Cyclooxygenase-2 expression in normal and neoplastic canine mammary cell lines

Mammary cancer is the most common cancer in female dogs. Induction of cyclooxygenase-2 (COX-2), a key enzyme in prostaglandins (PGs) biosynthesis, has been demonstrated in various cancers in humans and dogs, including mammary cancer. The objective of this study was to investigate the expression and regulation of COX-2 in canine mammary epithelial cells. Cell lines derived from normal and neoplastic canine mammary glands were cultured in the absence or presence of phorbol 12-myristate 13-acetate (PMA), and immunoblots, immunocytochemistry, radioimmunoassays, and a cell proliferation assay were used to study COX-2 expression and PGs production. Results showed that the neoplastic cell line CMT12 constitutively overexpressed COX-2 protein whereas other mammary cell lines expressed low to undetectable basal levels of COX-2 protein. Basal PGE(2) production was significantly higher (P < .05) in CMT12 compared to other cell lines. Levels of COX-2 protein in CMT12 decreased in a time-dependent manner with serum starvation, and PMA stimulation induced a strong time-dependent increase in COX-2 protein. Treatment of CMT12 cells with NS-398 (a specific COX-2 inhibitor) significantly blocked PGE(2) synthesis and reduced cell proliferation (P < .05). These results indicate that some neoplastic canine mammary cell lines constitutively overexpress COX-2, and that COX-2 inhibition decreases PGE(2) production and cell proliferation, supporting a role for COX-2 and PGs in canine mammary oncogenesis.     

Cyclooxygenase-2 expression is associated with histologic tumor type in canine mammary carcinoma

Cyclooxygenase-2 (COX-2) is an inducible member of the family of cyclooxygenase enzymes that has been implicated in the genesis of numerous cancers. The role of COX-2 in canine mammary neoplasia remains to be more clearly elucidated. The goal of the study reported here was to determine whether a direct association between levels of COX-2 expression and tumor histologic subtype exists in canine mammary carcinoma. Immunohistochemical analysis was performed using a polyclonal antiprostaglandin G/H synthase 2 IgG COX-2 antibody. Sections from the kidneys of young dogs, which stain positive for COX-2 in the macula densa, served as positive controls. Slides were reviewed by a single pathologist, and were evaluated for COX-2 expression according to previously established scales. Positive-staining tumors were given a COX-2 staining distribution (on the basis of the percentage of positive staining cells in five 400x fields) and intensity score according to previously established scales. The product of the COX-2 staining distribution and intensity scores was calculated to create a COX-2 staining index. COX-2 expression was detected in 28 of 50 (56%) samples evaluated. Anaplastic carcinomas had a significantly higher COX-2 staining distribution, intensity, and index, compared with those for adenocarcinomas (P < 0.0001). The overall percentage of positive tumors (56%) was consistent with that of prior studies. To the authors' knowledge, these results indicate, for the first time, a direct association between COX-2 expression and tumor histologic subtype in canine mammary carcinomas. Future research directed at measuring tumor response in canine mammary carcinoma patients treated with a selective COX-2 inhibitor is indicated.     

Cyclooxygenase-2 expression in canine appendicular osteosarcomas

Osteosarcoma is the most common primary bone tumor in dogs and it has a high mortality rate from distant metastatic disease. Targeted adjuvant therapies are needed to prolong currently achievable survival times. The role of cyclooxygenase-2 (COX-2) in carcinogenesis has been attributed to the production of prostaglandins and involvement in apoptosis, immune surveillance, and angiogenesis. COX-2 is up-regulated in a number of different human and animal epithelial tumors, but data about its function in mesenchymal tumors is lacking. The purpose of this study was to evaluate COX-2 expression in canine appendicular osteosarcomas and to identify if a relationship exists between the intensity of COX-2 expression and clinicopathologic outcome. Of 44 osteosarcomas analyzed, 34 (77.3%) were positive for COX-2 expression. Most of the positive cases (88%) had poor to moderate COX-2 staining. Dogs that had strong COX-2 expression had significantly decreased overall survival time (P = .0107). The median survival times for dogs with negative (n = 10), poor (n = 19), moderate (n = 11), and strong (n = 4) expression were 423, 399, 370, and 86 days, respectively. Additional studies are warranted to further evaluate COX-2 in osteosarcoma for its prognostic value and as a target for adjuvant therapy.     

Cyclooxygenase-2 expression in equine tumors

The enzyme cyclooxygenase-2 (COX-2) is expressed in some tumor and stromal tissues, and catalyzes production of prostaglandins with growth stimulatory, antiapoptotic, proangiogenic, and immunosuppressive properties. Pharmacologic inhibition of COX-2 is associated with antitumor activity in various human and canine malignancies. The purpose of this study was to assess COX-2 expression in a series of equine sarcoids, melanomas, and squamous-cell carcinomas (SCC). COX-2 expression was assessed in formalin-fixed paraffin-embedded tissues from 14 sarcoids, 11 melanomas, and 37 SCC that represent various anatomic sites by using standard immunohistochemical methods. COX-2 was expressed in 2 of 14 sarcoids, 7 of 11 melanomas, and 32 of 37 SCC, 56% of which demonstrated moderate-to-strong immunoreactivity. There were no differences in expression between anatomic sites. In conclusion, most equine SCC and many melanomas appear to express COX-2 and thus could respond to COX-2 inhibitor therapy.     

Alpha basic crystallin expression in canine mammary tumors

The aim of this study was to evaluate prognostic and/or diagnostic factors of canine mammary tumors by immunohistochemically analyzing the expression of alpha basic crystallin (αB-c). For this, formalin-fixed, paraffin-embedded blocks of 51 naturally-occurring canine mammary tumors (11 benign and 40 malignant) were used. Tissue from eight normal canine mammary glands were served as a control. Immunohistochemically, in the control mammary tissues, a few luminal epithelial cells were αB-c positive but myoepithelial cells were negative. In benign or simple type malignant tumors, αB-c expression was observed in luminal epithelial cells while the myoepithelial basal cells were negative. In benign or complex type malign tumors, positive staining was predominantly found in the cytoplasm of epithelial cells. Immunoreactivity of αB-c was also observed in neoplastic myoepithelial cells. Statistically, the number of cells immunolabeled with αB-c was found to be significantly different among tissues from normal canine mammary glands, benign lesions, and malignant tumors (p < 0.05). αB-c immunoreactivity was higher in malignant tumors than the control mammary tissues (p < 0.001). Data obtained in the current study revealed a strong association between high expression levels of αB-c and primary mammary gland tumors in canines.     

Amyloid in canine mammary tumors

In canine mammary carcinomas, amyloid was present as amyloid-containing corpora amylacea and as local deposits between neoplastic epithelial cells or in stromal tissue. Histochemical staining methods revealed that this amyloid was not of the AA-type amyloid and contained tryptophan. The possible pathogenesis of this amyloid deposition is discussed.     

Cyclooxygenase-2 (COX-2) expression in canine intracranial meningiomas

Meningiomas are the most common canine intracranial tumour. Neurologic disability and death from treatment failure remain problematic despite current surgical and radiotheraputic treatments for canine intracranial meningiomas. Cyclooxygenase-2 (COX-2) over-expression has been demonstrated in multiple canine malignancies, and COX-2 inhibitory treatment strategies have been shown to have both preventative and therapeutic effects in spontaneous and experimental models of cancer. The purpose of this study was to evaluate COX-2 expression in canine intracranial meningiomas. Immunohistochemical and Western blot (WB) analyses showed COX-2 expression in multiple tissues of the normal canine brain, and 87% (21/24) of intracranial meningiomas studied were immunoreactive to COX-2. No significant associations between COX-2 immunoreactivity and tumour grade were identified. Further studies are required to elucidate the physiologic roles of constitutive COX-2 expression in the central nervous system as well as its participation in meningioma tumourigenesis.     

犬乳腺肿瘤ER和C-erbB-2蛋白表达

犬乳腺肿瘤是犬常见的肿瘤之一,仅次于皮肤肿瘤,恶性肿瘤约为50%[1].尽管本病的病因尚未完全清楚,但类固醇、生长因子及其受体为阐明病因提供了重要的依据[2].     

Localized amyloidosis in canine mammary tumors

Histopathologic and immunohistochemical examinations were performed on localized amyloidosis associated with mammary tumors in two dogs. These tumors were identified as adenoma and adenocarcinoma. An acellular, amorphous pale eosinophilic material (amyloid) was observed in the lumina of acini lined by neoplastic cells and in the stroma of the tumors. Concentrically laminated pale eosinophilic bodies (corpora amylacea) were also found in the lumina of the acini. Amyloid and corpora amylacea stained positively with Congo red with and without 5% potassium permanganate pretreatment and revealed a green birefringence under polarized light. Corpora amylacea showed an occasional Maltese-cross pattern. Immunohistochemically, amyloid and corpora amylacea usually stained positively with anti-bovine alpha-casein antibody but negatively with anti-human amyloid AA, anti-bovine kappa-light and lambda-light chains, anti-human lactoferrin, anti-human transferrin, anti-human secretory component, and anti-human polyglucosan antibodies. These findings suggested that the amyloid deposition in these canine mammary tumors was related to lactating casein.     

The expression of intermediate filaments in canine mammary glands and their tumors

Monoclonal antibodies specific for different types of intermediate filaments (cytokeratin, vimentin, desmin and neurofilaments) were used to study the histogenesis of canine mammary glands and 57 canine mammary tumors by immunocytochemistry. The intra- and interlobular duct epithelium, acinar, and intralobular myoepithelial cells stained positively for cytokeratin. Peripheral ductal and acinar cells, as well as interstitial cells, stained positively for vimentin. A similar staining pattern was seen in adenomas, complex adenomas, benign mixed tumors, ductular carcinomas, and one myoepithelioma-like tumor. Additionally, cytokeratin positive cells were scattered interstitially in one single adenoma, most complex adenomas, some benign mixed tumors, complex carcinomas, and in the malignant mixed tumors. All stromal cells stained positively for vimentin. The fibrosarcomas were positive only for vimentin, while the following expressed both desmin and cytokeratin: epithelial-like cells in one adenoma, three complex adenomas, the myoepithelioma-like tumor, the single comedo carcinoma, two complex carcinomas, the single lobular carcinoma, one malignant mixed tumor, and three osteosarcomas. Epithelial-like cells in one adenoma, six complex adenomas, two benign mixed tumors, two complex carcinomas, the lobular carcinoma, and the malignant schwannoma stained for neurofilaments. Three tumors, one adenoma, one complex adenoma, and the lobular carcinoma expressed both desmin and neurofilaments in addition to cytokeratin and vimentin. The results show the expression of different types of intermediate filaments and indicate that there might be a stem cell origin in most of the canine mammary tumors.     

Loss of p27 expression in canine mammary tumors and their metastases

The p27 gene is a member of the cyclin-dependent kinase inhibitors, which arrest G1- to S-phase transition of the cell cycle. We have previously shown a significant reduction of p27 mRNA expression level in laser-microdissected mammary carcinomas and their lymph node metastases when compared to non-neoplastic mammary gland of the same dog. Here, p27 expression was analyzed on the protein level in non-neoplastic mammary gland, primary mammary carcinomas, their lymph node metastases and intravascular tumor cells of 49 dogs, adenomas of 49 dogs and non-neoplastic mammary gland of 98 dogs by immunohistochemistry. A significantly (p ? 0.05) decreased percentage of p27 positive tissue samples was found when normal gland was compared with adenomas, carcinomas and lymph node metastases. Specifically, 91% of normal gland epithelium displayed nuclear p27 expression. In contrast, only 22% of the adenomas, 20% of carcinomas, 12% of lymph node metastases and 32% of intravascular tumor cells had p27 reactivity. Cell cycle control by p27 is therefore lost in the majority of canine mammary tumors. The lack of significant differences between benign and malignant mammary tumors indicates that decreased p27 expression is an early step in carcinogenesis of canine mammary tumors and hinders the use of p27 as a marker of malignancy for this tumor type.     

E-cadherin immunoreactivity in canine mammary tumors.

The reduction or loss of E-cadherin (E-cad), a calcium-dependent epithelial cell adhesion molecule, has been associated with tumor dedifferentiation and invasiveness. The immunohistochemical pattern of E-cad expression was evaluated in formalin-fixed and paraffin-embedded sections of 6 normal mammary glands, 3 dysplasias, 12 benign tumors (8 benign mixed tumors, 4 adenomas), and 60 malignant tumors (12 stage 0, 29 stage I, 19 stage II) of the canine mammary gland. E-cadherin expression was classified as membranous, when on cell-cell boundaries, or as cytoplasmic, when in the form of a diffuse cytoplasmic staining. In addition, the percentage of E-cad-positive epithelial neoplastic cells was graded by a semiquantitative method, categorizing cases into a reduced (or -) type group, when showing less than 25% positivity, a reduced (or +/-) type group, when showing 25-75% positivity, and a preserved (or +) type group, when more than 75% positive cells were present. In the normal mammary gland, E-cad expression was evident in epithelial luminal cells. A stronger positivity was revealed in ductular than in alveolar luminal cells. The myoepithelial cells showed inconsistent, weak cytoplasmic positivity in the normal gland as well as in mammary tumors. In normal glands and benign and malignant noninvasive tumors, E-cad expression was mainly membranous and preserved in most of the epithelial cells. In stage I tumors, both membranous (38%) and cytoplasmic (62%) positivity were well represented, as well as preserved type (55%) and reduced type (45%) tumors. All stage II malignant tumors showed the highest frequency of cytoplasmic positivity (79%) and reduced type (62%) tumors.     

Microsatellite instability in canine mammary gland tumors

BACKGROUND: Genomic instability is a hallmark of cancer and may be required for the accumulation of cancer-causing mutations within cells. One form of genomic instability occurs in tandem nucleotide repeats and is known as microsatellite instability (MSI). HYPOTHESIS: We hypothesized that MSI can be observed in canine mammary gland tumors (MGT) and represents a potential carcinogenic mechanism in dogs. ANIMALS: Thirty-five dogs with MGTs and 9 dogs with other tumors were recruited from the University of Minnesota Veterinary Medical Center and referring veterinary clinics. METHODS: A panel of 21 canine microsatellite (MS) markers was amplified by polymerase chain reaction (PCR) from deoxyribonucleic acid obtained from blood and from fresh or formalin-fixed, paraffin-embedded tumor tissues. PCR products were evaluated by using capillary electrophoresis, and the chromatograms were analyzed by using genotyping software. MS genotypes obtained from fresh and formalin-fixed tumor tissues were compared, as were MS genotypes from normal tissue and tumor tissue. RESULTS: Genotypes obtained from formalin-fixed and fresh tissues were identical for all MS in 9 tumors evaluated, suggesting excellent concordance between the 2 sample types. For the 35 canine mammary tumors evaluated, 13 (37%) had stable genotypes; 22 (63%) exhibited aberrations in 1 or 2 MS; and 4 tumors (11%) demonstrated high-level instability, with aberrations in 29 to 61% of MS. CONCLUSIONS AND CLINICAL IMPORTANCE: Although some low-level MSI often is observed, high-level MSI is an infrequent finding in canine mammary tumors. Further evaluations are required to better characterize this phenomenon and to determine its relevance to canine carcinogenesis.     

Obesity,expression of adipocytokines,and macrophage infiltration in canine mammary tumors

Obesity influences the development, progression and prognosis of human breast cancer and canine mammary cancer (MC) but the precise underlying mechanism is not well-documented in the fields of either human or veterinary oncology. In the present study, the expression of major adipocytokines, including leptin, adiponectin, and leptin receptor (ObR) in benign (n = 28) and malignant (n = 70) canine mammary tumors was investigated by immunohistochemistry and on the basis of the subject's body condition score (BCS). To evaluate the relationship between obesity and chronic inflammation of the mammary gland, macrophages infiltrating within and around tumoral areas were counted.The mean age of MC development was lower in overweight or obese dogs (9.0 ± 1.8 years) than in lean dogs or optimal bodyweight (10.2 ± 2.9 years), and the evidence of lymphatic invasion of carcinoma cells was found more frequently in overweight or obese group than in lean or optimal groups. Decreased adiponectin expression and increased macrophage numbers in overweight or obese subjects were significantly correlated with factors related to a poor prognosis, such as high histological grade and lymphatic invasion. Leptin expression was correlated with progesterone receptor status, and ObR expression was correlated with estrogen receptor status of MCs, regardless of BCS. Macrophage infiltration within and around the tumor may play an important role in tumor progression and metastasis in obese female dogs and may represent a prognostic factor for canine MCs.     

The expression of sialyl Lewis X in canine and feline mammary gland tumors

The expression of sialyl Lewis X (sLe(x)) in 93 canine and 15 feline mammary gland tumors (MGT) obtained by surgical resection at Veterinary Medical Center, the University of Tokyo was examined by immunohistochemistry. Their clinicopathological features and prognosis were also reviewed. Approximately 60% of MGT tissues showed sLe(x) positive expressions, while all normal mammary gland tissues were negative. However, its expression was not correlated with clinicopathological features and prognosis significantly. This study suggests that sLe(x) may be a tumor-associated antigen in canine and feline MGTs.     

犬乳腺肿瘤组织病理学观察及腱糖蛋白C表达的检测

为了探讨腱糖蛋白C(TNC)表达与犬乳腺肿瘤生物学关系,对23例犬乳腺肿瘤临床病例进行病理学观察及分类,然后采用免疫组化SABC方法检测TNC蛋白在23例犬乳腺肿瘤和2例犬正常乳腺组织中的表达情况。结果显示:23例病例中,良性肿瘤有7例,占30.43%;恶性肿瘤有13例,占56.52%;增生病变有3例,占13.05%。在良性肿瘤中良性混合瘤最多,为3例,其余2例为导管内乳头状腺瘤,1例复杂腺瘤及1例纤维腺瘤。恶性肿瘤中,5例未分化癌,3例癌肉瘤,2例筛状癌,2例固体癌,1例纤维肉瘤,1例混合瘤引起的癌。TNC蛋白主要表达于细胞外基质。犬恶性乳腺肿瘤中,TNC蛋白阳性表达率为46.15%(6/13);良性乳腺肿瘤中,其阳性表达率为42.85%(3/7);乳腺增生病变和正常乳腺组织中,未见其表达。研究结果表明,TNC蛋白可以为犬乳腺肿瘤的鉴别诊断提供参考。     

Determination of estrogen receptors in canine mammary tumors

Dextran-coated charcoal assay with Scatchard plotting was used for the quantitative determination of high-affinity estradiol receptors (ER) in 10 canine mammary tumor cytosols. Mammary tumors diagnosed histopathologically as adenocarcinomas appeared to have a higher incidence of ER than those diagnosed as mixed mammary tumors. These receptors have a high affinity for estradiol with dissociation constants as determined by Scatchard analysis ranging from 1.2 to 2.0 X 10(-11) M. These dissociation constants are similar to those previously reported for the receptor-estradio interaction from target tissues in other species. The specific binding of the receptor for the hormone was diminished in the presence of an antiestrogenic compound. Our data suggest that canine mammary tumors with significant ER levels would respond favorably to antiestrogen therapy. With the demonstration of ER sites in canine mammary adenocarcinomas as presented herein, the potential for chemotherapeutic management appears to be encouraging.     

Classification and grading of canine mammary tumors

Mammary neoplasms are the most common neoplasm in female dogs. Two histologic classification systems for canine mammary tumors and dysplasias have been published: the first in 1974 and a modification in 1999. This article provides a brief overview of the two histologic classification systems. Since the publication of the second system, several new histologic subtypes of canine mammary neoplasms have been described. These have been incorporated into the proposed new classification system. This article also compares the grading systems for canine mammary carcinomas and their use for prognosis, along with the histologic classification.