Progesterone receptor isoform analysis by quantitative real-time polymerase chain reaction in formalin-fixed, paraffin-embedded canine mammary dysplasias and tumors.

Cloning and sequencing of the progesterone receptor gene in dogs have revealed 2 isoforms, A and B, transcribed from a single gene. Distribution of isoforms A and B in canine mammary lesions has hitherto been investigated only by Western blot analysis. This study analyzed progesterone receptor and its isoforms in formalin-fixed, paraffin-embedded tissue samples from canine mammary lesions (4 dysplasias, 10 benign tumors, and 46 carcinomas) using 1-step SYBR Green quantitative real-time polymerase chain reaction (RT-qPCR). Progesterone receptor was expressed in 75% of dysplasias, all benign tumors, and 59% of carcinomas. Carcinomas, and particularly simple epithelial-type carcinomas, displayed the lowest levels of expression. A high rate of agreement was recorded between RT-qPCR and immunohistochemical labeling. Isoforms A and B were successfully amplified, with correlation coefficients of 0.99 and amplification efficiencies close to 2, and were expressed in all lesion types analyzed. Predominance of A over B expression was observed in carcinomas and complex adenomas. Low-grade tumors exhibited higher progesterone receptor messenger RNA (mRNA) levels, but no difference was observed in the expression of isoform A versus B. Analysis of progesterone receptor mRNA isoforms by RT-qPCR was successful in routinely formalin-fixed, paraffin-embedded tissue samples and enabled the distribution of isoforms A and B to be identified for the first time in dysplasias, benign tumors, and malignant tumors of the canine mammary gland. These findings will facilitate future research into the role of progesterone receptor isoforms in the progression of canine mammary tumors.

Canine mammary tumors: a review and consensus of standard guidelines on epithelial and myoepithelial phenotype markers, HER2, and hormone receptor assessment using immunohistochemistry.

Although there have been several studies on the use of immunohistochemical biomarkers of canine mammary tumors (CMTs), the results are difficult to compare. This article provides guidelines on the most useful immunohistochemical markers to standardize their use and understand how outcomes are measured, thus ensuring reproducibility of results. We have reviewed the biomarkers of canine mammary epithelial and myoepithelial cells and identified those biomarkers that are most useful and those biomarkers for invasion and lymph node micrometastatic disease. A 10% threshold for positive reaction for most of these markers is recommended. Guidelines on immunolabeling for HER2, estrogen receptors (ERs), and progesterone receptors (PRs) are provided along with the specific recommendations for interpretation of the results for each of these biomarkers in CMTs. Only 3+ HER2-positive tumors should be considered positive, as found in human breast cancer. The lack of any known response to adjuvant endocrine therapy of ER- and PR-positive CMTs prevents the use of the biological positive/negative threshold used in human breast cancer. Immunohistochemistry results of ER and PR in CMTs should be reported as the sum of the percentage of positive cells and the intensity of immunolabeling (Allred score). Incorporation of these recommendations in future studies, either prospective or retrospective, will provide a mechanism for the direct comparison of studies and will help to determine whether these biomarkers have prognostic significance. Finally, these biomarkers may ascertain the most appropriate treatment(s) for canine malignant mammary neoplasms.

Malignant mesenchymoma of the heart base in a dog with infiltration of the pericardium and metastasis to the lung.

A 9-year-old male rottweiler was presented with abdominal distension, ascites and respiratory distress and marked bulging in the perineal region. At necropsy examination the animal had profuse ascites and hydropericardium and a multinodular mass in the right auricle of the heart infiltrating the epicardium and pericardium and metastasizing to the caudal lobe of the left lung. Microscopically and immunohistochemically the tumour was composed of neoplastic cells with muscular, cartilaginous and adipose differentiation. A diagnosis of malignant mesenchymoma with leiomyosarcomatous (≈ 50%), rhabdomyosarcomatous (≈ 30%), chondrosarcomatous (25%) and liposarcomatous (5%) components was made. Metastatic malignant mesenchymoma has not been reported previously at this site in the dog.

Activation of AKT in feline mammary carcinoma: a new prognostic factor for feline mammary tumours.

The PI3K/AKT/PTEN pathway is involved in the pathogenesis of several human cancers. This study investigated the biological and prognostic value of PI3K/AKT/PTEN pathway dysregulation in feline mammary tumours. Expression of p-AKT, HER2, PTEN and steroid receptors was assessed by immunohistochemistry (IHC) in 27 malignant and 12 benign mammary tumours from 39 female cats followed up over a 24-month period. Feline mammary carcinoma (FMC) cell lines were analyzed by Western blot and the feline AKT gene sequence was characterized. p-AKT expression statistically correlated with tumour malignancy, histological dedifferentiation and clinical recurrence. The animals with tumours expressing p-AKT had a shorter disease-free period than those with p-AKT-negative tumours. AKT activation was associated with HER2 expression and PTEN down-regulation, as occurs in human breast cancer, and feline AKT sequencing showed high homology with the human AKT gene. No AKT activation was observed in relation to either oestrogen receptor α (ERα) or progesterone receptor expression. Taken together, these data offer an explanation for AKT signalling and its role in FMC pathogenesis and prognosis, shedding new light on similarities between feline mammary tumours and hormone-independent breast cancer.

Human follicular fluid from superovulated women inhibits progesterone receptor-dependent gonadotropin-releasing hormone self-priming in an estrous cycle-dependent manner in the rat.

These experiments investigated the involvement of gonadotrope progesterone receptor (PR) in the effects of the putative gonadotropin surge-attenuating factor (GnSAF) on gonadotropin (LH and FSH) secretion. Human follicular fluids (hFF) used in this study were aspirated from follicles in gonadotropin-treated women for in vitro fertilization. Samples were subjected to two-fold charcoal extraction of steroid hormones and two-fold inhibin immunoprecipitation. Gonadotropin secretion parameters were assessed by specific radioimmunoassays. In the first experiment, the effects of hFF on both basal and GnRH-stimulated gonadotropin secretion and GnRH self-priming were studied in incubated hemipituitaries from rats on each day of the 4-day estrous cycle. hFF inhibited only GnRH self-priming in pituitaries from rats in diestrus. In the second experiment, immunohistochemical PR expression and action were evaluated in pituitaries from rats in diestrus. PR-positive (PR10A9 antibody) gonadotropes were detected (4-5/field 40x), and antiprogestins added to the incubation media blocked the ligand-independent (GnRH) activation of PR effects on GnRH selfpriming. Finally, the third experiment evaluated the effects of hFF on P-induced potentiation of GnRH-stimulated LH secretion. GnSAF bioactivity, as evidenced by inhibition of PR-induced potentiation of GnRH-stimulated LH secretion, was found in diestrous pituitaries incubated with hFF. The results indicate that GnSAF attenuated GnRH-dependent LH secretion in diestrus through the inhibition of PR-dependent GnRH self-priming.

Effects of aglepristone, a progesterone receptor antagonist, in a dog with a vaginal fibroma.

A 12-year-old, entire, nulliparous crossbreed female dog was presented with a history of vulval bleeding, bulging of the perineum and faecal tenesmus. A firm, non-painful perineal mass, measuring 9.11x5.4 cm, with erythema was detected. Abdominal radiography showed compression and elevation of the rectal ampulla. A dose of 10 mg/kg aglepristone was administered subcutaneously on days 1, 2, 8, 15, 28 and 35. An incision biopsy was taken on day 15 and immunohistochemical analysis showed that the majority of neoplastic cells expressed progesterone receptors. Both the cutaneous erythema and the faecal tenesmus had resolved by day 28. A 50 per cent reduction in size was observed by day 60 (surgical excision). This study shows that benign tumours of the vagina of the dog that contain progesterone receptors can be reduced in size in a palliative or neoadjuvant setting using the progesterone receptor antagonist aglepristone.

Steroid receptors in canine and human female genital tract tumours with smooth muscle differentiation.

The expression of oestrogen receptor-alpha (ERalpha) and progesterone receptor (PR) was examined in 32 canine genital tract tumours diagnosed as smooth muscle tumours (benign or malignant, pure or mixed). The immunohistochemical expression of calponin was used to assess the smooth muscle differentiation of the tumours. Nineteen human uterine leiomyomas were also examined. Calponin expression was detected in 89.3% of canine and 100% of human genital tract tumours diagnosed as leiomyomas, as well as in the majority of other tumours examined (canine or human, genital or extragenital, benign or malignant) with the exception of canine negative control tumours (cutaneous fibroma and hepatoid gland adenoma). ERalpha was found in 56.3% of canine and 52.6% of human leiomyomas, while PR was found in 84.4% of canine and 94.7% of human tumours. These results indicate that calponin is a good marker for differentiating neoplasia of the canine genital system of uncertain origin, as in human patients. They also show that canine tumours with smooth muscle differentiation of the genital tract of the bitch express steroid hormone receptors, a finding that opens up the possibility of hormone therapy.

Intestinal glandular inclusions (glandular choristoma) in the mesenteric lymph node of a goat.

A glandular choristoma found in the mesenteric lymph node of a goat would appear to represent the first reported case of non-neoplastic glandular inclusions in domestic animals. The origin of this type of lesion may be difficult to determine, but in the present case cytokeratin expression patterns suggested that the inclusions had an intestinal origin.

Gonadotropin-secreting cells in ovariectomized rats treated with different oestrogen receptor ligands: a modulatory role for ERbeta in the gonadotrope?

In the rat, oestrogen is a key regulator of gonadotrophin synthesis and release through activation of oestrogen receptors (ERs). Gonadotropes express alpha and beta isoforms of ER and both can activate transcription in response to oestrogen. These experiments were aimed at evaluating the relative contribution of ERalpha and ERbeta on gonadotrope morphology, progesterone receptor (PR) expression and LH secretion. Ovariectomized rats were daily injected over 3 days with 25 microg oestradiol benzoate, 0.3 or 1.5 mg of the selective ERalpha agonist propylpyrazole triol (PPT) with or without 1.5, 3.0 or 4.5 mg of the selective ERbeta agonist diarylpropionitrile (DPN), DPN alone, and 0.3 or 3 mg of tamoxifen. Controls were given 0.2 ml oil. Serum concentration and pituitary content of LH, gonadotrope PR expression, pituitary PR content, and gonadotrope morphology were analyzed by RIA, immunohistochemistry, Western blotting and light and electron microscopy, respectively. Results showed that PPT reversed all consequences of ovariectomy, DPN mimicked the effects of PPT except for its LH-releasing action and tamoxifen had ERalpha-like responses. When combined with PPT, DPN attenuated ERalpha effects without interfering with its LH-releasing activity. Oestradiol benzoate had similar effects to those of combined PPT and DPN. It is suggested that (i) the structural reorganization of the cytoplasmic organelles provided by oestrogen, and the shrinkage of the ovariectomy-induced hypertrophy of gonadotropes, which precedes the expression of PR, are evoked by ERalpha and modulated, in a ying-yang fashion, by ERbeta; and (ii) the oestrogen-dependent exocytosis of LH, the final step in the secretory process, is dependent on ERalpha exclusively.

A prospective analysis of immunohistochemically determined estrogen receptor alpha and progesterone receptor expression and host and tumor factors as predictors of disease-free period in mammary tumors of the dog.

The immunohistochemically determined estrogen receptor (ER) alpha (ERalpha) and progesterone receptor (PR) status, as well as recognized, well-accepted prognostic indicators and host factors were prospectively analyzed in 84 cases of primary canine mammary carcinoma for their effect on disease-free period (recurrence free, metastasis free, or combined) (DFP) after an observation period of 18 months. The presence of one or both receptors, as well as tumor size, lymph node status, histologic grading, intravascular growth, and necrosis, were of prognostic value for DFP. In multivariate analysis, only tumor size and histologic grading proved to be independent prognosticators. None of the host factors analyzed were of prognostic value for DFP. ERalpha, PR, or both were detected in 173 out of 228 tumors: 70 ERalpha and PR; 5 ERalpha only; 98 PR only. Statistically significant differences regarding the presence of one or both receptors were observed between benign and malignant tumors and between complex, mixed, and simple histologic subtypes of benign and malignant tumors. In the group of malignant tumors (n=155), the presence of one or both receptors was more frequent in tumors smaller than 3 cm, without lymph node metastasis, with tubulopapillary rather than solid patterns of growth among simple carcinomas, of histologic grades I and II, without both intravascular growth and necrosis, and with lymphocyte cell infiltrates. The most frequent groups of hormone receptors-positive tumors were the ERalpha-positive and PR-positive group among benign and the ERalpha-negative and PR-positive group among malignant tumors.

Gonadotrope oestrogen receptor-alpha and -beta and progesterone receptor immunoreactivity after ovariectomy and exposure to oestradiol benzoate, tamoxifen or raloxifene in the rat: correlation with LH secretion.

The selective oestrogen receptor modulator (SERM) tamoxifen (TX) has agonist/antagonist actions on LH secretion in the rat. Whereas in the absence of oestrogens TX elicits progesterone receptor (PR)-dependent GnRH self-priming, it antagonizes oestrogen-stimulatory action on LH secretion. The aim of these experiments was to explore whether TX treatment-induced differential expression of oestrogen receptor (ER)alpha and ERbeta in the gonadotrope may determine its agonist effect on LH secretion. In the first experiment, basal LH secretion, GnRH-stimulated LH secretion and PR-dependent GnRH self-priming were determined in incubated pituitaries from ovariectomized (OVX) rats treated with oestradiol benzoate (EB), TX or raloxifene (RX). Cycling rats in metoestrus or pro-oestrus were used as basic controls. As in pro-oestrus, pituitaries from OVX rats treated with EB exhibited GnRH-stimulated LH secretion, immunohistochemical PR expression and GnRH self-priming. While RX had no effect on these parameters, TX induced PR expression and GnRH self-priming. GnRH self-priming was absent in pituitaries incubated with the antiprogestin ZK299. In the second experiment, we evaluated the immunohistochemical expression of ERalpha and ERbeta in gonadotropes of cycling rats and OVX rats treated with EB, TX or RX. We found that while ERalpha expression was similar in all six groups, ERalpha expression was oestrous cycle dependent. Moreover, ERalpha expression in gonadotropes of TX-treated rats was as high as that found in pro-oestrus, while ERalpha expression in the gonadotropes of RX-treated rats was lower than in metoestrous or pro-oestrous pituitaries. These results suggest that, in the absence of the cognate ligand, TX, unlike RX, may regulate LH secretion through the ERalpha subtype in gonadotropes.

Immunohistochemical expression of progesterone receptors, growth hormone and insulin growth factor-I in feline fibroadenomatous change.

The immunohistochemical expression, tissue-specific and cell-specific distribution patterns of progesterone receptors (PR), growth hormone (GH) and insulin growth factor-I (IGF-I) have been studied in 22 cases of feline fibroadenomatous change (FFAC). PR and GH were detected in all cases and were distributed homogeneously throughout the lesion, while IGF-I was detected in 77% of the cases at the site of ductal budding. The simultaneous expression of PR, GH and IGF-I was detected in epithelial cells in 14 of 22 cases while PR and GH expression only was detected in epithelial cells in 11 cases. Cases that expressed GH and IGF-I without PR expression in the stroma were the most numerous. Double immunohistochemical staining showed the co-localisation of PR and GH in a subset of ductal epithelial cells located between basal/myoepithelial and luminal cells (probably undifferentiated stem cells). These results suggest that ligand-activated progesterone receptors may induce the local synthesis of GH which in turn may exert its proliferative action directly and also indirectly through the production of other growth factors, such as IGF-I, in an autocrine/paracrine manner.