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Dec 12, 2011 - noma of the mammary gland in a dog was used as a positive control. Negative ... arranged in nests within the stromal cystic spaces. (Figure 1(d)). .... 38(2):556–560. Cassali GD, Serakides R, Gartner F, Schmitt FC. 2002.
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Invasive micropapillary carcinoma of the mammary glands in a mare a

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C.O. Gamba , M.R. Araújo , M.S. Palhares , L.C. Garbin , R.P.A. Maranhão , G.D. a

Cassali & R. Ecco

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Laboratorio de Patologia Comparada, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil b

Escola de Veterinária, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil Available online: 12 Dec 2011

To cite this article: C.O. Gamba, M.R. Araújo, M.S. Palhares, L.C. Garbin, R.P.A. Maranhão, G.D. Cassali & R. Ecco (2011): Invasive micropapillary carcinoma of the mammary glands in a mare, Veterinary Quarterly, 31:4, 207-210 To link to this article: http://dx.doi.org/10.1080/01652176.2011.642618

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Veterinary Quarterly Vol. 31, No. 4, December 2011, 207–210

CASE REPORT Invasive micropapillary carcinoma of the mammary glands in a mare C.O. Gambaa, M.R. Arau´job, M.S. Palharesb, L.C. Garbinb, R.P.A. Maranha˜ob, G.D. Cassalia and R. Eccob* a

Laboratorio de Patologia Comparada, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil; b Escola de Veterina´ria, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, Brazil (Received 7 June 2011; final version received 16 November 2011)

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Keywords: mare; equine; immunohistochemistry; invasive micropapillary carcinoma; mammary gland; udder

An adult crossbred intact mare of unknown age was prostrated in poor body condition. Also, tachycardia and tachypnea were detected by the referring veterinarian. The owner reported that he had seen an increase in the size of the mammary glands two months before. On physical examination, the udder was enlarged and the skin was ulcerated, which oozed fetid and blood-stained exudate. Blood analysis was not carried out because a neoplasm was suspected, euthanization was recommended and authorized by the owner. Subsequently, necropsy was performed. At necropsy, poor body condition and pale mucous membranes were observed. Moderate subcutaneous edema was observed in the axillary, thoracic, and ventral abdominal areas. Both mammary glands were enlarged (20  40  16 cm), and the skin was diffusely and deeply ulcerated (Figure 1(a)). Abundant purulent and blood-stained exudate oozed from this area. Mammary papillae were not observed because there was marked cutaneous ulceration and fibrosis. At the cut surface, there was a neoplastic mass with multiple friable dark-red areas (hemorrhage and necrosis) (Figure 1(b)). Alternating these areas, there was a white and slightly firm tissue proliferation with a lobulated aspect (Figure 1(c)). A white, firm, thick layer of tissue (fibrosis) was observed close to the skin surface. The mammary, kidney, iliac, and inguinal lymph nodes were extensively enlarged. On the cut surface, white or grayish-white and slightly firm tissue was observed replacing most of the normal architecture. The afferent and efferent lymphatic vessels of the lymph nodes were tortuous, engorged, and viscous, and a reddish-yellow secretion flowed out from the cut surface. In the pericardiac space, there was approximately 300 ml of translucent liquid (moderated hydropericardium). Tissue samples of the mammary gland tumor and the lymph nodes were collected, formalin fixed, and paraffin embedded. Subsequently, the fourmicron-thick sections were either stained with hematoxylin and eosin (H&E) or submitted to *Corresponding author. Email: [email protected] ISSN 0165–2176 print/ISSN 1875–5941 online ß 2011 Taylor & Francis http://dx.doi.org/10.1080/01652176.2011.642618 http://www.tandfonline.com

immunohistochemistry (IHC). Mitotic index was calculated by evaluating 10 peripheral tumor areas with a microscope (BX-41; magnification, 400) (Dutra et al. 2008). A biotin-peroxidase system was used for the immunohistochemical procedure with identification of the secondary antibody by the polymer (Advance HRP enzymeÕ , DakoCytomation, Carpinteria, California, USA). Antigen retrieval was performed in a water bath at 98 C or pressurized heating at 125 C with a citrate buffer solution at pH 6.0 (DakoCytomation, Carpinteria, California, USA) (epithelial membrane antigen [EMA], Ki-67, Cytokeratin, and C-erb-B2) or EDTA buffer solution at pH 9.0 (estrogen receptor and progesterone receptor) (DakoCytomation, Carpinteria, California, USA). In order to block the endogenous peroxidase activity, slides were incubated with a solution of H2O2 (3%) in methyl alcohol. Reagents were applied manually, with 1 h incubation or 16 h for the primary antibody and 30 min for the other reagents, except for diaminobenzidine chromogen, incubated for 5 min. The IHC antibody panel is described in Table 1. Labeling for the estrogen receptor (ER) and progesterone receptor (PR) was evaluated, and the sample was considered positive for ER and PR if there were at least 1% positive tumor nuclei (Hammond et al. 2010). Human epidermal growth factor receptor (HER-2) expression was defined by epithelial cell membrane labeling and scored according to the American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) (Wolff et al. 2007). Labeling was scored as follows: 0, no labeling; (þ), faint, incomplete membranous pattern; (þþ), moderate, complete membranous pattern less than 30%; and (þþþ), strong membranous pattern of at least 30%. A score of 0 or (þ) was considered negative; (þþ), equivocal; and (þþþ), positive. The proliferative index was calculated by counting the positive nuclei for Ki-67 (anti-MIB-1) labeling in a total of

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Figure 1. (a) Mammary gland of mare. Markedly enlarged (20  40  16 cm) with extensive cutaneous ulceration and bloodstained exudate, (b) Same specimen as in Figure 1(a) at the cut surface. Neoplastic mass showing friable dark-red areas and peripheral fibrosis, (c) Magnification of the Figure 1(b). Neoplastic mass with a white, lobulated aspect interplaced with necrotic and hemorrhagic areas, (d) Mammary gland of mare. Invasive micropapillary carcinoma characterized by clusters of neoplastic epithelial cells within the cystic spaces. 200. H&E, (e) Mammary gland of mare. Invasive micropapillary carcinoma. Neoplastic epithelial cells PR-immunoreactives labeled in brown (nuclei) (þ). Counterstained with Harris’ hematoxylin. 3,30 -Diaminobenzidine tetrahydrochloride was used as a chromogen. 600 and (f) Mammary gland of mare. Invasive micropapillary carcinoma. Clusters of neoplastic epithelial cells with EMA labeled on the basal surface of the cells (stroma-facing surface). Counterstained with Harris’s hematoxylin. 3,30 -Diaminobenzidine tetrahydrochloride was used as a chromogen. 400.

1000 neoplastic cells from each lesion (Dutra et al. 2008). Qualitative analysis was performed using cytokeratin AE1AE3 and EMA. Micropapillary carcinoma of the mammary gland in a dog was used as a positive control. Negative controls were assessed using normal serum (Ultra V BlockÕ , Labvision, Fremont, California, USA) as the primary antibody. Histological analysis revealed that the tumor was composed of infiltrative and ‘‘in situ’’ micropapillary areas. The ‘‘in situ’’ areas were characterized by epithelial cells arranged in clusters in the neoplastic ducts, without fibrovascular stroma. In few areas, a lumen was present in the cluster of cells. The invasive areas were characterized by neoplastic epithelial cells arranged in nests within the stromal cystic spaces

(Figure 1(d)). The neoplasm was considered pure because it showed 100% invasive micropapillary areas. The clusters of epithelial cells were characterized as polygonal cells with eosinophilic cytoplasm and pleomorphic nuclei. Extensive fibrosis, multifocal to coalescing liquefactive necrosis, and hemorrhage were observed. The mitotic index was 3 mitosis/400 magnification. The lymph nodes showed neoplastic epithelial cells in a micropapillary pattern associated with multifocal to coalescing fibrosis. The immunohistochemical analysis for steroid receptors revealed positivity for ER (þ), with nuclear labeling in 10% of the cells, and PR (þ), with nuclear labeling in 25% of the cells (Figure 1(e)). Incomplete membrane labeling of moderate intensity was observed

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Veterinary Quarterly Table 1. Antibodies used in immunohistochemistry.

Target antigen

Clone/ manufacturer

Incubation time (h)

Dilution

Antigen retrieval (T C-min)

ER

1D5/Dako

1

1:20

Pressurized heating (125 C-2) Pressurized heating (125 C-2)

PR

HPRA2/ Neomarkers

1

1:20

EMA

E29/Dako

16

1:25

Water bath (98 C-20)

Ki-67

Mib-1/Dako

1

1:25

Cytokeratin

AE1AE3/Dako

1

1:100

Pressurized heating (125 C-2) Water bath (98 C-20)

C-erb-B2 (HER-2)

Polyclonal/Dako

16

1:200

Water bath (98 C-20)

Species Monoclonal mouse (anti-human) Monoclonal mouse (anti-human, rabbit, cow, horse, and chicken) Monoclonal mouse (anti-human) Monoclonal mouse (anti-human) Monoclonal mouse (anti-human) Polyclonal rabbit (anti-human)

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Notes: ER, estrogen receptor; PR, progesterone receptor; EMA, epithelial membrane antigen; HER-2/neu, human epidermal growth factor receptor.

for HER-2 (þ). Intense cytoplasmic labeling for cytokeratin was detected, and the basal surface of the cells was labeled for EMA (Figure 1(f)). The nuclear labeling for Ki-67 was observed in 8.7% of the neoplastic cells. Neoplasms of the mammary glands are common in carnivores but are very uncommon in herbivores (Moulton 1990). In horses, there are only a few reports of mammary neoplasms in the last 30 years (Schmahl 1972; Acland and Gillette 1982; Munson 1987; Foreman et al. 1990; Seahorn 1992; Reppas et al. 1996; Kato et al. 1998; Prendergast et al. 1999; Hirayama et al. 2003; Brito et al. 2008). Invasive micropapillary carcinoma (IMC) of the breast is an aggressive and distinctive variant of human infiltrative ductal carcinoma, which presents lymph tropism and frequently spreads to the lymph nodes (Luna-more´ et al. 1994). Micropapillary carcinoma has been described in dogs (Cassali et al. 2002; Gama et al. 2008) and cats (Seixas et al. 2007), but reports in equine species have not yet been found. This report described the clinical, pathological, and immunophenotypical characteristics of invasive micropapillary carcinoma of the mammary glands of an intact mare. Equine mammary carcinomas consist of solid carcinomas (Schmahl 1972; Acland and Gillette 1982; Munson 1987), papillary ductal adenocarcinomas (Foreman 1990; Reppas et al. 1996; Prendergast et al. 1999), and ductal carcinomas (Hirayama et al. 2003). Histopathological findings in this report correspond to the IMC in humans, dogs, and cats. ‘‘In situ’’ carcinomatous and infiltrative micropapillary areas were observed, and it is believed that in humans, these invasive regions may arise from the ‘‘in situ’’ areas (Siriaunkgul and Tavassoli 1993). This case displayed a 100% micropapillary pattern, defined as the pure invasive micropapillary carcinoma. Similar to the observations made in humans (Luna-more´ et al.

1994), dogs (Cassali et al. 2002), and cats (Seixas et al. 2007), the described neoplasm presented extensive lymph node metastasis that retained the architectural and histological appearance of the primary tumor. The equine IMC was positive for cytokeratin AE1AE3, confirming its epithelial histogenesis. Histopathological diagnosis was confirmed by the ‘‘inside-out’’ pattern (inversion of cellular polarity) identified through EMA labeling on the stroma-facing surface of the epithelial cell nests, similar to those in humans (Li et al. 2006) and dogs (Cassali et al. 2002) IMC findings. In human invasive micropapillary carcinomas, the expression of the EMA is predominantly detected on the basal surface of the cells (stroma-facing surface), in contrast to conventional adenocarcinomas in which EMA is mostly apical, intracytoplasmic, or intercellular. The inversion of cellular polarity may be one of the key factors in distinctive morphology of this tumor type, determining metastasis. Probably, the detachment of the neoplastic cells from the stroma allowed the dissection of the connective tissue, enhancing their spread and leading to early dissemination to the lymph nodes (Nassar et al. 2004). Some authors have described human IMCs with higher positivity for hormone receptors (Luna-More´ et al. 2000; Walsh and Bleiweiss 2001). The equine IMC showed positivity for hormonal receptors differently than for two others equine mammary carcinomas reported in the previous papers (Seahorn et al. 1992; Reppas et al. 1996). Similar to the canine findings (Cassali et al. 2002), an inverse relationship between positivity for hormone receptors and the cell proliferation index was observed. Characteristics observed in this report such as low cell proliferation index, positivity for hormone receptors, and negativity for HER-2 are related to better

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prognosis in humans (Pinder et al. 1995; Onitilo et al. 2009). However, metastasis to all lymph nodes was observed, supporting the described lymphatic dissemination in humans (Luna-more´ et al. 1994; De la Cruz et al. 2004), dogs (Cassali et al. 2002), and cats (Seixas et al. 2007). To the author’s knowledge, this is the first report of pure invasive micropapillary carcinoma in the equine species. Therefore, this neoplasm, though rare, should be included as a clinical differential diagnosis for mammary tumors in this animal species.

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Acknowledgments This work has been financially supported by ‘‘Fundac¸a˜o de Amparo a Pesquisa de Minas Gerais’’ (FAPEMIG), ‘‘Conselho Nacional de Desenvolvimento Cientı´ fico e Tecnolo´gico’’ (CNPq), ‘‘Pro-Reitoria de Pesquisa da UFMG,’’ and ‘‘Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´ vel Superior’’ (CAPES), Brazil.

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