,6-diamino-2-phenylindole (DAPI). Bar=50 µm.

Fig. 5. Immunohistochemical examination of the tumor portions composed of single-layered cystic ducts. The sections were im-munostained for (a) cytokeratin (CK) CAM5.2; (b) CK14; (c) p63 and (d) α-smooth muscle actin (SMA). The cells lining the ducts were constantly positive for CK 14, p63 and often positive for SMA. Bar=50 µm.

Fig. 7. Similar portion of Fig. 6. Cystic ducts were surrounded by a continuous basement membrane composed of type IV collagen (FITC, green). Immunostain for type IV collagen. Nuclei are colored purple with DAPI. Bar=50 µm.

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of normal myoepithelial cells [4]. Myoepithelioma and complex adenoma may be accompanied by deposition of extracellular basophilic myxoid matrices in the stroma, but not in the tubular lumens [4]. Ductal adenoma is composed of uniform bilayerd cords with two distinct rows [4]. The lobular hyperplasia consists of normal elements of mammary gland and does not exhibit aberrant structures as seen in this case. In conclusion, we diagnosed this tumor as an atypical mammary adenoma characterized by a single layer of cystic ducts comprising basaloid cells with frequent myoepithelial differentiation. The tumors may have originated from a bi-potent progenitor cell with predominant differentiation into the myoepithelal lineage than the luminal epithelial lineage.

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Fig. 8. Electron micrograph of a single-layered cystic duct in the mammary tumor. (a) Flat-shaped cell lined the duct with a few microvilli at the luminal surface. Bar=1 µm. (b) Fine filaments were identified within the cytoplasm of the flat-shaped cell. These filaments exhibited a parallel arrangement with focal densities (arrowheads) along their course. Bar=200 nm.