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.     

RADIOGRAPHIC APPEARANCE OF PRIMARY LUNG TUMORS IN CATS

The radiographic findings in 41 feline primary lung tumors are reported. Case material includes 29 adenocarcinomas, six bronchiolog-alveolar carcinomas, and six squamous cell carcinomas. Adenocarcinomas tended to occur as focal, solitary, well-circumscribed masses (eight cats), as localized consolidations (six cats), or as multiple, poorly circumscribed masses (five cats). Bronchiolo-alveolar cell carcinomas and squamous cell carcinomas were radiographically pleomorphic. Calcification was recognized in only five cats, four with adenocarcinomas and one with bronchiolo-alveolar carcinoma. Cavitation was identified in seven adenocarcinomas, one squamous cell carcinoma, and one bronchiolo-alveolar carcinoma. Nearly 50% (20 cats) of the patients had radiographic evidence of pleural involvement. Fifteen cats had evidence of regional lymph node involvement.     

Coordinate expression of cytokeratins 7 and 20 in feline and canine carcinomas.

Forty-seven feline and 60 canine epithelial tumors were studied to test the coordinate expression of cytokeratin 7 (CK 7) and cytokeratin 20 (CK 20) using commercially available monoclonal antibodies and an avidin-biotin immunoperoxidase staining technique. Previously, the distribution of both cytokeratins was examined in normal tissues from 4 cats and 4 dogs. The pattern of distribution of CK 7 in normal tissues was similar, with minor differences, to that described in humans, whereas the reactivity pattern of CK 20 in cats and dogs was wider than that in humans. The subset of tumors strongly expressing CK 7 and CK 20 included pancreatic adenocarcinomas (100%), transitional cell carcinomas (75%), and endometrial carcinomas (67%) in the cat. None of the canine tumors had this immunophenotype. Feline (50%) and canine (56%) mammary gland carcinomas and canine cholangiocarcinomas (67%) were the only tumors presenting the CK 7 +/CK 20- immunophenotype, whereas the CK 7-/CK 20+ immunophenotype included thyroid carcinomas (100%), intestinal adenocarcinomas (60%), bronchioloalveolar carcinomas (50%), and renal carcinomas (50%) in the cat and intestinal adenocarcinomas (56%), gastric adenocarcinomas (50%), and ovarian carcinomas (50%) in the dog. The CK 7-/CK 20- immunophenotype included the rest of the analyzed tumors. The immunohistochemical evaluation of coordinate expression of both CK 7 and CK 20 in feline and canine carcinomas using monoclonal antibodies provides important information that can help to discriminate among carcinomas from different primary sites and could be particularly helpful in the determination of the primary site of origin of carcinomas presenting as metastatic disease.     

Epidermal growth factor receptor expression in feline oral squamous cell carcinomas

Feline oral squamous cell carcinomas (SCC) have a poor prognosis despite aggressive treatment with surgery, radiation and anticancer drugs. Overexpression of the epidermal growth factor receptor (EGFR), a membrane‐bound tyrosine kinase receptor, has been found in many human epithelial neoplasms, including oral SCC. EGFR overexpression has been associated with advanced disease and a poor prognosis. The purpose of this study was to determine whether feline oral SCC express EGFR. Thirteen formalin‐fixed paraffin wax‐embedded biopsy samples from feline oral SCC were analysed for EGFR expression using immunohistochemistry. Nine of 13 tumours (69%) were positive for EGFR expression, suggesting that altered EGFR expression plays a role in feline oral SCC and provides a rationale for a potential clinical benefit using EGFR inhibitors in combination with conventional treatments.     

Cohort study of COX-1 and COX-2 expression in canine rectal and bladder tumours

OBJECTIVES: To determine the role that cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) play in malignant transformation in canine transitional cell carcinoma and rectal tumours. METHODS: Histological sections of 21 canine rectal adenocarcinomas and 18 canine transitional cell carcinomas were stained for COX-1 and COX-2. Mann-Whitney non-parametric tests were applied to determine if there was any relationship between the percentage of cells expressing COX-1 or COX-2, and between COX-1 and COX-2 staining intensity and age, breed or sex. RESULTS: For rectal adenocarcinomas, 19.0 per cent of the sections were negative for COX-1 and COX-2. A further 38.1 per cent of the sections were negative for COX-2 but positive for COX-1, and 38.1 per cent of the sections had rare or occasional single cells positive for COX-2. No significant differences were found in COX staining when compared with age, breed or sex. For transitional cell carcinomas, all of the sections were positive for COX-1 and COX-2. For COX-2 staining, 16.7 per cent had more than 30 per cent positive cells. For COX-1 staining, 38.9 per cent had more than 30 per cent positive cells. There was a significant increase in the percentage of COX-1 positive cells in small breed dogs (P = 0.0337). CLINICAL SIGNIFICANCE: The variations in COX expression reported in this study may explain the differences in the clinical response of transitional cell carcinomas and rectal adenocarcinomas following treatment with non-steroidal anti-inflammatory drugs.     

Cisplatin for treatment of transitional cell and squamous cell carcinomas in dogs

Thirteen dogs with tumors were treated with monthly infusions of cisplatin. Complete responses were not observed. Of 8 dogs with urinary tract transitional cell carcinomas, 1 (12.5%) had a partial response of 31 weeks' duration, and 4 (50%) had stable disease for 12, 30, 32, and 34 weeks. Three (60%) of 5 dogs with head and neck squamous cell carcinomas had partial responses for 2, 10, and 15 weeks. All 13 dogs were evaluated for signs of toxicosis. Transient episodes of vomiting were recorded for 7 dogs (54%), and 2 dogs (15%) had mild thrombocytopenia. Although renal function gradually decreased in 2 dogs (15%), none of the dogs had an episode of acute renal failure attributable to cisplatin. These findings suggest that cisplatin may be a safe and potentially effective agent for treatment of transitional cell and squamous cell carcinomas in dogs.     

Ki67 reactivity in nasal and periocular squamous cell carcinomas in cats treated with electron beam radiation therapy

Squamous cell carcinomas of sparsely haired skin are relatively common tumors in cats, and these tumors likely exhibit a rapid growth rate. Thus, we evaluated response and duration of response in relation to the Ki67 proliferative reactivity in such tumors. Seventeen cats with confirmed squamous cell carcinomas and treated with an accelerated, hypofractionated electron beam radiation protocol were included in the study. For all of them histologic grading, Ki67 reactivity, response, and disease-free interval (DFI) were evaluated. Response to therapy was excellent (94% complete response rate) with a median DFI of 414 days. Only moderate acute and few long-term adverse effects were seen. Cats with tumors with a low Ki67 reactivity had markedly shorter DFIs than cats with tumors with high Ki67 reactivity. We concluded that an accelerated, hypofractionated electron beam radiation therapy protocol is well suited for feline squamous cell carcinomas. The protocol appears especially efficacious in tumors with a high Ki67 reactivity.     

Expression of cyclooxygenase-2 in canine epithelial nasal tumors

Cyclooxygenase-2 (COX-2) is an enzyme upregulated in some human and animal tumors. Enzymatic products are associated with tumorigenic activities. Given the poor response of canine nasal tumors to radiation, we considered the possibility that some of this resistance may be associated with COX-2 expression. To test this, 21 formalin-fixed, paraffin-embedded, and archived biopsy samples from canine epithelial nasal tumors were analyzed for COX-2 expression using immunohistochemistry. The biopsies were collected from dogs prior to radiation therapy. COX-2 expression was present in 17 of 21 (81%) tumors. The expression was observed in several different tumor types, including nasal carcinomas, adenocarcinomas, and squamous cell carcinomas. Samples from five control dogs without nasal neoplasia were also analyzed for COX-2 staining. These specimens were characterized by varying degrees of lymphoplasmacytic rhinitis with scattered regions of COX-2 positive respiratory epithelial and stromal cells. Whether the intensity and distribution of COX-2 expression in nasal tumors can be used as a prognostic marker requires further investigation. A combination therapy of irradiation and a selective COX-2 inhibitor appears worthy of clinical investigation in the treatment of canine epithelial nasal tumors.     

Clinical features, survival times and COX-1 and COX-2 expression in cats with transitional cell carcinoma of the urinary bladder treated with meloxicam

Records of 11 cats with transitional cell carcinoma of the urinary bladder, which had been treated with meloxicam, were reviewed for signalment, duration of clinical signs prior to diagnosis, results of diagnostic imaging, whether or not concurrent surgery was performed and survival. Immunohistochemical expression of cyclo-oxygenase-1 (COX-1) and cyclo-oxygenase-2 (COX-2) was assessed in the tumours of seven cats. Tumour location varied greatly. The cats had a mean age of 13 years. Three cats had a previous diagnosis of feline idiopathic cystitis of up to 2008 days duration. Ten of the cats showed clinical improvement (reduction of haematuria and/or dysuria), with a mean survival time (MST) of 311 days (range 10-1064); 1-year survival of 50%. All seven bladders assessed for COX staining were COX-1 positive and five were COX-2 positive. The MST for the COX-2-positive cats was 123 days, the MST for the COX-2-negative cases was 375 days.     

Carcinoma in the urinary bladder of a cat: cytologic findings and a review of the literature

Percutaneous fine needle aspiration biopsy (FNAB) was used to diagnose a urinary bladder carcinoma in an aged cat. The cytologic appearance of specimens collected initially was similar to that reported for canine transitional cell carcinoma. However, impression smears of the tumor made at necropsy 7 weeks later consisted predominantly of atypical squamous epithelial cells compatible with squamous cell carcinoma. Histologically, the malignancy was noted to have intermixed areas of abnormal squamous and transitional cell proliferation. The neoplasia was interpreted as a transitional cell carcinoma with extensive transformation to squamous cell carcinoma. This report examines the use and limitations of FNAB in the diagnosis of feline urinary bladder carcinoma and the incidence and behavior of these tumors in the cat.     

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.     

Expression of cyclooxygenase-2 in canine nasal carcinomas

Cyclooxygenase(COX)-2 expression was evaluated in 24 paraffin-embedded canine nasal carcinoma tissue samples by immunohistochemistry. Several different tumor types were represented, including carcinomas, adenocarcinomas and squamous cell carcinomas. COX-2 expression was identified in 17/24 cases (71%). The proportion of positive cells expressing COX-2 ranged from 10 to 95% and COX-2 expression was predominantly localized in the cytoplasm. Treatment with a COX-2 inhibitor should be investigated, along with the utilization of COX-2 expression as a prognostic marker.     

Expression of podoplanin in various types of feline tumor tissues

Podoplanin is expressed in various human tumors where it promotes tumor progression, epithelial-mesenchymal transition, and distant metastasis. Podoplanin is also expressed in cancer-associated fibroblasts and induces tumor malignancy. The objective of this study was to evaluate podoplanin expression in various types of feline tumor tissues. Immunohistochemical analysis revealed that podoplanin was expressed in cells of 13/15 (87%) squamous cell carcinomas and 5/19 (26%) fibrosarcomas. Moreover, cancer-associated fibroblasts expressed podoplanin in most tumor types, including 18/21 (86%) mammary adenocarcinoma tissues. Our findings demonstrate that various types of feline tumor tissues expressed podoplanin, indicating the importance of the comparative aspects of podoplanin expression, which may be used as a novel research model for podoplanin biology.     

Survival of 54 cats with oral squamous cell carcinoma in United Kingdom general practice

OBJECTIVES: To determine the survival of 54 cats with histologically confirmed feline oral squamous cell carcinoma (FOSCC) treated in UK general practice and to determine factors predictive for survival. METHODS: Cases were identified from consecutive samples submitted for histological diagnosis. Observational and survival data were collated retrospectively from submitting practices. Immunohistochemical analysis of cyclooxygenase (COX) expression variables was available using previously published data. Kaplan-Meier product limit estimation for overall survival and Cox proportional hazards regression for potential explanatory variables were performed. RESULTS: The overall median survival time was 44 days [95 per cent confidence interval (CI): 31-79] and 1 year survival was 9.5 per cent. Variables associated with survival were whether the cat was pedigree [hazard ratio (HR)=8.17, 95 per cent CI: 1.96-34.12], whether the cat received non-steroidal anti-inflammatory drug (NSAID) therapy after diagnosis (HR=0.46, 95 per cent CI: 0.21-0.98) and whether the COX-1 staining distribution was patchy rather than diffuse (HR=0.25, 95 per cent CI: 0.08-0.014). CLINICAL SIGNIFICANCE: This study suggests that although the prognosis for inoperable FOSCC remains poor, palliative treatments may offer a survival advantage that compares favourably with more aggressive treatment methods. Further work is needed to evaluate NSAID therapy in this disease, in particular to determine whether the potential survival advantage is because of an analgesic or anticancer effect or both. COX-1 distribution patterns may have a role as a prognostic indicator in this disease.     

Immunohistochemical identification of B and T lymphocytes in formalin-fixed, paraffin-embedded feline lymphosarcomas: relation to feline leukemia virus status, tumor site, and patient age.

The lymphocyte phenotype of 70 formalin-fixed, paraffin-embedded feline lymphosarcomas (LSAs) was determined immunohistochemically using a T cell polyclonal antibody, and a B cell monoclonal antibody. Forty-seven of 70 (67%) tumors were T cell, 19/70 (27%) were B cell, and 4/70 (6%) did not stain with either marker. Thirty-eight of 70 (54%) tumors were positive for feline leukemia virus (FeLV) antigen by immunohistochemistry (IHC), and 52/70 (74%) tumors were positive for FeLV DNA using the polymerase chain reaction (PCR). B cell tumors were as frequently FeLV-positive as T cell tumors using either IHC or PCR. Intestinal tumors were more likely to be B cell than T. The incidence of B and T cell tumors was not different among young (< or = 3 y), middle-aged (> 3 y to < or = 8 y), and old (> 8 y) cats. Both B and T cell tumors from old cats were FeLV-positive more often by PCR than by IHC. Feline leukemia virus DNA but not antigen, was detected in B cell tumors and intestinal tumors from cats > 8 y as often as it was detected in B cell tumors and intestinal tumors from cats < or = 8 y. Previously, most B cell and intestinal tumors from old cats were considered to be negative for FeLV. Here, the results suggest involvement of latent or replication-defective forms of the virus in such tumors from old cats. This study supports a role for FeLV in feline B cell as well as T cell tumorigenesis.     

Dysregulation of beta-catenin is common in canine sporadic colorectal tumors.

Human colorectal tumorigenesis is often initiated by APC (adenomatous polyposis coli) or beta-catenin (CTNNB1) mutations, which result in dysregulation of beta-catenin expression, followed by alterations in E-cadherin and/or p53. We examined 32 canine intestinal tumors for expression and intracellular distribution of beta-catenin, E-cadherin, and p53 using immunohistochemistry. beta-Catenin in normal mucosal epithelial cells was restricted to lateral cell membranes, but 13/13 (100%) colorectal adenomas had intense cytoplasmic and/or nuclear reactivity. Three of six (50%) colorectal carcinomas, 2/13 (15%) small intestinal carcinomas, and dysplastic cells in 1/2 focal hyperplastic lesions in the small intestine had a similar pattern of staining; remaining tumors had normal membranous beta-catenin reactivity. There was a correlation (P = 0.007) between abnormal beta-catenin and E-cadherin staining with 11/13 (85%) colorectal adenomas, 3/6 (50%) colorectal carcinomas, and 3/13 (23%) small intestinal carcinomas showing decreased membranous reactivity compared with normal mucosal epithelium. E-cadherin staining was reduced more often in adenomas than in carcinomas (P = 0.04). There were two patterns of nuclear p53 staining: > 60% of nuclei in 2/26 (8%) carcinomas (one colorectal, one small intestinal) were strongly labeled, whereas three colorectal adenomas and one small intestinal carcinoma had fainter staining in 10-20% of cells. Dysregulation of beta-catenin appears to be as important in canine colorectal tumorigenesis as it is in the human disease and could be due to analogous mutations. Malignant progression in canine intestinal tumors does not appear to be dependent on loss of E-cadherin or beta-catenin expression or strongly associated with overexpression of nuclear CMI antibody-reactivity p53.     

Multimodality therapy for head and neck cancer.

The refinement of radiation therapy techniques should result in a decrease in morbidity in canine and feline nasal carcinoma patients and should further allow for the addition of adjuvant therapies. Patients with large oral tumors that are incompletely excised should have radiation therapy added to their treatment regimen. Tumors with significant metastatic potential, such as melanoma, should be considered for addition of chemotherapy. Carboplatin has activity in melanomas and is being added at several institutions, but trial results are not yet available. Chemoradiation has become the treatment of choice for human head and neck squamous cell carcinomas but remains largely unexplored in veterinary medicine. Hopefully, development of chemoradiation will benefit feline squamous cell carcinoma patients, because current treatment regimens are largely ineffective. Immunotherapy agents and targeted biologic therapeutics seem to hold promise for the future.     

Topographic distribution of bcl-2 protein in feline tissues in health and neoplasia

The bcl-2 family of genes encodes proteins that influence apoptosis. In the present immunohistochemical study, the topographic distribution of bcl-2 protein was examined in healthy feline fetal, neonatal, and adult tissues, a feline renal cell line, and feline tumors obtained from a veterinary hospital. The topographic distribution of bcl-2 in healthy tissues was similar to that described in human tissues. In lymphoid tissues, follicular mantle cells strongly expressed bcl-2. In complex and differentiating epithelium, bcl-2 expression was detected in stem cell and proliferation zones. Bcl-2 expression was also detected in lower crypts of the intestine and in skin basal layers. The feline Crandell kidney cells expressed bcl-2 diffusely throughout the cytoplasm. Of 180 tumors examined, bcl-2 was expressed almost uniformly in cutaneous basal cell tumors, thyroid adenomas, and mammary carcinomas and in 50% of the lymphomas examined. Bcl-2 may play a role in blocking apoptotic cell death in a broad range of normal feline tissues, whereas dysregulated bcl-2 may extend the life of certain tumors or render certain tumors resistant to therapy because most chemotherapeutic and radiotherapeutic agents eliminate tumor cells by triggering apoptosis.     

Detection of papillomaviral DNA sequences in a feline oral squamous cell carcinoma

Oral squamous cell carcinomas (OSCCs) are common and often fatal feline neoplasms. Factors that predispose to neoplasm development in cats are poorly defined. Around 25% of human OSCCs are caused by papillomaviruses (PVs). To determine if PVs are associated with OSCCs in cats, three sets of consensus primers were used to evaluate 20 feline OSCCs and 20 non-neoplastic feline oral lesions for the presence of PV DNA. Papillomaviral sequences were detected within one OSCC, but no non-neoplastic lesion. Sequencing of the amplified DNA revealed a previously unreported PV that was most similar to human PV type 76. This is the first time PV DNA has been amplified from the oral cavity of a cat. However, while these results suggest that feline gingival epithelial cells can be infected by PVs, they do not support a causal association between viral infection and the development of feline OSCCs.