Metallothionein expression in benign and malignant canine mammary gland tumours

The presence of metallothioneins (MTs) were demonstrated immunohistochemically using a monoclonal antibody (E9) against a conserved epitope of I and II isoforms in canine mammary tumours. In a semiquantitative analysis MT expression in the tumour cells was observed in 54/54 cases of benign and 32/40 malignant mammary neoplasms. A statistically significant difference at the level of P<0.01 was observed for MT expression between benign and malign mammary tumours in terms of immunoreactivity score. It is concluded that immunohistochemically demonstrated MT expression is significantly associated with benign canine mammary tumours.     

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.     

The antiproliferative effect of bovine lactoferrin on canine mammary gland tumor cells

Lactoferrin has several biological activities, including antitumor activities in some human and animal tumor cells. Clinical trials have been carried out in human medicine based on these effects. However, the antitumor effects of lactoferrin in veterinary medicine remain unknown. In this in vitro study, we demonstrated that co-incubation of canine mammary gland tumor cells (CIPp and CHMp) and bovine lactoferrin induced growth arrest of tumor cells. This growth arrest was associated with induction of G1 arrest. Furthermore, this effect was stronger in tumor cells than in normal cells. These findings demonstrate that bovine lactoferrin has anti-tumor activity in canine mammary tumors and has the potential for use in tumor-bearing dogs.     

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。     

实时荧光定量RT-PCR检测犬乳腺肿瘤血管内皮生长因子(VEGF)基因方法的建立及初步应用

为了准确地分析血管内皮生长因子（Vascular endothelial growth factor,VEGF）在犬乳腺肿瘤形成过程中的作用,试验设计了针对犬VEGF基因的特异性引物,提取乳腺肿瘤组织RNA并扩增出目的片段,将PCR产物克隆到pGEM-T Easy载体,经质粒PCR扩增、酶切和测序鉴定重组质粒,构建标准曲线等,成功建立了乳腺肿瘤VEGF基因实时荧光定量PCR检测方法。运用实时荧光定量PCR检测了40例不同的犬乳腺肿瘤病例、2例正常乳腺组织。结果发现：在犬恶性乳腺肿瘤组织中,VEGF基因表达量明显高于良性乳腺肿瘤和正常乳腺组织（P〈0.001）;在伴有淋巴结转移的犬乳腺癌组织中,VEGF基因表达量明显高于没有发现转移的乳腺癌组织（P〈0.01）,但VEGF基因的表达量与肿瘤的大小和发病动物年龄无关（P〉0.05）。VEGF基因在犬乳腺癌组织中的表达量远高于其在正常乳腺和良性乳腺肿瘤组织中的表达,且与淋巴结是否发生转移相关。因此,VEGF可作为犬乳腺癌转移、复发的预测指标之一。     

Estrogen receptors in canine mammary gland tumours

The aim of the present study was to establish the relationship between estrogen receptor expression in neoplastic parenchymal cells and the type of canine mammary gland tumour. The research material included mammary gland tumours obtained from 66 dogs. Paraffin sections were stained with hematoxylin-eosin and immunohistochemically with monoclonal antibodies and LSAB/Peroxidase/Universal Kit. The estrogen receptor expression was observed in nuclei of neoplastic cells in about 59% of the cases and in the cytoplasm in about 89% of the cases. In about 20% of the cases the expression in the nuclei and in the cytoplasm was extremely weak. No correlation was found between the expression of estrogen receptors and the value of mitotic indexes in the neoplasms investigated.     

Immunohistolocalization of carbonic anhydrase isozyme (CA-VI) in bovine mammary glands

The localization of bovine carbonic anhydrase isozyme VI (CA-VI) was examined immunohistochemically in bovine mammary glands during early lactation period (after 2-3 days of postpartum) and dry period (at about 2 months preparturition in adults), and young calves (at 30 and 150 days after birth) using specific CA-VI antiserum. The immunoreaction for anti-CA-VI antiserum was very weak in the mammary glands in young (prepubescent) calves. In dry period, CA-VI was also weakly expressed in secretory epithelial (acinar) and ductal cells. In contrast, the reaction was intense in mammary gland cells in early lactation period. Dot blotting analysis indicated that anti-CA-VI reacted positively to beastings and mature saliva, but weakly or not at all to milk during the dry period or calf saliva, respectively. The intense expression of CA-VI in the mammary glands in early lactation period might compensate for low levels of secretion from functionally and structurally immature salivary glands in young calves.     

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.     

Significance of tumor-infiltrating immune cells in spontaneous canine mammary gland tumor: 140 cases

The numbers of tumor infiltrating T lymphocytes, B lymphocytes and antigen presenting cells were evaluated in an immunohistochemical manner in 140 canine spontaneous mammary gland tumor (MGT) tissues. As a result, we found a statistically significant increase in the number of intratumoral T lymphocytes (23.2 ± 23.8) in the malignant MGT group (n=51) compared with the benign MGT group (14.0 ± 16.0, n=89; P<0.05). Moreover, the high T lymphocyte infiltration in the malignant group correlated with poor prognosis in multivariate analysis (P<0.05). This study indicated the relationship between increased infiltrating T lymphocytes and canine MGT malignancy.     

Erythropoietin receptor expression in canine mammary tumor: an immunohistochemical study

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of the erythropoiesis. Recently, it has been demonstrated that EPO and its receptor (EPOR) are expressed in several neoplastic cell lines and solid tumors. Furthermore, in vitro and in vivo studies have shown that EPO could promote human breast carcinoma growth by means of the binding with its receptor, although a clear function for EPO in this setting has not been yet established. While the human medical literature has been accumulating strong evidence on EPO's role in oncogenesis, to date, there are no veterinary reports focusing on such an issue. The aim of the present study was to investigate the immunohistochemical expression of EPOR in canine mammary gland dysplastic and neoplastic lesions. Our results show a weak to moderate EPOR expression in dysplastic glands, being immunoreactivity enhanced as the lesion shows an increasing malignant pattern. On the basis of these findings, this study describes, for the first time, the evidence for EPOR expression in canine mammary gland tumor and suggests a feasible EPO's role for canine mammary tumor progression.     

Secretory carcinoma of the canine mammary gland

Secretory carcinoma is an uncommon variant of breast cancer, characterized by the presence of intracellular and extracellular eosinophilic secretion. Here, we report the cytologic, histologic, and immunohistochemical findings of a secretory carcinoma diagnosed in the left inguinal mammary gland of a 3-year-old female German Shepherd Dog. The fine-needle aspiration cytology showed numerous large branching sheets of neoplastic cells and isolated cells with cytoplasmic vacuoles. Histologically, the tumor was composed of cells with clear cytoplasm and prominent vacuoles that pushed the nuclei to the periphery, resembling signet ring cells. These cells were arranged in solid or tubular structures with lumenal spaces filled with eosinophilic secretion. Immunohistochemical reactions to cytokeratin (CAM 5.2) and alpha-lactalbumin were strongly positive in all neoplastic cells, and staining for vimentin and S100 protein was negative. The cytomorphologic and immunohistochemical features of this tumor are similar to those seen in tumors in women, hence enabling the diagnosis of a rare case of primary secretory carcinoma of the canine mammary gland.     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

Expression and significance of PTEN in canine mammary gland tumours

To explore the expression and clinical importance of the anti-oncogene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in canine mammary gland tumours, PTEN expression was compared in 50 cases of canine mammary tumour and four examples of normal mammary tissue using real-time quantitative PCR. PTEN expression was similar in benign mammary tumours and normal mammary tissues (P>0.05), but was lower in malignant tumours than in normal mammary tissues or benign mammary tumours (P<0.001). PTEN expression was also low in the lymph node metastases of malignant mammary tumours. The expression profile of PTEN in malignant mammary tumours compared to those without lymph node metastasis varied significantly. Low-level PETN expression might play an important role in carcinogenesis and the progression of canine mammary tumours, and PTEN protein detection might be useful in evaluating tumour development and prognosis.     

Oncolytic reovirus synergizes with chemotherapeutic agents to promote cell death in canine mammary gland tumor

The oncolytic effects of reovirus in various cancers have been proven in many clinical trials in human medicine. Oncolytic virotherapy using reovirus for canine cancers is being developed in our laboratory. The objective of this study was to examine the synergistic anti-cancer effects of a combination of reovirus and low doses of various chemotherapeutic agents on mammary gland tumors (MGTs) in dogs. The first part of this study demonstrated the efficacy of reovirus in canine MGTs in vitro and in vivo. Reovirus alone exerted significant cell death by means of caspase-dependent apoptosis in canine MGT cell lines. A single injection of reovirus impeded growth of canine MGT tumors in xenografted mice, but was insufficient to induce complete tumor regression. The second part of this study highlighted the anti-tumor effects of reovirus in combination with low doses of paclitaxel, carboplatin, gemcitabine, or toceranib. Enhanced synergistic activity was observed in the MGT cell line treated concomitantly with reovirus and in all the chemotherapeutic agents except toceranib. In addition, combining reovirus with paclitaxel or gemcitabine at half dosage of half maximal inhibitory concentration (IC₅₀) enhanced cytotoxicity by activating caspase 3. Our data suggest that the combination of reovirus and low dose chemotherapeutic agents provides an attractive option in canine cancer therapy.