L. Peña

Immunohistologic Detection of Estrogen Receptor Alpha in Canine Mammary Tumors: Clinical and Pathologic Associations and Prognostic Significance

Eighty-nine canine mammary tumors and dysplasias of 66 bitches were investigated to determine the immunohistochemical expression of classical estrogen receptor (ER-α) and its clinical and pathologic associations and prognostic value. A complete clinical examination was performed and reproductive history was evaluated. After surgery, all animals were followed-up for 18 months, with clinical examinations every 3–4 months. ER-α expression was higher in tumors of genitally intact and young bitches (P < 0.01, P < 0.01) and in animals with regular estrous periods (P = 0.03). Malignant tumors of the bitches with a previous clinical history of pseudopregnancy expressed significantly more ER-α (P = 0.04). Immunoexpression of ER-α decreased significantly with tumor size (P = 0.05) and skin ulceration (P = 0.01). Low levels of ER-α were significantly associated with lymph node involvement (P < 0.01). Malignant tumors had lower ER-α expression than did benign tumors (P < 0.01). Proliferation index measured by proliferating cell nuclear antigen immunostaining was inversely correlated with ER-α scores (P = 0.05) in all tumors. Low ER-α levels in primary malignant tumors were significantly associated with the occurrence of metastases in the follow-up (P = 0.03). Multivariate analyses were performed to determine the prognostic significance of some follow-up variables. ER-α value, Ki-67 index, and age were independent factors that could predict disease-free survival. Lymph node status, age, and ER-α index were independent prognostic factors for the overall survival. The immunohistochemical detection of ER-α in canine mammary tumors is a simple technique with prognostic value that could be useful in selecting appropriate hormonal therapy.

Immunohistochemical Detection of Ki-67 and PCNA in Canine Mammary Tumors: Relationship to Clinical and Pathologic Variables

The objectives of this study were to measure the proliferation indices in canine mammary tumors using immunohistochemical detection of Ki-67 and proliferating cell nuclear antigen (PCNA), to determine the relationship of these antigens to clinical and pathologic variables, and to investigate the usefulness of these antigens as prognostic indicators. Ninety-six female dogs with 115 primary nonmetastasized spontaneous mammary tumors and dysplasias were included in the study. Immunostaining was performed using MIB-1 and PC10 monoclonal antibodies against Ki-67 and PCNA, respectively. Ki-67 and PCNA proliferation indices were determined. Dogs were followed for 18 months, with clinical examinations every 3–4 months. There was a significant correlation between Ki-67 and PCNA indices in the dogs with dysplasias and benign tumors but not in the dogs with malignant tumors. The clinical stage at first presentation was related to the proliferative index measured with Ki-67 but not to that measured with PCNA. Proliferation indices were significantly lower in the nonmalignant tumors and dysplasias than in the malignant tumors. In malignant tumors, the PCNA index had a positive correlation with the histologic malignant grade and the nuclear grade. High index values of Ki-67 were positively correlated with metastasis, death from neoplasia, low disease-free survival rates, and low overall survival rates. PCNA displayed no significant association with these variables. Multivariate analyses concerning metastasis, disease-free survival, and overall survival revealed that the Ki-67 index had prognostic value.

Canine inflammatory mammary carcinoma: histopathology, immunohistochemistry and clinical implications of 21 cases.

Human inflammatory breast carcinoma (IBC) is the most malignant type of breast cancer with an extremely poor prognosis. The dog is the unique animal species in which spontaneous inflammatory mammary carcinoma (IC) has been reported, although it is not well documented. The purpose of this study was to characterize histopathologically and immunohistochemically the canine IC, considering associated clinical features. Twenty-one dogs diagnosed with IC and with known clinical and necropsy data were included in the study. Tissue samples from necropsies underwent a histopathological review and an immunohistochemical study (Ki-67, estrogen receptor (ER), progesterone receptor (PR), and P53 tumor suppressor protein). The histological study revealed several types of carcinomas (solid, tubular, papillary, and adenosquamous) and three lipid-rich carcinomas. All tumors were ER negative. Two histological patterns of neoplastic dermal infiltration were observed: tubular/papillary and sarcomatous-like. Dermal sarcomatous-like infiltration was significantly related to previous treatments with progestagens (p=0.006), primary type of IC (p=0.03), extreme local pain (p=0.02), reduced observation of emboli in dermal lymphatic vessels (p=0.01), and increased expression of p53 (p=0.001). PR expression was significantly higher in secondary post-surgical IC (p=0.04). The absence of PR was related to the existence of pulmonary metastases at necropsy (p=0.04). Canine primary IC is the most aggressive form of this disease with distinct histopathological and immunohistochemical characteristics. Progestins and endocrine-related mechanisms seem to be involved in canine IC development. Canine IC could serve as a spontaneous model for human IBC, particularly in studies concerned with new therapeutics approaches.

Histological, Immunohistological, and Ultrastructural Description of Vasculogenic Mimicry in Canine Mammary Cancer

Canine inflammatory mammary cancer (IMC) and human inflammatory breast cancer (IBC) are the most aggressive and lethal type of mammary cancer in female dogs and in women. The generation of microvascular channels by malignant tumor cells (endothelial-like cells [ELCs]) without endothelial cell participation (vasculogenic mimicry) has been reported in human breast cancer, including IBC, and is considered a new type of tumor angiogenesis. The aim of this study was to investigate the presence of ELCs in highly malignant canine mammary tumors (IMC and non-IMC) by histology, inmunohistochemistry (pancytokeratin, cytokeratin 14, vimentin, actin, desmin, vWF, CD31, and CD34), and electron microscopy. This retrospective study included 21 female dogs with diagnoses of IMC and 20 animals with metastatic grade III noninflammatory malignant mammary tumors (MMT). IMC tumors (33.33%) and MMT (5%) showed ELCs forming structures similar to small capillaries. The histological, immunohistochemical (positive to AE1/AE3 and cytokeratin 14, mostly negative to endothelial markers), and ultrastructural characteristics of these cells indicated vasculogenic mimicry. The higher frequency of this phenomenon in inflammatory versus noninflammatory canine mammary cancer is in agreement with previous studies in experimental and spontaneous human IBC, and it could be in relation with the extremely high lymphangiogenic capacity and metastatic lymphangiotropism characteristics of inflammatory breast cancer.

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

In this prospective study, a canine-adapted histological grading method was compared with histopathological and clinical characteristics and was evaluated as a prognostic indicator in canine mammary carcinomas (CMCs). Recruited dogs with at least 1 malignant mammary tumor (n = 65) were clinically evaluated, surgically treated, and followed up (minimum follow-up 28 months, maximum 38 months). Histopathological diagnoses were performed according to Goldschmidt et al (2011). Tumors were graded as grade I (29/65), grade II (19/65), and grade III (17/65). The tumor size, clinical stage, histological diagnosis, presence/absence of myoepithelial proliferation, and regional lymph node metastases at diagnosis were significantly associated with histological grade. The histological grade, age, clinical stage, tumor subtype group, and lymph node metastases at time of diagnosis were significantly associated with the development of recurrences and/or metastases, cancer-associated death, and survival times (disease-free survival and overall survival) in univariate analyses. A subdivision of clinical stage I (T1N0M0) into stages IA and IB was proposed in terms of prognosis. The clinical stage, histological grade, and spay status were selected as independent prognostic variables (multivariate analyses) with disease-free survival as the dependent variable. When overall survival was evaluated as a dependent variable, clinical stage and histological grade were selected as the independent covariates. This grading system is a useful prognostic tool, facilitates histological interpretation, and offers uniform criteria for veterinary pathologists. Comparative studies on CMCs performed in different countries should take into account possible changes in the prognoses due to different proportions of spayed females among the selected dog population.

Role of steroid hormones and prolactin in canine mammary cancer

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.

Steroids and receptors in canine mammary cancer

The aims of this study were to investigate the serum and tissue content of androgens and estrogens in canine inflammatory mammary carcinomas (IMC) as well as in non-inflammatory malignant mammary tumors (MMT), and assessed the immunoexpression of estrogen and androgen receptors using immunohistochemistry. Profiles for the androgens dehydroepiandrosterone (DHEA), androstenedione (A4), and testosterone (T), and for the estrogens 17beta estradiol (E2) and estrone-sulphate (SO4E1) were measured both in tissue homogenates and in serum of MMT and IMC by EIA techniques in 42 non-inflammatory malignant mammary tumors (MMT) and in 14 inflammatory mammary carcinomas (IMC), prospectively collected from 56 female dogs. Androgen receptor (AR) and estrogen receptor alpha (ERalpha) and beta (ERbeta) expression was studied using immunohistochemistry (strepavidin-biotin-peroxidase method) in samples of 32 MMT and 14 IMC, and counted by a computer image analyzer. IMC serum and tissue levels of androgens were significantly higher than MMT levels. Tissue content of estrogens was also significantly higher in IMC than in MMT. Serum values of SO4E1 were significantly higher in IMC, but serum levels of E2 were significantly lower in IMC compared to MMT cases. Medium-high androgen receptor intensity was observed in 64.28% of IMC and 40.62% of MMT. No important differences were found between ERalpha expression in IMC (100% negative) and MMT (90% negative). ERbeta and AR were intensely expressed in highly malignant inflammatory mammary carcinoma cells. To our knowledge, this is the first report relative to AR immunohistochemistry in canine mammary cancer and to estrogens or androgens in serum of dogs with benign or malignant mammary tumors.

First description of feline inflammatory mammary carcinoma: clinicopathological and immunohistochemical characteristics of three cases

IntroductionInflammatory breast cancer is a special type of locally advanced mammary cancer that is associated with particularly aggressive behaviour and poor prognosis. The dog was considered the only natural model in which to study the disease because, until now, it was the only species known to present with inflammatory mammary carcinoma (IMC) spontaneously. In the present study we describe clinicopathological and immunohistochemical findings of three cats with IMC, in order to evaluate its possible value as an animal model.MethodsWe prospectively studied three female cats with clinical symptoms of IMC, identified over a period of 3 years. Clinicopathological and immunohistochemical evaluations of Ki-67, and oestrogen, progesterone and androgen receptors were performed.ResultsAll three animals presented with secondary IMC (postsurgical) characterized by a rapid onset of erythema, severe oedema, extreme local pain and firmness, absence of subjacent mammary nodules, and involvement of extremities. Rejection of the surgical suture was observed in two of the cats. Histologically, highly malignant papillary mammary carcinomas, dermal tumour embolization of superficial lymphatic vessels, and severe secondary inflammation were observed. The animals were put to sleep at 10, 15 and 45 days after diagnosis. Metastases were detected in regional lymph nodes and lungs in the two animals that were necropsied. All tumours had a high Ki-67 proliferation index and were positive for oestrogen, progesterone and androgen receptors.ConclusionOur findings in feline IMC (very low prevalence, only secondary IMC, frequent association of inflammatory reaction with surgical suture rejection, steroid receptor positivity) indicate that feline IMC could be useful as an animal model of human inflammatory breast cancer, although the data should be considered with caution.

Crosstalk between GH/IGF-I axis and steroid hormones (progesterone, 17β-estradiol) in canine mammary tumours

Growth hormone (GH), insulin-like growth factor I (IGF-I), progesterone (P4) and 17beta-estradiol (17-E2) concentrations have been studied in 84 mammary tumours (44 dysplasias and benign tumours and 40 malignant neoplasias) from 33 female dogs. Thirteen normal mammary glands from 80 healthy female dogs were also analysed as controls. GH concentrations were determined in mammary homogenates by radio-immunoassay. IGF-I, P4 and 17-E2 tissue levels were determined by enzyme-immunoassay (EIA) techniques. The potential correlations between GH/IGF-I concentrations and P4 and 17-E2 mammary tissue levels were investigated. Tissue GH (p<0.01) and IGF-I concentrations (p<0.01) were significantly higher in malignant tumours than in benign neoplasms. Likewise, malignant tumours were the mammary lesions that displayed the highest P4 and 17-E2 tissue levels. Strong correlations between GH/IGF-I (n=84; r=0.436; p<0.001), P4/GH (n=84; r=0.562; p<0.001) and 17-E2/IGF-I (n=84; r=0.638; p<0.001) were observed in tumoral tissue homogenates. Our study provides evidence that P4 might increase autocrine GH production which might directly stimulate local or systemic IGF-I secretion. Additionally, the IGF-I effect might be influenced by local levels of 17-E2. These results suggest that all these hormones and factors might act as local growth factors stimulating the development and/or maintenance of canine mammary tumours in an autocrine/paracrine manner.

Steroid hormone profile of canine inflammatory mammary carcinoma: a preliminary study

Inflammatory mammary carcinoma (IMC) is the most aggressive spontaneous type of mammary malignant tumor both in women and dogs. Latest studies in dogs indicate that different endocrine mechanisms seem to be involved in inflammatory carcinomas (IMCs). The aim of the present study was to characterize the steroid hormone profile of inflammatory carcinoma, and to compare it with mammary dysplasias, benign tumors and other malignant tumors. Eighty-six mammary samples (10 normal mammary tissue, 21 dysplasias, 26 benign, 22 malignant, and 7 IMC) from 30 female dogs were used. Hormone levels of progesterone (P4), 17beta-estradiol (E2), androstenedione (A4), dehydroepiandrosterone (DHEA), and estrone sulphate (E1SO4) in tissue homogenates were measured by enzyme immunoassays (EIAs) techniques, previously validated for this species. IMC displayed the following steroid profile: P4: 13.80+/-0.56 microg/g; E2: 675.19+/-33.00 ng/g; A4: 631.73+/-70.73 microg/g; DHEA: 702.22+/-89.93 microg/g, and E1SO4: 2.84+/-0.32 mg/g. All of these hormones were significantly higher (P<0.001) compared with the hormone steroid profile determined for malignant, benign, dysplasias, and normal mammary tissue. The most relevant finding was the increased levels, two or three times, of both DHEA and E1SO4 in IMC respect to other groups (P<0.001). These results, together with the highest immunohistochemical expression of P450scc found in IMC, suggest the hypothesis that an autocrine mechanism could be especially involved in the development of canine inflammatory carcinoma.