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.     

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.     

Relationship between receptors for insulin-like growth factor- I, steroid hormones and apoptosis-associated proteins in canine mammary tumors

In the veterinary literature there are few data concerning the expression of insulin-like growth factor type I (IGF-IR) in the canine mammary gland tumors. The aim of the present study was the evaluation of IGF-IR expression and its correlation to the expression of estrogen receptor alpha (ERalpha) and progesterone receptor (PR), proteins: Bcl-2, Bax, p53 in canine mammary gland tumors, and also a correlation with other features: bitch's age, tumor diameter, histologic type of tumor, degree of histologic malignancy, proliferate activity. The study was done on 112 epithelial neoplasms: 21 (19%) were adenoma, 38 (34%) complex carcinoma (adenocarcinoma), 47 (42%) simple carcinoma (adenocarcinoma) and 6 (5%) solid carcinoma. Histochemistry and immunohistochemistry methods were employed. It was shown that more common and/or higher IGF-IR expression in cells of canine mammary gland tumors was related to the histologic type of cancer of worse prognostic (solid and simple carcinoma), high histologic degree of malignancy (III degrees) but the statistical analysis did not reveal any significant differences. We observed the high degree of IGF-IR expression in tumors which displayed the high ERalpha and PR expression. These results suggest the involvement of IGF-IR in the development of hormonosensitive canine mammary tumors. Additionally, the significant positive correlation between expression of IGF-IR and p53, Bax was found. Our study provides some evidence that interactions exist between the IGF-IR and these apoptosis-associated proteins may contribute to the development and progression of canine mammary gland tumors. These results require further investigations.     

Immunolocalization of the smooth muscle-specific protein calponin in complex and mixed tumors of the mammary gland of the dog: assessment of the morphogenetic role of the myoepithelium

The immunohistochemical expression of the smooth muscle-specific protein calponin was studied to assess the contribution of myoepithelial cells to the histogenesis of spindle cells of complex and mixed tumors of the mammary gland of the dog and the origin of cartilage and bone in mixed tumors. Formalin-fixed tissues from 55 benign and malignant tumors (49 also containing surrounding normal mammary gland) were evaluated. Periacinar and periductal myoepithelial cells of all the 49 normal mammary glands were diffusely stained by the anti-human calponin monoclonal antibody. Calponin was found in 53 (98%) of the tumors studied, reacting with the myoepithelium-like cells of 86% of benign tumors and their remnants in 85% of malignant tumors. Five different types of calponin-immunoreactive myoepithelial cells were identified: hypertrophic myoepithelial cells. fusiform cells, stellate myoepithelial cells, rounded (myoepithelial) cells, and chondroblasts. Differences in staining intensity and staining pattern among these five types of cells suggested a transition of myoepithelial cells to chondroblasts. Stromal myofibroblasts also showed calponin immunoreactivity, but they did not react with a cytokeratin 14 monoclonal antibody, which recognizes myoepithelial cells in mammary gland. Calponin appears to be a very sensitive marker of normal and neoplastic myoepithelium in the canine mammary gland, and its identification in different cell types of complex and mixed tumors of the mammary gland of the dog suggests a major histogenetic role for myoepithelial cells.     

Immunohistochemical analysis of c-yes and c-erbB-2 oncogene products and p53 tumor suppressor protein in canine mammary tumors

In order to evaluate the involvement of c-yes and c-erbB-2 oncogene products, and p53 tumor suppressor protein in canine mammary neoplastic lesions, sections of archived paraffin-embedded samples of 79 mammary tumors were analyzed immunohistochemically using antibodies against human c-yes p62 and c-erbB-2 products and p53. These 79 tumors were divided into 2 groups: 32 benign (2 adenosis, 7 simple adenomas, 14 complex adenomas, and 9 benign mixed mammary tumors) and 47 malignant tumors (26 simple adenocarcinomas, 7 complex adenocarcinomas, 5 solid carcinomas, 2 sclerosing carcinomas, 6 malignant mixed mammary tumors, and 1 malignant myoepithelioma). As a result of immunostaining, 40.6% (13/32) of the benign tumors and 21.3% (10/47) of the malignant tumors expressed the c-Yes oncogene product, ErbB-2 expression was detected in 50% (16/32) of the benign tumors and in 19.1% (9/47) of the malignant tumors. P53 expression was detected in 16% (4/25) of the benign tumors and in 30.6% (11/36) of the malignant tumors. Co-expression of c-Yes and ErbB-2, ErbB-2 and p53, and all 3 products was detected in 6, 1 and 7 tumors, respectively.     

PTEN 基因在犬乳腺肿瘤组织中的表达及临床意义

有关酪氨酸磷酸酶基因（Phosphatase and tensin homolog deleted on chromosometen,PTEN）在乳腺肿瘤中的检测在人医早有报道。为了研究PTEN基因在犬乳腺肿瘤组织中的表达情况,笔者运用实时荧光PCR定量检测了38例不同的犬乳腺肿瘤组织（包括15例良性乳腺肿瘤和23例恶性乳腺肿瘤）、4例正常犬乳腺组织。结果发现：PTEN基因在犬恶性乳腺肿瘤组织中表达量明显低于其在良性乳腺肿瘤和正常乳腺组织中的表达量,两者差异极显著（P〈0.001）;PTEN在良性乳腺肿瘤组织中的表达与正常犬乳腺组织相比,差异不显著（P〉0.05）;发生了淋巴结转移的乳腺癌PTEN基因的表达量与未发生转移的乳腺癌组织的表达量间差异亦不显著（P〉0.05）,且PTEN的表达量与肿瘤组织的大小和发病动物年龄无关。结论：PTEN蛋白表达异常可能与乳腺肿瘤发生、发展相关,可考虑作为判断犬乳腺肿瘤生物学行为和预测的指标。     

Cyclooxygenase-2 expression in canine mammary tumors

Mammary tumors are the most common neoplasms in female dogs. Induction of cyclooxygenase-2 (COX-2) has been implicated in various cancers in humans. However, expression of COX-2 has not been investigated in canine mammary tumors. Normal mammary gland (n = 4), simple or complex adenomas (n = 63), and simple or complex adenocarcinomas (n = 84) were studied by immunohistochemistry. Results showed that COX-2 was not expressed in the normal gland but was detected in 24% of adenomas and in 56% of adenocarcinomas (P < 0.001). The incidence of COX-2 expression and the intensity of the COX-2 signal were higher in adenocarcinomas than in adenomas (P < 0.001). These results demonstrate for the first time that COX-2 is induced in a proportion of canine mammary tumors and that COX-2 expression is more frequent and more intense in malignant than in benign tumors, suggesting a potential role for COX-2 in canine mammary tumorigenesis.     

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.     

Cytologic differentiation of benign from malignant canine mammary tumors

Cytologic and histologic examination of 91 canine mammary masses was performed by two cytologists and two histopathologists. Ten important cytologic criteria of malignancy for canine mammary tumors were identified. A cytologic grading system for differentiation of benign from malignant mammary tumors was proposed using these criteria. With this system, approximately one fourth of the malignant mammary tumors were given a concordant cytologic diagnosis. Approximately one-half of the benign masses were given a concordant cytologic diagnosis by the two cytologists. One-half of all the tumors examined were given inconclusive cytologic diagnoses by both cytologists. The cytologic identification of spindle cells did not differentiate complex and mixed mammary tumors from simple tumors. Only five of the animals studied died of mammary cancer, precluding a critical analysis of the cytologic criteria for prediction of cancer mortality.     

Expression of bone morphogenetic protein-6 (BMP-6) in myoepithelial cells in canine mammary gland tumors.

Seventy-three mammary tumors and three mammary tissue specimens were examined to elucidate the expression of bone morphogenetic protein (BMP)-6 in the myoepithelial cells of canine mammary gland tumors. Morphologically, the myoepithelial cells were classified into four types: resting and proliferating cells inside the basement membrane, and spindle- and star-shaped cells proliferating in the outer area of the basement membrane. The characteristics of these myoepithelial cells were confirmed by immunohistochemistry using antibodies raised against keratin, cytokeratin 19, alpha-smooth muscle actin, and vimentin. In simple adenoma, a small number of resting myoepithelial cells was immunopositive for BMP-6. In complex adenomas and benign mixed tumors, all types of myoepithelial cells, depending in some cases on their specific location within the tumor, were immunopositive for BMP-6, but almost all of the tubular epithelial cells were immunonegative. Foci consisting of a proliferation of BMP-6-positive star- and spindle-shaped cells had mucinous stroma with marked hyaline and chondroid changes. In contrast, the foci with BMP-6-negative spindle- and star-shaped cells tended to have mucinous stroma without chondroid change. Several types of mesenchymal cells including chondrocytes, osteoblasts, and fibroblastlike cells in the mixed tumors, showed an intense immunopositive reaction for the BMP-6 antibody, and were located close to the ectopic cartilage and bone matrix. No significant immunoreactivity for BMP-6 was observed in most of the malignant mammary tumors; only one malignant mixed tumor was examined. All of these findings indicate that BMP-6 expression in myoepithelial cells may increase in complex adenomas and benign mixed tumors in canine mammary glands, and that BMP-6 expression is most intense in the vicinity of chondroid matrix in these tumors.     

Expression of bone morphogenetic protein-6 and bone morphogenetic protein receptors in myoepithelial cells of canine mammary gland tumors

To study the ectopic chondrogenesis in canine mammary mixed tumors, the expression of bone morphogenetic protein-6 (BMP-6) and specific BMP receptors (BMPRs), BMPR-IA, BMPR-IB, and BMPR-II, was examined using immunohistochemical and immunoblot analysis in 39 canine mammary gland tumors. Immunohistochemically, BMP-6 and all three types of BMPRs were coexpressed in the myoepithelial cells and chondrocytes in six of eight benign mixed tumors. In complex adenomas, myoepithelial cells showed an expression pattern of BMP-6, BMPR-IA, and BMPR-II similar to those in benign mixed tumors, whereas immunoreactivity for BMPR-IB was very mild. The myoepithelial cells proliferating within the basement membrane showed more intense immunoreactivity for BMP-6 and all BMPRs as compared with those proliferating in the interstitial areas. Western blotting analysis revealed immunopositive bands at 40-45 kDa for BMP-6 in the samples from simple and complex adenomas and benign mixed tumors. The BMPR-IB-specific bands at 45 kDa were most detected in benign mixed tumors. Because among BMPRs, BMPR-IB is thought to be the major receptor for BMP-6 for primary chondrogenesis, these findings suggest that the expression of BMP and its receptors on the myoepithelial cells might play a role in the ectopic cartilage formation in canine mammary gland tumors, especially in benign mixed tumors.     

The expression of p63 and cytokeratin 5 in mixed tumors of the canine mammary gland provides new insights into the histogenesis of these neoplasms

Cytokeratin 5 and p63 have been described as basal and myoepithelial cell markers in human breast. Mixed tumors of the canine mammary gland have been associated with a myoepithelial origin. Cytokeratin 5 expression has not been evaluated in these tumors. We investigated the relation between cytokeratin 5 and p63 double-immunohistochemical expression in 23 mixed tumors of the canine mammary gland (10 benign mixed tumors and 13 carcinomas arising from benign mixed tumors) and their origin. Cytokeratin 5 and p63 co-expression was observed in myoepithelial cells of benign mixed tumors, as well as in squamous differentiation of carcinoma arising from benign mixed tumors. Though a few interstitial spindle cells of the mesenchymal components expressed both p63 and cytokeratin 5, the basal epithelial cells were labeled only by cytokeratin 5. The co-expression of p63 and cytokeratin 5 in myoepithelial cells and squamous differentiation suggest that, like in human breast, cytokeratin 5 can also be considered a myoepithelial- and squamous-cell differentiating marker in canine tumors. The presence of some interstitial spindle cells stained for p63 and cytokeratin 5 might be associated with a myoepithelial origin of the mesenchymal component of mixed tumors of the canine mammary gland. Moreover, contrary to p63, basal epithelial cells were labeled by cytokeratin 5, indicating that cytokeratin 5 may not represent an exclusive myoepithelial cell marker but also a basal epithelial cell marker in canine mixed tumors. According to these data, basal epithelial cells may be related to the origin of the epithelial component of mixed tumors of the canine mammary gland.     

Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERalpha and p53

TopBP1 is aberrantly expressed in human and feline mammary carcinomas, but expression of this BRCA1-related protein has not been investigated in canine mammary carcinomas. In this study, 132 canine mammary tumours (46 benign, 86 carcinomas) were examined immunohistochemically for expression of TopBP1, oestrogen receptor α (ERα), Ki67 and p53. Positive staining for TopBP1 was evident in all canine mammary lesions, although five samples had <20% positive cells. The number of samples with high levels of staining increased in different categories from benign mixed tumour to adenoma to carcinoma. Most TopBP1 staining was nuclear, but both nuclear and cytoplasmic staining were observed as the degree of malignancy increased, similar to human and feline mammary carcinomas. Benign mixed tumours, however, had more cytoplasmic staining than adenomas. Expression of p53 and the proliferation marker Ki67 increased from benign mixed tumour to adenoma to carcinoma, but the differences between benign and malignant tumours were more distinct than for TopBP1 expression. ERα expression decreased from malignant to benign tumours, although over half of the benign mixed tumours were negative. TopBP1 was expressed in canine mammary tumours at higher levels than has been reported previously for cats, although the shift in cellular localisation with malignancy was similar.     

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.     

Cellular proliferative and telomerase activity in canine mammary gland tumors

In canine mammary tumors, we examined the telomerase activity, proliferative activity by proliferative cell nuclear antigen (PCNA) immunohistochemistry, and percentage of apoptotic cells by the deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The relationship between these measures and histopathologic malignancy was also investigated. PCNA index was highest in malignant tumors (adenocarcinoma: 27.0%; malignant mixed tumor: 15.7%), followed by benign tumors (adenoma: 4.4%; benign mixed tumor: 5.3%), hyperplasia (2.1%), and normal mammary gland (0.9%). In adenoma and adenocarcinoma, papillary and solid types showing higher cellularity tended to have higher PCNA indices than did cystic and tubular types. Although the TUNEL index was <1% in all cases, the relationship between this measure and histopathologic diagnosis showed the same tendency as observed in PCNA immunostaining. Telomerase activity was detectable in all adenomas, benign mixed tumors, and adenocarcinomas examined. In contrast, all normal mammary glands, hyperplasias, and malignant mixed tumors were negative for telomerase. Relative telomerase activity (RTA) of adenocarcinoma (56.5) was significantly higher than that of adenoma (27.8) and benign mixed tumor (33.9), and a significant positive correlation (P < 0.001) was noted between RTA and PCNA index. No significant correlations were noted between either PCNA or TUNEL index and clinical features such as metastasis and tumor diameter. PCNA index and telomerase activity may be useful markers for judging malignancy of canine mammary tumors.     

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.     

p63: a novel myoepithelial cell marker in canine mammary tissues

Several immunohistochemical markers have been used to demonstrate the presence of myoepithelial cells in order to determine their role in the histogenesis of mammary tumors. p63, a recently characterized p53 homologue, is consistently expressed in myoepithelial cells of the human breast; however, no assessment of its immunoreactivity has been reported so far in canine mammary tissues. We investigated p63 immunohistochemical expression, as a novel myoepithelial cell nuclear marker, in 81 samples of normal (n = 2), hyperplastic (n = 11), and neoplastic (n = 68) canine mammary tissues. Myoepithelial phenotype was confirmed by using complementary monoclonal antibodies: alpha-smooth muscle actin, cytokeratin 14, cytokeratin AE1/AE3, and vimentin. p63 expression was observed in 91.4% (74/81) of the samples evaluated. Normal mammary glands, mammary hyperplasias, and benign tumors showed 100% immunoreactivity, with p63 expression restricted to myoepithelial cell nuclei. In general, benign mixed tumors showed a basal cell compartment immunoreactive to p63, with a gradual decrease of its expression during myoepithelial transformation. p63 expression was found in 72% of malignant tumors, allowing myoepithelial or basal cell identification in spindle-cell carcinomas (2/2), tubulopapillary carcinomas (8/9), solid carcinomas (7/10), and carcinosarcomas (1/3). The osteosarcoma analyzed was p63 negative. In our series, stromal components were consistently nonreactive to p63. In conclusion, the present study reveals p63 as a sensitive and highly specific marker of myoepithelial cells in canine mammary tissues, and the authors suggest p63 as an additional marker for defining myoepithelial histogenesis.     

The activity of matrix metalloproteinases (MMPS) and tissue inhibitors of metalloproteinases (TIMPs) in mammary tumors of dogs and rats

We conducted zymography for detecting the activity of matrix metalloproteinases (MMPs) and reverse zymography for the activity of tissue inhibitors of metalloproteinases (TIMPs) in canine spontaneous and rat 7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor tissues. The activities of MMPs of canine mammary tumors were quite higher than those of the rat chemically induced tumors. The activities of MMPs were significantly higher in malignant tissues than in benign ones of canine tumors, whereas the activity of only MMP-2 was higher in both benign and malignant rat tumors compared to normal tissues. There were no differences of MMPs activities between benign and malignant rat tumors. The results of reverse zymography indicated that the activities of TIMP-1, -2 and -3 were strikingly higher in rat tumors than in canine tumors. The activities were higher in malignant tissues than in benign ones of dogs, and higher in tumor tissues than in normal mammary tissues of rats. The results of film in situ zymography for tissue localization of gelatinolytic activity showed that the digested area was more extended in malignant tumors than in benign ones of dogs. However, the area was similarly extended in both benign and malignant rat tumors. These results may indicate that the canine spontaneous malignant mammary tumors possess more aggressive nature than the rat chemically induced counterpart, resulting from the high level of MMPs and low level of TIMPs activities of the tumor tissues.     

A statistical assessment of the biological relationship between simultaneous canine mammary tumours

Simultaneous canine mammary tumours (CMTs) are frequently reported in the literature, but few studies have addressed their biological relationship in detail or performed statistical assessments. In this study, 269 canine mammary gland tumours from 216 dogs were categorized using an extended histopathological classification, where semiquantitative and binomial scales enumerated morphological parameters of the tumours. The classification facilitated a statistical study of the biological relationship between simultaneous within‐dog tumours. Seventy‐seven percent of the dogs had single tumours and 23% had simultaneous tumours. Sixty‐one percent of the neoplasias were benign, with complex adenoma as the most frequent diagnosis and 39% were malignant, with complex carcinoma as the most common malignancy. Simultaneous tumours within dogs more often had equal diagnoses and neoplastic level (benign or malignant) than would be expected by chance alone, as compared with random pairs of single tumours from different dogs. This statistically supported finding indicated the presence of a biological relationship between simultaneous tumours.     

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.