Nuclear cytomorphometry in feline mammary gland epithelial tumours

Stained cytological specimens from 35 feline mammary gland epithelial tumours (4 adenomas, 11 tubulopapillary carcinomas 13 solid carcinomas and 7 cribriform carcinomas) were analysed by computer-assisted nuclear morphometry. In each case, the nuclei of at least 100 neoplastic cells were measured, and the mean nuclear area (MNA), mean nuclear perimeter (MNP), mean nuclear diameter (MND) and nuclear roundness (NR) were calculated. The study aimed to evaluate (1) the possibility of using nuclear cytomorphometry as an auxiliary diagnostic method to differentiate between benign and malignant feline mammary gland epithelial tumours, and (2) the prognostic value of nuclear morphometry in feline mammary carcinomas. The results indicated that MNA, MNP, MND and NR could be a useful adjunct in diagnosis but are not reliable prognostic indicators for feline mammary gland carcinomas.     

Mammary invasive micropapillary carcinoma in cats: clinicopathologic features and nuclear DNA content

Invasive micropapillary carcinoma (IMC) is a variant of infiltrating ductal carcinoma of the breast associated with poor outcome. In this study, we report 16 carcinomas of the feline mammary gland displaying histologic features that correspond to IMC of the breast in women. The clinicopathologic findings, overall survival time, disease-free survival time, and nuclear DNA content of these cats were compared with 65 more common invasive mammary carcinomas (other feline mammary carcinoma [FMC]) of nonspecified type. IMC was associated with larger tumor size, higher histologic grade (P < .0001), deeper muscle invasion (P = .004), and more frequent lymphovascular invasion and nodal metastases (P = .009 and P = .001, respectively) than other FMCs. The aneuploid pattern was more frequent in IMC lesions. IMCs were also associated with lower survival rates. In summary, all cases of feline IMC were associated with clinicopathologic features of high biologic aggressiveness and should be classified as independent histologic types of FMC.     

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.     

MIB-1 labeling index in feline dysplastic and neoplastic mammary lesions and its relationship with postsurgical prognosis

Samples from feline normal, dysplastic, and neoplastic mammary tissues were used to investigate the usefulness of MIB-1 labeling index (MIB-1 I) as a prognostic indicator. Forty-eight queens bearing invasive carcinomas were included in a 2-year follow-up study. Mammary lesions were classified according to the World Health Organization system, and invasive carcinomas were further graded on the basis of the degree of tubule formation, the degree of nuclear and cellular pleomorphism, and mitotic count. Additional sections were immunostained using MIB-1 antibody, and MIB-1 I was expressed as a percentage of positive nuclei. In normal mammary gland tissues, the mean MIB-1 I was <1%. A low proliferation rate was found in all mammary adenosis and in situ carcinomas, and the highest rates were observed in feline mammary hypertrophy and invasive carcinomas. Twenty-one (43.7%) of the queens bearing invasive carcinomas were still alive at the end of the trial, and 27 (56.2%) had died. The MIB-1 I was not significantly correlated with clinical outcome, age, histologic type, or grading of the tumors, but a borderline correlation was observed with invasion of lymphatic vessels. Univariate analysis showed that high MIB-1 I was also not associated with decreased overall survival, whereas the grading system of the tumors had high predictive value (P = 0.0040) for postsurgery survival. The lack of correlation between MIB-1 I and postsurgery survival suggests that this marker alone is not sufficient to determine a correct prognosis in feline mammary carcinomas, even if it is a useful proliferation marker.     

Immunohistochemical analysis of cyclin A, cyclin D1 and P53 in mammary tumors, squamous cell carcinomas and basal cell tumors of dogs and cats

The involvement of cyclin A, cyclin D1 and p53 proteins in canine and feline tumorigenesis was analyzed immunohistochemically. In the present study, a total of 176 cases were examined, among which there were 108 canine cases (75 mammary lesions, 16 squamous cell carcinomas and 17 basal cell tumors) and 68 feline cases (43 mammary lesions, 20 squamous cell carcinomas and 5 basal cell tumors). Speckled nuclear staining for cyclin A was observed in 19/38 (50%) canine malignant mammary tumors and 18/37 (48.6%) feline mammary carcinomas, while this was not seen in benign mammary tumors of either dogs or cats. Marked intense nuclear cyclin A staining was seen in 7/16 (43.8%) canine squamous cell carcinomas and 18/20 (90.0%) feline squamous cell carcinomas. Only 3/17 (17.6%) canine basal cell tumors showed slight and scattered staining for cyclin A. Expression of cyclin D1 was very rare in both canine and feline tumors. Nuclear staining of p53 was found in 7/37 (18.9%) feline mammary carcinomas. Intense immunoreactivity for p53 was found in 6/16 (37.5%) canine squamous cell carcinomas and 8/20 (40%) feline squamous cell carcinomas. These results suggest that cyclin A may have a role in the proliferation of canine malignant mammary tumors, feline mammary carcinomas and squamous cell carcinomas of dogs and cats, and p53 may associate with the tumorigenesis of feline mammary carcinomas and squamous cell carcinomas of dogs and cats.     

Tissue culture of feline normal mammary gland

To study the growth and morphology of feline mammary epithelial cells in vitro, normal feline mammary tissues as well as cells collected from feline milk were cultured and examined using phase-contrast microscopy and immunohistochemistry. The method employed was a modification of Hiratsuka et al., and was found to be useful for the culture of dissociated feline mammary tissue. Small polygonal cells and large spindle-shaped cells that were positively stained with anti-keratin and anti-actin antibodies formed colonies with a pavement-like structure in culture of mid-pregnant mammary gland. Cultures of involuting mammary gland were composed mainly of small spindle-shaped cells, which were negative for keratin immunostaining and thought to be fibroblasts. Myofibroblasts, which participate in feline mammary carcinogenesis, and show keratin negative, actin positive, were not encountered in the present culture system of normal mammary gland. The cells from milk formed small colonies, but did not grow to reach confluent monolayer.     

Amplification of the cyclin A gene in canine and feline mammary tumors

DNAs from 33 canine mammary tumors and 8 feline mammary carcinomas were examined by Southern blot analysis to clarify genomic abnormalities of the cyclin A gene. Amplification of cyclin A was detected in 27.3% (9/33) of canine mammary tumors and 87.5% (7/8) of feline mammary carcinomas. It was suggested that amplification of cyclin A do not correlate directly with the tumorigenesis of canine mammary tumors, because there was no significant difference of incidence of cyclin A amplification between the benign and malignant tumors. In feline mammary carcinomas, the high frequency of cyclin A amplification raised the possibility that the amplification lead to the protein overexpression and play an important role in the tumorigenesis.     

Properties of the feline tumour suppressor reduced expression in immortalized cells (REIC/Dkk‐3)

Reduced expression in immortalized cells (REIC/Dkk‐3), a member of the human Dickkopf (Dkk) family, is a growth suppressor in human and canine mammary tumours. Mammary gland tumours are common neoplasms with high malignancy in female cats. The purpose of this study was to clone the feline REIC/Dkk‐3 homolog, investigate its expression in cell lines established from feline mammary gland tumours, and test its tumour suppressor function. Western blot analysis revealed that expression of the REIC/Dkk‐3 protein was reduced in feline mammary carcinoma cell lines. Forced expression of REIC/Dkk‐3 induced apoptosis in feline mammary tumour cell lines. These results demonstrate that REIC/Dkk‐3 expression, which is downregulated in feline mammary tumour cell lines, results in the induction of apoptosis in these cells. Our findings suggest that feline REIC/Dkk‐3 represents a potential molecular target for the development of therapies against feline mammary cancers.     

基于LC-MS技术的患乳腺癌猫血清代谢组学分析

本试验旨在研究患乳腺癌猫与健康猫体内差异代谢物的变化情况,并探讨差异代谢物与猫乳腺癌发生之间的关系。本试验选取临床收集的经组织病理学确诊的猫乳腺癌血清样本6例作为试验组（T组）,同时选取年龄相似,品种相同的健康猫血清6例作为对照组（C组）。运用超高效液相色谱质谱联用技术（liquid chromatography-mass spectrometry,LC-MS）对两组猫的血清样本进行检测,采用无监督的主成分分析（principal component analysis,PCA）、正交偏最小二乘法-判别分析（orthogonal projections to latent structures-discriminant analysis,OPLS-DA）及学生t检验（Student's t-test）筛选出差异代谢物,然后再对筛选出的差异代谢物进行层次聚类分析（hierarchical clustering analysis,HCA）,并进行差异代谢物的KEGG注释及差异代谢物的代谢通路分析。结果表明,在两组样本中共定性到159种差异代谢物。与C组相比,在T组中有49种差异代谢物出现下调,110种差异代谢物出现上调。最终共筛选出5种与猫乳腺癌发生发展密切相关的差异代谢产物。与C组相比,T组中麦角硫因（ergothioneine,EGT）和肌酸（creatine）出现下调,吲哚乙酸（indolelactic acid,IAA）、胆碱（choline）和尿酸（uric acid）出现上调。这些差异代谢物表明,在猫乳腺癌的发生过程中,机体变化涉及了甘氨酸、丝氨酸和苏氨酸代谢、精氨酸和脯氨酸代谢、组氨酸代谢、色氨酸代谢、嘌呤代谢异常和甘油磷脂代谢等多个代谢途径,为今后深入研究猫乳腺癌的发病机制开辟了一个新的思路。     

Serum Metabolomics Analysis of Feline Mammary Carcinomas Based on LC-MS Techniques

The purpose of this study was to investigate the variation of differential metabolites in mammary carcinomas bearing cats and healthy cats, and to explore the relationship between differential metabolites and the occurrence of feline mammary carcinomas. In this study, 6 feline mammary carcinomas serum samples were selected as test (T) group. Meanwhile, 6 healthy cats with similar age and same breed were selected as control (C) group. Serum samples were detected by ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (LC-MS). Differential metabolites were screened by principal component analysis (PCA), orthogonal projections to latent structures-discriminant analysis (OPLS-DA) and Student's t-test. Then the hierarchical clustering analysis (HCA) was carried out for the screened differential metabolites. The KEGG annotation and the metabolic pathway of differential metabolites were analyzed. The results showed that a total of 159 differential metabolites were identified in the 2 groups. Compared with C group, 49 differential metabolites were down-regulated and 110 differential metabolites were up-regulated in T group. Finally, a total of 5 differential metabolites which closely related to feline mammary carcinomas were selected. Ergothioneine (EGT) and creatine in T group were down-regulated, while indolelactic acid (IAA), choline and uric acid were up-regulated compared to C group. These differential metabolites indicated that during the development of feline mammary carcinomas, the body changes involved multiple metabolic pathways, such as glycine, serine and threonine metabolism, arginine and proline metabolism, histidine metabolism, tryptophan metabolism, purine metabolism and glycerophospholipid metabolism. This study provides a new idea for further research of the pathogenesis of feline mammary carcinomas.     

Fractal dimension of canine mammary gland epithelial tumors on cytologic smears

BACKGROUND: Fractal geometry is a tool that can be used for describing, modeling, analyzing, and processing irregular and complex figures. Past investigations in medicine have revealed that fractal analysis could also be applied in tumor pathology to characterize irregular boundaries of the nuclei of tumor cells. OBJECTIVE: The aim of this study was to define whether the fractal dimension parameter could be used on cytologic specimens to differentiate benign from malignant canine mammary gland epithelial tumors. METHODS: The fractal dimension of nuclear surface was determined by computer-assisted morphometry on Hemacolor-stained cytologic smears obtained by fine needle aspiration of normal canine mammary gland epithelial cells, and cells in mammary adenomas, tubulopapillary carcinomas, solid carcinomas, and anaplastic carcinomas. Data were analyzed by Mann-Whitney U test. RESULTS: Significant differences (P <.001) were observed in mean fractal dimension among all tumor types and in comparison with normal canine mammary gland epithelial cells (except for the fractal dimension between solid carcinomas and anaplastic carcinomas). CONCLUSION: The morphometric parameter, fractal dimension, could help in the diagnostic discrimination between benign and malignant canine mammary gland epithelial tumors on cytologic specimens.     

Ultrasonographic assessment of the feline mammary gland

The aim of this study is to characterise the feline mammary echotexture using B-mode ultrasonography, which is not routinely used to examine the feline mammary gland. Using a 5-9 MHz linear transducer the ultrasonographic appearance of non-stimulated and stimulated mammary glands was determined in 35 mature intact non-pregnant, pregnant and lactating queens aged from 16 months to 8 years. In intact non-pregnant queens, mammary glands are fairly underdeveloped and on the ultrasonograms they appear with a regular hypoechoic texture and generally show a thickness of less than 2.0mm. The stimulated mammary tissue typically presents a more hyperechoic appearance compared to the non-stimulated gland and a fine granular echotexture. Maximum echogenicity of the mammary gland is reached during lactation. In late pregnancy, the mammary glands reach 6-9 mm in thickness. During lactation, the size of the glands depends on the existence of a suckling stimulus, with the suckled glands reaching about 11 mm in thickness. Ductal structures can only be imaged during late pregnancy and lactation. Ultrasonographic evaluation of the feline mammary gland can become a valuable diagnostic tool to characterise physiological changes and may further contribute to a better characterisation of diseased mammary tissue.     

Lectin histochemistry on squamous metaplasia in different epithelial tumors of dogs.

Biotinylated lectins and avidin-biotin-peroxidase complex were used to study the correlation between cellular glycoconjugates' expression and squamous maturation in normal canine skin and in various epithelial neoplasms. Normal skin tissue was obtained from five, male, random-source dogs, 5 to 7 years old. The tumors tested, selected from the files of our Department, were fifteen squamous cell carcinomas from different tissue origin, five hepatoid perianal gland adenocarcinomas with squamous metaplasia, and fourteen solid mammary carcinomas with and without histologic evidence of squamous metaplasia. Except for mammary gland carcinomas, all tumors had been surgically excised from male dogs. Intermediate filament aggregation of twelve solid mammary gland carcinomas were studied electron microscopically. The basal and the lower spinous cells in normal skin and the less differentiated cells in squamous cell carcinomas stained moderately with Griffonia simplicifolia agglutinin-I. Spinous and granular cell layers stained strongly with Phytolacca americana mitogen and Arachis hypogaea agglutinin. Both lectins stained well-differentiated cells in squamous cell carcinomas. The electron microscopic study carried out in solid carcinomas of mammary glands revealed some relationship between the presence of intracytoplasmic tonofibrils and the binding of Griffonia simplicifolia agglutinin-I and Phytolacca americana mitogen to the tumors tested. Our results suggest that the glycosylation pattern occurring during normal keratinocyte differentiation is conserved in squamous cell carcinomas and that Griffonia simplicifolia agglutinin-I and Phytolacca americana mitogen may represent useful tools in distinguishing poorly differentiated squamous cell carcinomas from other poorly differentiated mammary epithelial tumors.     

Histologic features of mammary carcinomas in zoo felids treated with melengestrol acetate (MGA) contraceptives

Melengestrol acetate (MGA), a potent synthetic progestin, has been used as a contraceptive in zoo felids since 1975. Mammary gland carcinomas have been linked to MGA treatment in zoo felids, but the histologic features of these tumors and steroid receptor expression have not been described. Zoo felid mammary tumors were requested from participating zoos from 1986 through 1998, and 31 mammary carcinomas from 28 MGA-treated and 3 untreated felids were received. The carcinomas were evaluated on the basis of histologic pattern, tumor grade, and occurrence of metastasis; then features of the tumors were compared to determine if carcinomas in MGA-treated felids differed from those that occur spontaneously. Estrogen- and progesterone-receptor expression was evaluated in 17 of the 31 carcinomas. Of the 31 tumors, 22 (70.9%) had multiple histologic patterns, 29 (93.5%) were high grade, and 28 (90.3%) had metastasized. Within tumors, the tubulopapillary pattern was most common (87.1%, n = 27); solid (61.3%, n = 19), cribriform (38.7%, n = 12), and comedone (25.8%, n = 8) patterns were less common; and the mucinous (3.2%, n = 1) pattern was rare. Both MGA-treated and untreated zoo felids had similar patterns and grades of mammary gland cancer as well as prevalence of metastasis. These results indicate that mammary carcinomas in zoo felids are high grade with a predominant tubulopapillary pattern and aggressive behavior. Five of 17 carcinomas expressed progesterone receptors, and 1 of 17 expressed estrogen receptors. Although more zoo felids with cancer had been exposed to MGA in this study, mammary carcinomas were similar in appearance and behavior in untreated and MGA-treated zoo felids. The association of MGA with the development of malignant mammary gland tumors should be considered when using this contraceptive in zoo felids.     

Comparison of steroid receptor expression in normal, dysplastic, and neoplastic canine and feline mammary tissues

Steroid receptor expression was assessed by immunohistochemistry in neoplastic, hyperplastic/dysplastic, and normal mammary tissue samples removed from 68 queens and 47 bitches, using monoclonal antibodies against human oestrogen-alpha (ER) and progesterone receptors (PR). Mammary lesions were classified according to World Health Organization (WHO) criteria, and all animals with invasive carcinomas were clinically followed for 2 years. Stromal and/or lymphatic invasion and histological grading were also recorded. In both species, ER expression was significantly higher in healthy tissues, hyperplastic/dysplastic lesions, and benign tumours than in carcinomas. The loss of ER expression was more marked in feline than in canine carcinomas. In queens, PR expression increased in dysplastic lesions and "in situ" carcinomas and decreased in invasive carcinomas, even if parts of these tumours were still PR-positive. In bitches no significant variation in PR expression was observed between normal tissue, dysplasias, and benign neoplasms, but was significantly lower in carcinomas. In both species ER and PR expression in invasive carcinomas did not correlate either with histological parameters or overall survival time. This study demonstrates several differences in steroid hormone dependency between the two species. The percentage of PR-positive feline carcinomas suggests a possible role of progesterone in promoting early tumour cell growth in queens. The low percentage of ER-positive invasive carcinomas further demonstrated the aggressive phenotype and behaviour of feline mammary tumours.     

Correlation of vascular endothelial growth factor expression to overall survival in feline invasive mammary carcinomas

Samples from feline invasive mammary carcinomas (FMCs) were used to determine the prognostic significance of the immunohistochemical expression of vascular endothelial growth factor (VEGF) and microvessel density (MVD). Forty-eight queens bearing FMCs were included in a 2-year follow-up study. Mammary tumors were classified according to the World Health Organization system and graded on the basis of histologic criteria. Tumor sections were immunostained using anti-VEGF and anti-von Willebrand factor (vWf) antibodies. VEGF expression was quantified on the basis of the percentage of positive cells. MVD of vWf-positive microvessels was determined by both mean microvessel counts and highest microvessel counts. Normal mammary gland tissues showed an inconspicuous VEGF staining. In FMCs the proportion of VEGF-positive cells was significantly higher in papillary and solid carcinomas than in tubular and papillary cystic tumors. An increased number of cells expressing VEGF was also observed in poorly differentiated FMCS. Sixteen (33.3%) of the queens bearing invasive carcinomas were still alive at the end of the 2-year follow-up period, and 32 (66.7%) had died. The VEGF expression was significantly correlated with the clinical outcome, but no correlation was observed with the invasion of lymphatic vessels. A correlation between the higher percentage of VEGF-positive cells and the unfavorable prognosis was demonstrated by the estimation of curves for overall survival (P = 0.03). Univariate analysis showed that MVD did not correlate with the overall survival. The results of our study demonstrated that VEGF expression, although not associated with increased angiogenesis, is a prognostic indicator in feline mammary tumors. In contrast, there is no support for a role of neovascularization as an indicator of survivability.     

COX‐2, mPGES‐1 and EP2 receptor immunohistochemical expression in canine and feline malignant mammary tumours

Prostaglandin (PG) signalling is involved in human and animal cancer development. PG E₂ (PGE₂) tumour‐promoting activity has been confirmed and its production is controlled by Cyclooxygenase‐2 (COX‐2) and microsomal PGE synthase‐1 (mPGES‐1). Evidence suggests that mPGES‐1 and COX‐2 contribute to carcinogenesis through the EP2 receptor. The aim of our study was to detect by immunohistochemistry COX‐2, mPGES‐1 and EP2 receptor expression in canine (n = 46) and feline (n = 50) mammary tumours and in mammary non‐neoplastic tissues. COX‐2 positivity was observed in 83% canine and 81% feline mammary carcinomas, mPGES‐1 in 75% canine and 66% feline mammary carcinomas and the EP2 receptor expression was observed in 89% canine and 54% feline carcinomas. The frequency of COX‐2, EP2 receptor and mPGES‐1 expression was significantly higher in carcinomas than in non‐neoplastic tissues and adenomas. COX‐2, mPGES‐1 and EP2 receptor expression was strongly associated. These findings support a role of the COX‐2/PGE2 pathway in the pathogenesis of these tumours.     

Calponin expression and myoepithelial cell differentiation in canine, feline and human mammary simple carcinomas

Calponin is a 34‐kDa smooth muscle‐specific protein that has been shown to be a highly sensitive marker of myoepithelial cells in canine, feline and human mammary tissue and tumours. The expression of calponin was studied in 15 canine, 32 feline and 28 human simple mammary carcinomas using a monoclonal mouse antihuman calponin antibody and the avidin–biotin peroxidase complex (ABC) immunohistochemical technique. Calponin expression was compared with the expression of cytokeratin 14, a marker of normal mammary myoepithelial cells in the three species. Four different types of calponin‐positive cells were identified: (1) Type 1: cytokeratin‐14‐positive pre‐existing myoepithelial cells forming a continuous layer with images of focal disruptions; (2) Type 2: cytokeratin‐14‐positive isolated nests of fusiform, polygonal or round cells without atypia; (3) Type 3: cytokeratin‐14‐positive atypical cells indistinguishable from non‐reactive atypical cells, which should have never been detected in haematoxylin and eosin‐stained sections and (4) Type 4: cytokeratin‐14‐negative stromal fusiform cells around the neoplastic growth or cell nests, identified as myofibroblasts. Calponin‐negative and cytokeratin‐14‐positive atypical neoplastic cells were observed in three canine, 28 feline and two human carcinomas. The latter were indicative of altered expression of high‐molecular‐weight cytokeratins in luminal epithelial‐type simple carcinomas. Our findings show that calponin is a good marker of myoepithelial cell differentiation in feline, human and, particularly, canine simple carcinomas. The high number (six out of 15) of canine tumours with type 3 cells points to the need of both introducing calponin examination in the routine diagnostic schedule and performing further studies on its prognostic significance.     

Prognostic studies of canine and feline mammary tumours: the need for standardized procedures

For several years, veterinary oncologists have been struggling with the prognosis of mammary tumours in dogs and cats. Translation of tumour characteristics into prognostic information is an invaluable tool for the use of the most appropriate therapies, as well as for planning innovative therapeutic trials. Moreover, canine and feline spontaneous mammary gland tumours are good models for the study of human breast cancer. Collecting and interpreting information regarding the prognosis of canine and feline mammary tumours is difficult due to the fact that different methods have been applied to study various components and characteristics. This review identifies some of the challenges of prognostic studies of spontaneous canine and feline mammary tumours and suggests standardized procedures to overcome these challenges and facilitate reproducibility and assessment of results.