P. Dias Pereira

COX-2 expression in canine normal and neoplastic mammary gland.

COX-2 expression was examined immunohistochemically in samples of normal canine mammary tissue (n=22) and benign (n=36) and malignant (n=45) mammary tumours including metastases (n=12). COX-2 was constitutively expressed in normal mammary tissue with membranous apical labelling of glandular epithelium, suggesting a role for this molecule in normal mammary physiology. By contrast, in neoplastic lesions and in adjacent non-neoplastic mammary tissue COX-2 was expressed in the cytoplasm of epithelial cells, suggesting that internalization of the molecule is associated with oncogenesis. Marked expression of COX-2 was observed in 8.3% of benign neoplasms and in 42.2% of malignant neoplasms, mainly in poorly differentiated areas. The majority of metastatic lesions (58.3%) exhibited strong COX-2 labelling and in almost all cases (83.3%) the labelling intensity was similar or stronger to that of the primary neoplasm. This finding is consistent with the hypothesis that COX-2 metabolites are important promoters of angiogenesis and invasiveness and therefore contribute to metastatic spread.

An immunohistochemical study of feline endometrial adenocarcinoma.

Feline endometrial adenocarcinomas are uncommon malignant neoplasms that have to date been poorly characterized. The present immunohistochemical study describes the expression of the pancytokeratins AE1 and AE3, cytokeratin-14, vimentin, alpha-actin, cyclo-oxygenase-2, E-cadherin, beta-catenin, the progesterone receptor, the oestrogen receptor and caveolin-1 within normal feline uterine tissue and tissue from six cats with endometrial adenocarcinoma. Synthesis of cyclo-oxygenase-2 and reduced expression of progesterone receptors may be involved in the neoplastic transformation of feline endometrium. The loss of cellular adhesion that occurs within these tumours does not require down-regulation of E-cadherin expression and nuclear translocation of beta-catenin is not a feature of these neoplasms.

Expression of E-cadherin in normal, hyperplastic and neoplastic feline mammary tissue

This paper describes an immunohistochemical study of the expression of E-cadherin in four samples of normal, eight samples of hyperplastic and 19 samples of neoplastic feline mammary tissue. In the normal tissues, the luminal epithelial cells showed a strong pattern of staining for E-cadherin at the cell-cell boundaries, whereas the myoepithelium showed no immunoreactivity. In the hyperplastic tissues and the five benign neoplasms, there were disturbances in the expression of E-cadherin in the luminal epithelium, in the form of the coexistence of membranous and cytoplasmic staining, together with immunoreactivity in a small percentage of myoepithelial cells. In 11 of 14 carcinomas, there was a reduction or absence of E-cadherin expression and abnormalities in the pattern of immunostaining; these changes were more pronounced in cribriform and solid carcinomas.

Reduced expression of claudin-2 is associated with high histological grade and metastasis of feline mammary carcinomas.

Claudins (CLDNs) are a family of tight junction (TJ) proteins that play an important role in maintaining cell polarity, in controlling paracellular ion flux and in regulating cell proliferation and differentiation. There is a growing body of evidence that associates changes in CLDN expression with the development of human breast cancer. In the present study CLDN-2 expression was examined immunohistochemically in samples of normal feline mammary tissue (n = 5) and mammary carcinomas (n = 52), including metastatic lesions (n = 29). Seventy-seven percent of carcinomas showed reduced CLDN-2 expression compared with that observed in normal mammary gland. Reduced expression of CLDN-2 was significantly associated with a high histological grade of carcinoma (P = 0.011), with 88.6% of grade II/III carcinomas showing decreased expression. Furthermore, CLDN-2 down-regulation was significantly associated with metastatic disease (P = 0.0027), with 93.1% of cases with signs of metastasis showing decreased expression of this protein. CLDN-2 may constitute a molecular marker for identification of a subgroup of feline mammary carcinomas characterized by high histological grade and the development of metastasis.

Caveolin-1 in Diagnosis and Prognosis of Canine Mammary Tumours: Comparison of Evaluation Systems

Caveolin-1 (Cav-1) is a major structural protein of caveolae, plasma membrane invaginations related to several cellular processes including regulation of signal transduction. In recent years there has been some controversy regarding the distribution of Cav-1 in normal and neoplastic mammary cell types, which may be attributed to different scoring systems adopted in different studies. The present study compares Cav-1 immunoexpression in normal (n=17) and neoplastic (n=79) canine mammary tissues assessed by two different scoring methods (previously reported by others with conflicting results) and associates Cav-1 expression with metastasis and overall survival (OS). Results obtained with both scoring methods were similar, revealing absence of immunoreactivity in normal luminal epithelium and in benign neoplasms and clearly associating Cav-1 expression with malignant transformation. The data suggest that Cav-1 expression is associated with highly malignant subtypes of mammary tumours (i.e. basal-like carcinoma), invasion and metastasis, thus supporting the hypothesis that it may play a major role in the epithelial-mesenchymal transition process. Furthermore, one of the scoring systems employed associated Cav-1 expression with unfavourable prognosis in canine mammary carcinomas, showing a strong correlation between Cav-1-positive carcinomas and shorter OS.

Immunohistochemical expression of caveolin-1 in normal and neoplastic canine mammary tissue.

Caveolins are the major structural proteins of caveolae and play a role in tumour surveillance. The expression of caveolin-1 (Cav-1) was investigated immunohistochemically in samples of normal canine mammary tissue (n=5), benign (n=23) and malignant (n=45) mammary tumours, tumour emboli (n=10) and metastatic lesions (n=10). Cav-1 was not expressed by normal luminal epithelium, but was consistently expressed by normal myoepithelial cells. There was no epithelial expression of Cav-1 by the benign mammary lesions, but the molecule was detected in 35.6% of the malignant lesions, 80% of the tumour emboli and in 40% of the metastatic lesions. These findings link increased expression of Cav-1 to neoplastic transformation and suggest that Cav-1 expression is associated with more malignant canine mammary tumours and their vascular invasion and regional lymph node metastasis.

Recurrent and metastatic canine urethral transitional cell carcinoma without bladder involvement

LOWER urinary tract tumours are rare in dogs, comprising less than 1 per cent of all canine tumours; among them, transitional cell carcinoma (TCC), arising from the transitional epithelium of the urinary tract, is the most frequently occurring (Nielsen and Moulton 1990, Norris and others 1992, Meuten 2002). The most common site of TCC development is the trigone area of the bladder (Henry 2003). Primary urethral TCCs are rare in dogs (Meuten 2002), being more frequent in older female dogs (mean age 10·4 years) (Klausner and Caywood 1995). However, it is known that the urethra, as well as the vagina, prostate and rectum, may be secondarily involved by extension of a bladder TCC (Nielsen and Moulton 1990). Distant metastases, including regional lymph nodes, occur in approximately half of all bladder TCCs and onethird of urethral TCCs (Nielsen and Moulton 1990, Norris and others 1992, Klausner and Caywood 1995). Haematuria, dysuria and pollakiuria are the most common clinical signs associated with a bladder and/or urethral TCC (Meuten 2002). This short communication describes a case of urethral TCC in a seven-year-old, mixed breed, neutered, obese, female dog with clinical signs of dysuria and haematuria. Physical examination of the dog revealed an irregular, erythematous, vaginal mass protruding from the urethral orifice. Smears were made from the surface of the mass using a cotton swab. Slides were air-dried, stained with Diff-Quik (Hemacolor; Merck) and submitted for cytological examination. Cytological analysis revealed a sample with a high cell density, consisting of a unique population of epithelial cells with moderate cell-to-cell adhesion. The cells had distinct outlines, with a variable amount of cytoplasm presenting diverse tinctorial characteristics. The degree of anisocytosis and anisokaryosis, and the variation in nucleus:cytoplasm ratio between cells was marked. The nuclei were large, bizarre and multiple, with reticular or clumped chromatin, and had either a large, prominent nucleolus or multiple nucleoli of variable size. Some cells showed signet-ring formation and, occasionally, the vacuoles were filled with a granular magenta material (Fig 1). Rare degenerative neutrophils involved in the phagocytosis of bacteria (cocci) were also present. The epithelial cell morphology was highly suggestive of a TCC. Ultrasound examination of the bladder and the upper urinary tract detected large calculi in the bladder, with regular thickening of the bladder wall. Cytological examination of the urinary sediment, collected by cystocentesis, revealed neutrophils phagocytosing bacteria, as well as individual, atypical, transitional epithelial cells. A partial vaginectomy was performed, and the vaginal mass was removed and submitted for histological examination. Microscopic analysis revealed a tumour composed of pleomorphic epithelial cells arranged in a papillary (Fig 2a) and nodular fashion. The cells were large, irregular in shape, and the majority had an extremely vacuolated cytoplasm. Some of these vacuoles contained an eosinophilic, granular material that stained positively with periodic acid-Schiff. Nuclear pleomorphism was marked, with large, pale nuclei and, most often, one large, central, prominent nucleolus. On average, four mitotic figures were observed per high-power field, with some atypical figures. Invasion of the basement FIG 1: Cytological preparation of the vaginal mass revealed marked anisocytosis and anisokaryosis, and signet-ring formation of the neoplastic cells. Diff-Quik. x 400

Influence of Catechol-O-Methyltransferase (COMT) Genotypes on the Prognosis of Canine Mammary Tumors

Catechol-O-methyltransferase (COMT) is an important enzyme involved in inactivation of catechol estrogens, which are metabolites with carcinogenic properties. Some investigations in human breast cancer associate a genetic polymorphism in the COMT gene (COMT val158met) with an increased risk and poor clinical progression of the disease. In dogs, there are 2 recognized single nucleotide polymorphisms in the COMT gene (COMTG216A and COMTG482A); however, their influence on the outcome of mammary neoplasms has never been investigated. The purpose of this study is to investigate the influence of COMT in the clinical progression of canine mammary tumors, namely in recurrence, metastasis and survival by testing 2 SNPs (G216A and G482A), and 2 genotypes of the COMT gene. A case series was conducted analyzing genomic DNA samples by polymerase chain reaction-restriction fragment length polymorphism from 80 bitches with mammary tumors. Animals were submitted to an active follow-up study for a period of 24 months after surgery. We observed that bitches carrying both genetic variations simultaneously are more likely to develop recurrence of mammary lesions. Our results demonstrate a possible role for COMT genotypes in the outcome of mammary neoplasms in the dog. Identifying a genetic factor predictive of recurrence may be useful in selecting the most effective surgical approach for canine mammary neoplasms.

Estrogens Metabolism Associated with Polymorphisms: Influence of COMT G482a Genotype on Age at Onset of Canine Mammary Tumors

Catechol-O-methyltransferase (COMT) is an important enzyme participating in inactivation of carcinogenic oestrogen metabolites. In humans there is a single nucleotide polymorphism in COMT gene (COMT va1158met) that has been associated with an increased risk for developing breast cancer. In dogs, there is a single nucleotide polymorphism in COMT gene (G482A), but its relation with mammary carcinogenesis has never been investigated. The aim of this study was to focus on the evaluation of such polymorphism as a risk factor for the development of mammary tumors in bitches and on the analysis of its relationship with some clinicopathologic features (dogs age and weight, number and histologic type of the lesions, lymph node metastasis) of canine mammary neoplasms. A case-control study was conducted analyzing 90 bitches with mammary tumors and 84 bitches without evidence of neoplastic disease. The COMT G482A polymorphism was analyzed by PCR-RFLP. We found a protective effect of the polymorphism in age of onset of mammary tumors, although we could not establish a significant association between COMT genotype and other clinicopathologic parameters nor with mammary tumor risk overall. Animals carrying the variant allele have a threefold likelihood of developing mammary tumors after 9 years of age in comparison with noncarriers. The Kaplan-Meier method revealed significant differences in the waiting time for onset of malignant disease for A allele carrier (12.46 years) and noncarrier (11.13 years) animals. This investigation constitutes the first case-control study designed to assess the relationship between polymorphic genes and mammary tumor risk in dogs. Our results point to the combined effect of COMT genotype with other genetic and/or environmental risk factors as important key factors for mammary tumor etiopathogenesis.

Case of malignant biphasic mesothelioma in a dog.

indirect fluorescent antibody test (IFAT) (Yoon and others 1992). Approximately 75,000 doses were distributed to 10 different farms between January and December 2000. Six farms were originally PRRS positive, while four were PRRS virus negative before receipt of any semen from this source. A thorough history of the farm, an outline of the protocol employed, and all previously collected diagnostic information were shared with farm owners and veterinarians before the semen was purchased. Clinical or diagnostic evidence of the introduction of a new PRRS virus has not been detected in any of the farms to date. Disadvantages of the protocol discussed included occasional jowl abscessation in the boars due to repeated venipuncture, the cost of multiple diagnostic tests and the labour involved in testing. Another inherent disadvantage was the potential loss of genetic value secondary to the culling of seropositive boars which may no longer have harboured PRRS virus. Therefore, it is planned to extend the isolation and testing period for animals with evidence of declining ELISA S/p ratios to determine if they eventually become seronegative. Furthermore, there is unmeasured risk with the continual introduction of boars. Since replacement boars enter the Al centre every four months and follow the same protocol before entry, strict monitoring of serum and semen is required to alleviate that risk. In order to reduce risk, it may be necessary to reduce the required s/p ratio, for example, to less than 0-3, for entry into the Al centre. The major limitation of this study was that it involved one only farm. Therefore, it is unknown whether similar results would be obtained on other farms, particularly if the infection was to occur later in the life of the pig. Also, no controls were included in the study. Therefore, before it can be accepted as an established, efficacious protocol, it must be replicated across a number of farms at different sites with differing PRRS virus strains, facilities and genetic lines. Due to the detection of seropositive samples following stocking of the Al centre, the use of a whole herd test and removal protocol (Dee and others 2001) immediately following population may enhance the success of future projects (Table 2). Also, since a 2 per cent false-positive rate has been reported following the use of the ELISA, auxiliary diagnostics such as the IFAT, or postmortem examination and tissue submissions may be necessary to better understand the PRRS virus status of individual boars (Dee and others 2001). Finally, this study used animals which were infected at an early age; therefore, PRRS virus transmission in the breeding herd at the source farm was not a significant issue. If repeatable, it may provide a means to produce groups of PRRSseronegative breeding stock from previously infected animals, protect valuable genetic lines and to avoid the need for the early weaning and segregation of offspring.