Columnar cell lesions of the canine mammary gland: pathological features and immunophenotypic analysis
© Ferreira et al; licensee BioMed Central Ltd. 2010
Received: 28 April 2009
Accepted: 23 February 2010
Published: 23 February 2010
It has been suggested that columnar cell lesions indicate an alteration of the human mammary gland involved in the development of breast cancer. They have not previously been described in canine mammary gland. The aim of this paper is describe the morphologic spectrum of columnar cell lesions in canine mammary gland specimens and their association with other breast lesions.
A total of 126 lesions were subjected to a comprehensive morphological review based upon the human breast classification system for columnar cell lesions. The presence of preinvasive (epithelial hyperplasia and in situ carcinoma) and invasive lesions was determined and immunophenotypic analysis (estrogen receptor (ER), progesterone receptor (PgR), high molecular weight cytokeratin (34βE-12), E-cadherin, Ki-67, HER-2 and P53) was perfomed.
Columnar cell lesions were identified in 67 (53.1%) of the 126 canine mammary glands with intraepithelial alterations. They were observed in the terminal duct lobular units and characterized at dilated acini may be lined by several layers of columnar epithelial cells with elongated nuclei. Of the columnar cell lesions identified, 41 (61.2%) were without and 26 (38.8%) with atypia. Association with ductal hyperplasia was observed in 45/67 (67.1%). Sixty (89.5%) of the columnar cell lesions coexisted with neoplastic lesions (20 in situ carcinomas, 19 invasive carcinomas and 21 benign tumors). The columnar cells were ER, PgR and E-cadherin positive but negative for cytokeratin 34βE-12, HER-2 and P53. The proliferation rate as measured by Ki-67 appeared higher in the lesions analyzed than in normal TDLUs.
Columnar cell lesions in canine mammary gland are pathologically and immunophenotypically similar to those in human breast. This may suggest that dogs are a suitable model for the comparative study of noninvasive breast lesions.
The development of human breast cancer is believed to be a complex multistep process originating in terminal duct lobular units (TDLUs) and progressing towards invasive cancer. Various precursor breast lesions have been implicated in cancer development: atypical ductal hyperplasia (ADH), atypical lobular hyperplasia (ALH), lobular carcinoma in situ (LCIS), ductal carcinoma in situ (DCIS) and more recently columnar cell lesions (CCLs) [1, 2].
Columnar cell lesions (CCL) of the human breast comprise a group of conditions characterized by varying degrees of acinar dilation in the TDLUs, lined by several layers of columnar epithelial cells with uniform, ovoid nuclei oriented perpendicular to the basement membrane. The number of cellular layers enable CCLs to be divided into two broad categories: columnar cell change (CCC) [1-2 cell layers] or columnar cell hyperplasia (CCH) [>2 cell layers]. CCC and CCH with cytological atypia are further subclassified as flat epithelial atypia (FEA) [3–5].
Recent observational studies and emerging genetic evidence suggest that some CCLs, particularly those with low-grade/monomorphic-type cytological atypia, represent precursors to, or an early stage in the development of, low-grade ductal carcinoma in situ (DCIS) and invasive carcinoma [1, 6].
The canine mammary gland bears significant pathological lesions similar to the human breast [7, 8]. Breast lesions in dogs show cellular changes involved in the progression to invasive carcinoma. They are known as atypical hyperplasia and carcinoma in situ [9–11]. These descriptions may suggest that dogs are a promising model animal for comparative oncology. Therefore, a clearer account of the alterations in canine mammary cancer will help to better understand the key steps in the formation of human tumors.
In this paper we describe the presence of CCLs in canine mammary gland specimens, their association with other breast lesions and immunohistochemical findings in a series of specimens.
Specimens from one hundred and twenty-six consecutive cases of canine mammary gland, with previous diagnosis of epithelial lesions, were selected from the archives of the Laboratory of Comparative Pathology of the Biological Science Institute of the Federal University of Minas Gerais. The mammary gland samples were obtained after clinical diagnosis of mammary tumor and surgical removal of the lesion. Hematoxylin and eosin-stained sections were reviewed to search for columnar cell and associated lesions. The ages of the animals at the time of surgery ranged from 3 to 16 years (mean 9.8 years ± 2.2).
Histologic features of the different categories of columnar cell lesions in canine mammary gland.
Columnar Cell Change without atypia
Columnar Cell Hyperplasia
Flat Epithelial Atypia (FEA)
Flat Epithelial Atypia (FEA)
CCC with atypia
CCH with atypia
One to two columnar cell layers with uniform ovoid to elongated nuclei; nucleoli absent or inconspicuous.
Cellular stratification more than two columnar cell layers with uniform ovoid to elongated nuclei; nucleoli absent or inconspicuous.
One to two columnar cell layers with complex architectural patterns present; Mild to moderate cytologic atypia present (usually low-grade): round-to- ovoid, mildly pleomorphic and hyperchromatic nuclei, with inconspicuous nucleoli.
Cellular stratification more than two columnar cell layers with complex architectural patterns present; Mild to moderate cytologic atypia present (usually low-grade): round-to- ovoid, mildly pleomorphic and hyperchromatic nuclei, with inconspicuous nucleoli.
Considering the small size of the lesions, the immunohistochemical analysis were performed only on cases with enough material. Paraffin blocks were selected from six cases containg columnar cell lesions. Consecutive 5 μm thick sections were obtained and mounted on silanated slides for immunohistochemical study. Sections were stained for rabbit polyclonal antibodies: HER-2 (c-erbB-2; Dako; dilution: 1:40), P53 (clone CM1; Covance; diluition: 1:80); and mouse monoclonal antibodies: E-cadherin (clone 4A2C7; Zymed; dilution: 1:100), ER-LH2 (clone CC4-5; Novocastra; dilution: 1:25), PgR (clone hPRa2; Neomarkers; dilution: 1:20), Ki-67 (clone Mib-1; Dako; dilution: 1:25), cytokeratins 1, 5, 10 and 14 (clone 34βE-12; Dako; dilution: 1:40). Heat-induced epitope retrieval (20 min) using Dako antigen retrieval solution, pH 6.0 (Dako), was previously performed in a water bath. The slides were then cooled to room temperature for 20 min in the antigen retrieval buffer. The sections were incubated at room temperature in 3% (vol/vol) H2O2 for 15 min, in primary antibodies for 16 h, in reagent contained anti-mouse and anti-rabbit secondary antibodies (Biotinylated Goat Anti-polyvalent, Laboratory Vision) for 15 min and streptavidin peroxidase (UltraVision Large Volume Detection System, HRP, Laboratory Vision) for 15 min. Between incubations, the slides were washed for 2 × 5 min in phosphate-buffered saline containing 1% (vol/vol) Tween 20. Immunoreactivity was visualized by incubating the slides for 10 min with diaminobenzidine (DAB Substrate System; Laboratory Vision). The slides were then counterstained with Harris hematoxylin. Positive and negative control slides were included in each batch. As a positive control we used human breast cancer tissue known to express of the antibodies. Negative controls were assessed using normal serum (Ultra V Block, Laboratory Vision) as the primary antibody.
Staining for ER, PgR, P53, CK34βE-12 and E-cadherin was evaluated semi-quantitatively and scored into five categories: negative (-), <5% of cells stained; positive (+), 5% to 25% of cells stained; positive (++), 25% to 50% of cells stained; positive (+++), 50% to 75% of cells stained; diffusely positive (++++), >75% of cells stained . The proliferative index was calculated by counting the positive nuclei for Ki-67 staining in a total of 500 columnar cells from each lesion. HER-2 expression was defined as epithelial cell membrane staining and scored according to the American Society of Clinical Oncology, College of American Pathologists .
All procedures were performed under the guidelines and with the approval of the Ethics Committee in Animal Experimentation (CETEA/UFMG), protocol 192/2006.
The intraluminal microcalcifications were detected in columnar cell changes (6 cases without atypia and 6 cases with atypia) and in columnar cell hyperplasia (only 1 case without atypia).
Types of canine mammary columnar cell lesions and associated neoplastic alterations.
Tumor types (**)
carcinoma in benign tumor
ductal carcinoma in situ
13 (50.0) (*)
18 (46.1) (*)
benign mixed tumor
Types of canine mammary columnar cell lesions and associated ductal hyperplasia in presence of different tumors types.
ductal carcinoma in situ
The proliferation rate as measured by Ki-67 appeared higher in CCLs (mean 4.7%) than normal TDLU. Ki-67-positive cells were generally present but in low numbers in normal internal control tissue epithelium (<1% positive cells) (Figure 4D).
The CCLs were negative for cytokeratin 34βE-12, P53 and HER-2 in all six cases studied. Interestingly, P53 and HER-2 negative results were similar to those obtained with the associated benign and malignant tumors (two benign mixed tumors, one duct papilloma, one solid carcinoma and two ductal in situ carcinoma).
Columnar cell alterations of the canine mammary gland have been documented during microscopic analysis of breast tissues. A common component of most hyperplastic ductal lesions was columnar alteration (also referred to as columnar cell metaplasia) . However, histopathological criteria that characterize columnar lesions in dogs and their malignant potential when detected as the sole histopathological abnormality following breast biopsy are undocumented.
The morphological appearance of the canine CCLs was very similar to the human breast. Hyperplastic breast lesions, such as ductal hyperplasias without and with atypia, have previously been described in the canine mammary gland . To our knowledge, our study is the first to describe CCLs in the mammary glands of female dogs. In the present study we found CCLs in 53.1% of consecutive canine breast specimens. Lesions ranged from CCCs without atypia (61.1%) to FEA (38.8%).
Observational studies have revealed a relationship between CCLs and tubular carcinoma [15, 16]. Columnar cell lesions, in particular those with nuclear atypia, may also co-exist with lobular in situ neoplasia. Flat epithelial atypia is associated with established DCIS more frequently than by chance [17, 18].
Microcalcifications were also frequently found in association with canine CCLs, similar to human lesions. Although CCLs have long been recognized in the human breast under different names, their importance increased after more were diagnosed in mammographically detected lesions owing to microcalcifications [16, 18]. Although mammography is not routinely used in canine species we supposed that such microcalcifications would have a radiological appearance similar to that in the human breast.
Myoepithelial/basal and some epithelial cells in human CCLs are positive for CK5/6 . Though columnar cell lesions in human breast are usually negative for HER-2/neu, p53 and basal CK5/6 and CK14. Similar expression was found in all six cases studied, supporting the use of breast tumor biomarkers in this model. The immunohistochemical similarities between human and dog columnar cells also included, increased epithelial expression of the proliferation marker Ki-67/MIB1 and strong expression of hormonal receptors (ER and PR) and E-cadherin [13, 20, 21].
The canine species could be the most adequate model for new studies for cancer, due to the morphological and genotypic similarities of human lesions [20, 22, 23]. Spontaneous epithelial mammary lesions are common in dogs well before the age of onset of palpable mammary tumors [9, 7]. Thus, spontaneous tumours of canine mammary glands have been proposed as comparative models for the study of human breast cancer, since these lesions share epidemiological, clinical, behavioural and antigenic features [23–25].
Columnar cell lesions in canine mammary gland are pathologically and immunophenotypically similar to those in human breast. This may suggest that dogs are a suitable model for the comparative study of noninvasive breast lesions. Additional studies are needed to analyze the frequency of columnar cells lesions and their relationship to in situ carcinomas in canine mammary glands to provide a model for testing treatment modalities for mammary lesions and ultimately for clarifying patient management.
Atypical Ductal Hyperplasia
Atypical Lobular Hyperplasia
Columnar Cell Lesions
Columnar Cell Change
Columnar Cell Hyperplasia
Ductal Carcinoma In Situ
Ductal Hyperplasia without atypia
Flat Epithelial Atypia
Lobular Carcinoma In Situ
Terminal Duct Lobular Units
This work was supported in part by Fundação de Amparo a Pesquisa de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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