Background
Breast cancer is the second leading cause of death in women in the United States, with about 1,479,350 expected cases in 2009. This accounts for 27% (192,370) of all new cancer cases among women and 562,340 deaths per annum [
1]. With the implementation of mammographic screening, great progress in early breast cancer diagnosis has been achieved. Nowadays, many women are diagnosed with preinvasive intraepithelial lesions (IELs). Approximately half a million breast IELs are diagnosed yearly; these include 360,000 cases of usual hyperplasia (UH), 60,000 of atypical ductal hyperplasia (ADH) [
2], and 57,604 of ductal carcinoma
in situ (DCIS) [
1].
Detection and evaluation of IELs are used routinely to estimate a woman's risk of developing breast cancer and to aid physicians in designing optimal therapeutic strategies. It was postulated, based on epidemiological studies, that the risk for breast cancer ranges from 1.5-2, 4-5, and 8-10, respectively, for women diagnosed with UH, ADH, and DCIS [
3,
4]. Understanding the histopathological and molecular characteristics of the IELs will assist in elucidating the pathogenesis of breast cancer and identifying specific therapeutic targets [
5]. Several transplantable or chemically induced rodent models have been developed to study human cancers, including osteosarcoma, melanoma, bladder and intestinal tumors, non-Hodgkin lymphoma and mammary tumors [
6]. However, these models lack many aspects of human cancers [
7]. An animal model that develops spontaneous mammary tumors that resemble human breast cancer in many aspects is needed [
8,
9].
Feline mammary carcinoma is similar to human breast cancer in the age of onset, incidence, histopathologic features, biologic behavior, and pattern of metastasis [
9,
10]. The annual incidence of feline mammary neoplasia was estimated at 12.8-25.4 per 100,000 female cats [
11]. About 85% - 93% of feline mammary tumors are malignant, and there is little breed-associated predilection, except that Siamese cats appear to have a 2-fold increased risk. Mammary neoplasia has been reported to occur in cats from 9 months to 23 years of age (mean, 10 to 12 years) [
11,
12]. Hormonal influences are probably involved in the pathogenesis of feline mammary neoplasia. Cats that are ovariohysterectomized before 6 months or 1 year of age had 91% and 86% reduction in risk of developing mammary tumors, respectively, when compared to intact female cats [
13].
These data implicate ovarian hormones in the development of feline mammary tumors [
14]. The influence of hormonal factors is emphasized by Misdorp
et al., who demonstrated that the regular and prolonged administration of progestins (used for estrus prevention in cats) increased the risk of mammary tumor development [
15]. The influence of ovarian hormones is also well-established in humans. Early menarche, before age 12, increases the risk four-fold, as does late menopause [
16].
In humans, ER+ tumors have a better prognosis, and 50-60% of cases tend to respond to hormonal treatments [
17,
19]. ER+ breast carcinomas are usually (70-80%) well-differentiated with low expression of proliferation markers [
18]. However, 30% of human breast cancers are ER-negative with a worse prognosis than ER-positive tumors [
19]. Most cats (80%) tend to have ER-negative, highly aggressive mammary tumors; thus, they may be particularly suitable as animal models of human hormone-unresponsive breast cancer [
20].
Human epidermal growth factor receptor 2 (HER-2/
neu) is a cell-membrane receptor tyrosine kinase, normally involved in the signal transduction pathways leading to cell growth and differentiation [
21]. Approximately 15-20% of breast cancers have amplification of the HER-2/
neu gene or over-expression of its protein product. HER-2/
neu protein over-expression is associated with increased disease recurrence and has been used to predict patient response to treatment [
22]. De Maria
et al. reported that HER-2 gene kinase domain in cats and humans has 92% homology [
23]. HER-2 protein was highly expressed in feline carcinomas when compared to human breast carcinoma, suggesting its possible role as a prognostic marker [
24,
25].
To the best of our knowledge, feline mammary IELs have not been compared with pre-invasive lesions of the human breast. Thus, this study was undertaken to investigate the prevalence and types of IELs in feline mastectomy specimens and compare them to human breast IELs, and to determine the expression of ER-α, PR, HER-2/neu, and Ki67 by immunohistochemistry.
Methods
Tissue samples
Two hundred five formalin-fixed, paraffin-embedded specimens from 203 female cats with clinical mammary disease were retrieved from the archives of the Purdue University Animal Disease Diagnostic Laboratory and Veterinary Teaching Hospital (West Lafayette, IN) and the Department of Pathology and Veterinary Clinic, School of Veterinary Medicine, (Sassari, Italy). Eighty cats had been spayed before diagnosis; 122 were sexually intact; the sexual status of 1 cat was unknown. The cats' age ranged from 0.5-18 years (median, 10 years). Cats of different breeds were included (82 domestic shorthair, 18 domestic long-hair, 21 European, 10 Siamese, 8 Persian, 4 mixed-breed, 1 Himalayan, 1 Burmese); 58 cats had no breed designation.
Histology
Histologic sections, stained with hematoxylin and eosin (HE), were evaluated for the presence of IELs in tissue adjacent to excised mammary tumors. Lesions were classified according to criteria for IELs of the human breast [
26,
27]. In this study, we focused on the best-characterized IELs that arise in the terminal duct-lobular units; these included UH, ADH and DCIS (low-, intermediate-, and high-grade) [
5].
Lesions were classified in consultation with an MD pathologist (VM) and compared with IELs in women. Human samples were obtained from the Institute of Anatomy and Histopathology, Sassari University School of Medicine. The study protocol was approved by the Ethical Committee at the University of Sassari. Features applicable to usual ductal hyperplasia (UH), also called epitheliosis, consisted of ducts partially filled by a mixed population of epithelial and myoepithelial cells that exceeded 3 or 4 layers in thickness. The diagnosis of atypical ductal hyperplasia (ADH) was made when the mixed population of epithelial and myoepithelial cells had nuclear atypia. In some cases, cords of epithelial cells formed bridges with irregular fenestrations. In IELs in which cytologic features of low- or intermediate-grade DCIS were observed, but confined to 1 duct cross-section, the IEL was classified as ADH [
26,
28]. Ductal carcinoma
in situ (DCIS) was diagnosed when the IEL was composed purely of epithelial cells with cytologic and architectural atypia. Based on these cytological and architectural characteristics, DCIS was subdivided into 3 categories. Low-grade DCIS was composed of a proliferation of monomorphic cells with hyperchromatic central nuclei, inconspicuous nucleoli and few mitotic figures. Intermediate-grade DCIS was distinguished by the lack of the monotonous aspect and moderate nuclear pleomorphism. Finally, high grade DCIS was composed of pleomorphic atypical cells with large nuclei, prominent nucleoli, and frequent and/or atypical mitotic figures. Different patterns were observed (cribriform, papillary, micropapillary, solid, and solid with comedo-type necrosis). Tumors were classified according to WHO Histological Classification of Mammary Tumors of the Dogs and Cats [
29] and graded according to a semi-quantitative scheme, originally developed in humans [
30] and applied to feline mammary carcinoma by Castagnaro [
31]. Mammary carcinomas were graded as well (WDC), moderately (MDC) and poorly differentiated (PDC) carcinoma. Percentage of tubule formation, mitotic index, cellular and nuclear morphology were each assigned an individual score from 1 to 3 and then added, classifying carcinoma as follows: grade I (WDC), 3-5 points; grade II (MDC), 6-7 points; grade III (PDC), 8-9 points.
Immunohistochemistry
Immunohistochemistry was performed using the labeled streptavidin biotin (LSAB) method. Histologic sections (5 μm thick) from formalin-fixed, paraffin-embedded feline mammary tissue with IELs and without IELs (control tissue) were mounted on positively charged Superfrost slides (Fisher Scientific). Tissue sections were deparaffinized and rehydrated through a series of graded alcohols. Antigens were retrieved by a high-temperature heating method (slides were immersed in target retrieval solution at pH 6 [Dako Cytomation], in a steamer (90-95°C) with a 20-minute incubation for all antigens except for Her-2 neu, for which slides were kept in a water bath for 40 min at 97°C. Tissues were then blocked for endogenous peroxidase in 3% hydrogen peroxide in water, and for nonspecific binding in PBS containing 0.25% casein, stabilizing protein and 0.015 mol/L sodium azide (Protein Block Serum-Free, DakoCytomation). Tissues were incubated overnight at 4°C in the following antisera: ER-α monoclonal mouse anti-human antibody clone NCL-ER-6F11 at 1:40 dilution (Novocastra Ltd.), progesterone receptor monoclonal antibody PR 10A9 at 1: 50 dilution (Immunotech, Marselle, France), Ki67 monoclonal mouse anti-human antibody clone MIB-1 at 1:50 dilution (DakoCytomation), and HER-2/neu polyclonal rabbit anti-human antibody at 1:100 dilution (DakoCytomation), followed by biotinylated goat anti-mouse or goat anti-rabbit secondary antibodies (DakoCytomation). The chromogen was 3,3'-diaminobenzidine (DakoCytomation). Sections were counterstained with Mayer's hematoxylin and then cover-slipped in 50:50 xylene/Permount (Fisher Scientific). Negative control slides were treated with isotype-matched IgG serum. Control slides, known to be positive for each antibody, were incorporated into each run. Nuclear immunostaining for ER, PR and Ki-67 was evaluated counting a total of 1000 cells in 10 representative fields at high magnification (400×) whereas for smaller lesions, the entire lesion was considered. The number of immunopositive cells was expressed as a percentage (mean, median, minimum and maximum values).
The intensity of ER, PR, and Ki67 immunoreactivity was graded on a scale of 0 to 3, in which 0 = no reactivity, 1 = weak, 2 = moderate, and 3 = strong reactivity.
The over-expression of HER-2/neu was defined as increased cell membrane reactivity of epithelial cells. The scoring system according to the HercepTest™ can be summarized as follows: 0 = no staining or weak and incomplete membrane staining in less than 10% of the neoplastic cells; 1+ = incomplete and faint membrane staining in more than 10% of the neoplastic cells; 2+ = moderate and complete membrane staining in more than 10% of the neoplastic cells; 3+ = strong and complete membrane staining in more than 10% of the neoplastic cells. Scores of 0 or 1 were considered negative, whereas 2 or 3 were considered positive for HER-2/neu over-expression.
SDS-PAGE and Western Immunoblotting
Protein was extracted from fresh-frozen biopsy specimens from 5 feline mammary carcinomas and 5 normal mammary glands. Liver was used as a control. Replicate 5-μm-thick slices were cut from frozen tissue blocks. Five sections of each sample were placed in 2-mL Eppendorf safe-lock tubes and immersed in Laemmli buffer for lysis. After incubation on ice for 20 min, tissue lysates were clarified for 10 min at 12,000 × g at 4°C, denatured at 95°C for 5 min, and stored at -80°C until needed. For electrophoresis, protein extracts from fresh-frozen mammary and liver specimens were subjected to SDS-PAGE in 8% polyacrylamide gels according to Laemmli [
32]. Electrophoresis was stopped when the tracker dye reached the end of gels. Proteins were then stained with Coomassie Brilliant Blue R 250 (Sigma-Aldrich, St. Louis, MO) according to Westermeier [
33], decolorized and digitized with an Image Scanner (GE Healthcare).
For western immunoblots, electrophoresed proteins were transferred to nitrocellulose membranes and blocked in phosphate buffered saline, 0.05% Tween 20 (PBS-T), plus 5% skim milk for 1 hour to overnight. The membrane was then incubated with the HER-2/neu polyclonal rabbit anti-human antibody at 1:1000 dilution (DakoCytomation) in PBS-T plus 2% skim milk for 2 hours, washed five times with PBS-T, and incubated for 1 h with peroxidase-conjugated goat anti-rabbit secondary antibodies (Sigma-Aldrich) in PBS-T plus 2% skim milk. After washing the membrane five times with PBS-T, immunoreactivity was visualized by incubation with a chemiluminescent peroxidase substrate (Sigma-Aldrich).
Statistical analysis
Differences in IHC expression of ER, PR, Her-2/neu and Ki67 between types of IELs (e.g., ADH and UH) was obtained by a standard t-test for two-group comparison. Correlation between IELs and the adjacent tumors for ER, PR, HER-2/neu and Ki67 was obtained by simple regression analysis.
Discussion
Atypical lesions (ADH; DCIS) are predictors of invasive breast cancer [
3,
4]. However, monitoring the progression and invasion of these lesions in humans is not practical because the current standard therapy for DCIS is complete excision [
35]. Thus, establishing an animal model for IELs that correlates with invasive mammary carcinoma is important to develop preventive measures and effective treatments as well as for understanding the pathogenesis of the breast cancer.
Mammary IELs have not been well characterized in genetically engineered mouse models [
36], principally because they are not spontaneous, but rather induced by chemicals, radiation, or genetic modification. As in humans, and in contrast to mice and rats, spontaneous mammary tumors are quite common in cats [
9,
11]. Even though cats may not develop mammary neoplasia as frequently as dogs, their tumors more closely resemble those in women. For example, the benign mixed tumor that is so common in dogs almost never develops in cats or women [
37].
Although feline IELs (ductal hyperplasia and carcinoma in situ) have been reported, these lesions were not described in detail or compared with human IELs. Consequently, we evaluated mammary IELs and expression of ER, PR, and HER-2/neu in feline mastectomy specimens. Ki67 proliferation index was also estimated.
IELs were observed in 28% of mastectomy specimens from female cats with clinical mammary disease; 79% were associated with malignant neoplasms. DCIS was the most common lesion, as in human mammary biopsy specimens [
26]; 89% of DCIS lesions were adjacent to malignant tumors. ADH was detected less commonly than DCIS; nevertheless, 95% of these lesions were adjacent to malignant tumors. In contrast, about 50% of UH lesions were adjacent to benign tumors, duct ectasias or fibroadenomatous change, consistent with its only slightly elevated cancer risk in women [
38]. In our study, the prevalence of IELs in feline mammary gland may have been underestimated because only minimal peritumoral tissue was available for histologic evaluation.
Estrogen receptor expression in benign mammary epithelium could be a risk factor for malignancy by rendering cells susceptible to the proliferative stimulus of estrogens [
39]. In this study, ER was expressed in 62.5% of UH and in 7% of ADH, whereas all DCIS and 93% of tumors were negative. These data confirm that some feline mammary dysplasias and most neoplasms are estrogen receptor-negative as reported by Martin de las Mulas [
20] and Millanta [
40]. In cats, ER expression dramatically decreased as the IELs increased in grade; almost all neoplasms were negative for this marker. Most preinvasive lesions were ER-negative. Allred suggested that human ER-negative IELs could be involved in the development of ER-negative DCIS and its evolution into the 30% ER-negative human breast cancers [
19].
PR immunoreactivity was low in non-lesional mammary gland, IELs, and tumors in contrast to the findings of Millanta and de las Mulas [
40,
41]. This disparity may be due to a different proportion of ovariectomized cats, different stages of the estrus cycle, administration of exogenous progestins, or different PR immunohistochemical technique. Positivity was observed in only 6% of UH, in 7% of ADH, and in 17% of low-grade DCIS. No immunoreactivity was detected in intermediate-grade or high-grade DCIS, or in any of the 44 tumors. As for ER, PR expression decreased with increasing grade of IEL.
In agreement with Millanta and Dias Pereira [
42,
43], the Ki67 proliferative index increased from normal mammary tissue through IELs to malignant tumors. In our study, the expression of Ki67 correlated with the grade of malignant lesions and was inversely associated with ER expression. In fact, highly proliferative lesions tended to lose ER expression. In humans, Ki67 expression increased with increasing tumor grade and correlated with decreased overall survival rates and poor response to hormonal therapy. In cats, use of Ki67 as a prognostic factor for survival with mammary carcinoma has produced conflicting results. Studies by Castagnaro
et al revealed an association between Ki-67 index and biological behavior [
44]; however, Millanta
et al reported no significant prognostic importance in feline mammary carcinomas [
42]. In a recent investigation by Dias Pereira, the Ki67 index correlated positively for different histologic lesions and tumor types with grade [
43].
HER-2/
neu IHC results were surprising and differed from those of De Maria, Ordas, and Millanta [
23‐
25]. Those authors reported no immunoreactivity [
24,
25], or a faint, barely perceptible signal in part of the cell membrane [
23] in normal mammary ducts and acini. A number of normal tissues, including breast, express this receptor, which probably has a role in normal cell function, regulating growth and proliferation [
45]. However, we found HER-2/
neu protein expression in normal mammary epithelium with strong, complete membrane staining (3+), contrary to what is observed in humans [
46]. Immunohistochemical HER-2 protein overexpression was found in 27% of IELs and in 27% of tumors. HER-2/
neu expression was confirmed by Western Blot, in which both normal and neoplastic tissue showed a 185 kDa band, corresponding to human HER-2/
neu. Differences in signal intensity, however, were observed at comparable total protein loads. This result could reflect a higher expression of HER-2/
neu in some neoplastic tissues, as in the case of samples corresponding to lanes 3-4 of Figure
4, characterized by a stronger signal compared to neoplastic samples in lanes 1-2, and to healthy tissues (lanes 5 to 8). However, the presence of HER-2/
neu signal in adjacent histologically normal tissues, although constantly observed throughout this study, is unexpected, and needs explanation. If the DAKO antibody cross-reacts with a physiological epidermal growth factor normally expressed in the feline mammary gland, an increase in antibody specificity could overcome this issue. If the polyclonal antibody reacts with both HER-2/
neu and another epidermal growth factor receptor (EGFR) normally expressed in ducts and acini of non neoplastic mammary feline tissue, that would explain the differences in signal intensity both within neoplastic samples, and between neoplastic and healthy tissue samples. Furthermore, the HER-2/
neu protein could be present at higher levels in the normal feline mammary gland compared to the normal human mammary gland. Further investigations will be necessary to clarify the exact nature of this unexpected reactivity.
Similar to what was reported by Antuofermo in dogs, about half the feline IELs without atypia (UH) were associated with benign disease, whereas atypical IELs (ADH and DCIS) were generally associated with mammary cancer [
47]. The histologic grades of feline mammary carcinomas in our study were similar to those reported by Castagnaro [
31]. Like Seixas [
34], we recognized cases of micropapillary carcinoma.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
GPB: designed the study, performed the histopathological evaluation and IHC experiment and drafted the manuscript.
SIM.: coordinated, supervised and critically revised the manuscript.
MAM: performed the first histopathological evaluation and critically revised the manuscript.
VM: performed the second histopathological evaluation and critically revised the manuscript.
SP: performed the histopathological evaluation and critically revised the manuscript.
MFA: performed Western Blot experiments and critically revised the manuscript.
SU: revised the manuscript for important intellectual content.
EA: designed, coordinated, founded the study, drafted and critically revised the manuscript.
All authors read and approved the final manuscript.