Establishment of four pairs of canine mammary tumour cell lines derived from primary and metastatic origin and their E-cadherin expression

Four new pairs of canine mammary carcinoma cell lines derived from both primary and metastatic lesions were established. The cells were cultured in RPMI‐1640 with 10% fetal bovine serum and they showed stable growth for more than 120 passages. Using these cell lines, the expression of E‐cadherin was measured by flow cytometry and the function of E‐cadherin was evaluated by cell aggregation assay and results from the primary and metastatic lesions were compared statistically. E‐cadherin was strongly expressed in all of the cell lines, without a notable difference between cells of primary and metastatic origin. In the cell aggregation assay, the function of E‐cadherin was significantly weaker in the cells of primary origin (p < 0.05), as compared with cells of metastatic origin. The present results suggest that a reduction in E‐cadherin function may be implicated in the invasive and metastatic potential of canine mammary tumour cells; however, further study will be needed to clarify E‐cadherin function in the context of the metastasis of canine mammary carcinoma.     

Expression of E‐cadherin in Canine Anal Sac Apocrine Gland Adenocarcinoma and its Association with Survival

Introduction:  Inverse associations have been shown between E‐cadherin expression and degree of differentiation in canine mammary tumours and with the acquisition of malignancy in human epithelial cancers. The purpose of this study was to examine whether there was an association between prognosis, and the distribution or intensity of immunohistochemical staining for E‐cadherin in anal sac adenocarcinoma.
Methods:  Formalin‐fixed paraffin‐wax embedded canine anal sac adenocarcinoma specimens were obtained for 36 patients for whom clinical data, including survival statistics, were available. Canine mammary adenoma tissue was employed as the positive and negative controls with antibody diluent replacing the primary antibody in the latter. Samples were scored according to proportion of cells showing positive cytoplasmic membranous staining, intensity of cytoplasmic membranous staining and proportion of nuclei staining. As quality control, a sample of 10 slides was randomly selected for repeat evaluation; results exactly matched those obtained in the first assessment.
Results:  All anal sac adenocarcinoma cells expressed E‐cadherin to varying degrees. There was no evidence of an association between survival time and the intensity of staining or the proportion of nuclei staining. Patients whose tumours exhibited >75% positive cytoplasmic membranous staining survived significantly longer than those exhibiting <75% staining with median survival times of 1100 and 479 days respectively (log rank test: χ² = 3.89, 1 DF, p = 0.049).
Conclusion:  Immunohistochemical evaluation of the proportion of cells in anal sac adenocarcinoma samples exhibiting positive cytoplasmic membranous staining for E‐cadherin may allow improved determination of prognoses and could aid clinical decision making.     

MIB‐1 immunoreactivity correlates with biologic behaviour in canine cutaneous melanoma

The growth fraction of 68 canine cutaneous melanomas was determined by immunostaining with MIB‐1, a monoclonal antibody to a Ki‐67 epitope that recognizes all proliferating cells. Fifty tumours were classified histologically as benign and 18 as malignant. The Ki‐67 proliferative index (percentage of positive cells over 500 neoplastic cells) was low (< 15%) in 55 cases and high ( 15%) in 13 cases. High Ki‐67 proliferative index and histological malignancy were both associated with significantly poorer 2‐year survival (P < 0.0001). However, the predictive value of the Ki‐67 proliferative index (97%) was higher than the predictive value of classical histology (91%). The evaluation of the growth fraction by the Ki‐67 proliferative index is highly predictive of the biological behaviour of canine cutaneous melanoma.     

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.     

Overexpression of HER‐2 via immunohistochemistry in canine urinary bladder transitional cell carcinoma ‐ A marker of malignancy and possible therapeutic target

Transitional cell carcinoma (TCC) is the most commonly diagnosed neoplasm in the urinary bladder. Distant metastases to the regional lymph nodes, lungs, abdominal organs or bones are noted in up to 50% of dogs at time of death. Surgical excision is often not practical as TCC typically involve the trigone of the bladder and/or occurs multifocally throughout the bladder with field cancerization. Therapeutic approaches are very challenging and the requirement to evaluate alternative therapeutic protocols that may prolong survival times in dogs bearing these tumours is compelling. We assessed the immunohistochemical expression of HER‐2 in 23 cases of canine TCCs of the urinary bladder and compare it with non‐neoplastic urothelium in order to evaluate a rationale for targeted therapies and gene‐based vaccines. HER‐2 positivity was recorded in 13/23 (56%) neoplastic lesions. The receptor was significantly overexpressed in neoplastic than in non‐neoplastic samples (P = .015). According to our preliminary results, it would be of interest to further evaluate the role of HER‐2 in canine TCCs as a marker of malignancy and a therapeutic target for cancer vaccine and antibodies. Moreover, the significantly different overexpression of HER‐2 in TCCs than in non‐neoplastic urothelium further supports to investigate its role in the progression toward malignancy of non‐neoplastic lesions.     

Two canine Merkel cell tumours: immunoexpression of c‐KIT,E‐cadherin, β‐catenin and S100 protein

Canine Merkel cell tumours are rare neuroendocrine neoplasms that show a relatively benign biological behaviour when compared with their human counterparts. To date, little information is available on their immunohistochemical properties. This report describes the histopathological and immunohistochemical features of two such tumours. The tumours’ immunoreactivity profile was studied with respect to different cellular molecules including chromogranin A (CGA), neurone‐specific enolase (NSE), S100 protein, c‐KIT, the cytokeratins (CKs) detected by pancytokeratin (AE1/AE3) antibodies (i.e. high molecular weight CKs 1, 2, 3, 4, 5, 6, 10, 14, 15 and 16, and low molecular weight CKs 7, 8 and 19) and three markers proposed to correlate with increased malignancy in human tumours: E‐cadherin, β‐catenin and p63 protein. In both lesions, tumour cells were positive for cytokeratins, CGA, NSE, S100 and c‐KIT. No immunostaining was observed for p63 protein, and there was no loss or change in E‐cadherin or β‐catenin immunoexpression. These results suggest that the generally benign behaviour of canine Merkel cell tumours, when compared with their human counterparts, may be partly explained by the conservation of important intercellular adhesion molecules such as E‐cadherin and β‐catenin. Additionally, expression of S100 but not of the p63 protein suggests that these canine tumours present a trend towards neural, rather than basal, epithelial differentiation and do not readily compare with human Merkel cell tumours.     

Influence of E‐cadherin genetic variation in canine mammary tumour risk,clinicopathological features and prognosis

E‐cadherin is a cell adhesion molecule that participates in several cellular processes that guarantee the maintenance of structural and functional integrity of epithelial tissues. E‐cadherin plays an important role in mammary carcinogenesis, and various studies have demonstrated the effect of CDH1 genetic variation in risk, progression and biological behaviour of human breast cancer. Although there are some recognized genetic variations in canine CDH1 gene, their influence in canine mammary tumour development and progression has not been previously evaluated. In this study, we aim to assess the influence of CDH1 SNPs rs850805755, rs852280880 and rs852639930 in the risk, clinicopathological features and clinical outcome of canine mammary tumours. A case‐control study was conducted involving 206 bitches with mammary tumours and 161 bitches free of mammary neoplasia. CDH1 SNPs rs850805755 and rs852280880 were associated with a decreased risk and a later onset of mammary tumour development. Furthermore, these SNPs were related to the development of small size carcinomas, of low histological grade and low nuclear pleomorphism. SNP rs852639930 was associated with the development of small size tumours with a non‐infiltrative, non‐invasive growth pattern. Data from the present investigation demonstrate that these CDH1 genetic variants could have a protective role in canine mammary tumours, by being associated with low risk of tumour development, delayed onset of the disease and less aggressive clinicopathological features.     

Serum and Tissue Steroid Hormone Levels in Canine Mammary Tumours: Clinical and Prognostic Implications

Hormonal dependency of canine mammary tumours (CMT) has been studied over the last few decades. However, studies assessing the prognostic and predictive potential of serum and/or tissue steroid hormone levels are still scarce in CMT. To the best of our knowledge, this is the first report relating serum and tissue levels of steroid hormones and prognosis in dogs. Serum and tumour tissue from 45 female dogs with spontaneous CMT were included in the study. Moreover, serum and normal mammary tissue from 13 healthy female dogs were also included as controls. Steroid hormones were determined by competitive enzyme immunoassay. Overall, levels of steroid hormones in serum and tissue homogenates were significantly different between malignant and benign mammary tumours (p < 0.01), except for progesterone (P4) serum levels that revealed no statistical differences between groups. In malignant tumours, oestrone sulphate (SO4E1), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T) and P4 elevated tissue concentrations were significantly associated with tumour relapse and/or distant metastasis during follow‐up. A significant association was found between elevated tissue SO4E1 (p = 0.003), 17β‐oestradiol (E2) (p = 0.036), DHEA (p = 0.022), A4 (p = 0.001) and P4 (p = 0.013) concentrations and shorter disease‐free survival and overall survival in female dogs with malignant mammary tumours. The high levels of tissue steroids found in cases of poor prognosis open the possibility of additional new therapeutic approaches. Future clinical trials will be needed to clarify the usefulness of targeting steroid hormones in the treatment of this neoplastic disease.     

Expression and Hormonal Regulation of E‐Cadherin in Canine Uterus During Early Pregnancy

E‐cadherin, a Ca^{2 +} ‐dependent cell adhesion molecule, is necessary for endometrial receptivity to blastocyst implantation. The aim of this study was to investigate the differential expression of E‐cadherin in canine uterus during early pregnancy and its regulation under different conditions by in situ hybridization. E‐cadherin mRNA expression was at a low level in the glandular epithelium on days 6, 12 and 17 of pregnancy. On days 20 and 23 of pregnancy, E‐cadherin mRNA was highly expressed in the glandular epithelium surrounding the embryo, but not in the luminal epithelium and declined in villi and placenta on day 28 of pregnancy. During oestrous cycle, a moderate level of E‐cadherin mRNA expression was found in the luminal and glandular epithelium of canine uteri at oestrus stage. The same expression was also found at anoestrus stage. Progesterone slightly induced the expression of E‐cadherin mRNA in the luminal and glandular epithelium of ovariectomized canine uterus. These results suggest that E‐cadherin expression is closely related to canine implantation and can be up‐regulated by progesterone.     

p62/SQSTM1 expression in canine mammary tumours: Evolutionary notes

Recent studies highlighted the role of autophagy as a cardinal regulatory system for homeostasis and cancer‐related signalling pathways. In this context, the deregulated expression of p62 – Sequestosome1 (p62/SQSTM1) – a protein acting both as an autophagy receptor and signalling hub, has been associated with tumour development and chronic inflammation. Multiple clinical studies test drugs targeting autophagy, and even more research is on the way to clinical trials. However, no comparative investigations have been carried out to identify adequate preclinical models to assess p62‐based medicine. In veterinary oncology the role of p62 in cancer‐related pathways has been largely ignored. We compared p62 sequences in multiple organisms and found that canine p62 significantly diverges from the humans and from other animals sequences. Then, we chart by immunohistochemistry the expression levels of p62 in canine mammary tumours. A total of 66 tumours and 10 non‐neoplastic mammary samples were examined. The expression of p62 was higher in normal tissue and adenomas than carcinomas, with lowest levels of p62 protein detected in high grade carcinomas. In all cases examined the tumour stroma appeared to be p62‐negative. Taken together our results would suggest that in dogs the association between p62 expression and cancer cells overturns that reported in human breast carcinoma, where p62 accumulates in malignant cells as compared to normal epithelium. Thus, at least in canine mammary tumours, p62 should be not considered a tumour‐rejection antigen for an anti‐cancer immunotherapy.     

High lysyl oxidase expression is an indicator of poor prognosis in dogs with cutaneous mast cell tumours

Mast cell tumour (MCT) is one of the most frequent skin tumours in dogs. Due to their unpredictable biological behaviour, MCTs often cause several therapeutic frustrations, leading to investigation regarding prognostic markers. Lysyl oxidase (LOX) is an enzyme that promotes extracellular matrix stability and contributes to cell migration, angiogenesis and epithelial-mesenchymal transition. Its expression positively correlates with poor prognoses in several human and canine mammary cancers. The aim of this study was to characterise the immunohistochemical expression of LOX in MCT samples and compare it with histological grading and post-surgical survival. Twenty-six tumours were submitted to immunohistochemistry for LOX expression evaluation. All samples were positive for LOX, with variable percentages of cytoplasmic and nuclear positivity. Cytoplasmic positivity was significantly higher in high-grade MCTs (P = .0297). Our results indicate that high expression of cytoplasmic LOX in neoplastic mast cells is an indicator of poor prognosis for canine cutaneous MCTs.     

Expression of the glutamine metabolism‐related proteins glutaminase 1 and glutamate dehydrogenase in canine mammary tumours

Glutamine metabolism is an important metabolic pathway for cancer cell survival, and there is a critical connection between tumour growth and glutamine metabolism. Because of their similarities, canine mammary carcinomas are useful for studying human breast cancer. Accordingly, we investigated the correlations between the expression of glutamine metabolism‐related proteins and the pathological features of canine mammary tumours. We performed immunohistochemical and western blot analysis of 39 mammary tumour tissues. In immunohistochemical analysis, the expression of glutaminase 1 (GLS1) in the epithelial region increased according to the histological grade (P < .005). In the stromal region, complex‐type tumours displayed significantly higher GLS1 intensity than simple‐type tumours. However, glutamate dehydrogenase expression did not show the same tendencies as GLS1. The western blot results were consistent with the immunohistochemical findings. These results suggest that the expression of GLS1 is correlates with clinicopathological factors in canine mammary tumours and shows a similar pattern to human breast cancer.     

Molecular markers of prognosis in canine cortisol‐secreting adrenocortical tumours

Hypercortisolism is caused by a cortisol‐secreting adrenocortical tumour (ACT) in approximately 15%‐20% of cases in dogs. Little is known about which molecular markers are associated with malignant behaviour of canine ACTs. The objective of this study was to identify molecular markers of prognosis, which could be useful to refine prognostic prediction and to identify potential treatment targets. Cortisol‐secreting ACTs were included from 40 dogs, of which follow‐up information was available. The ACTs were classified as low risk of recurrence tumours (LRT; n = 14) or moderate‐high risk of recurrence tumours (MHRT; n = 26), based on the novel histopathological Utrecht score. Normal adrenals (NAs) were included from 11 healthy dogs as reference material. The mRNA expression of 14 candidate genes was analysed in the 40 ACTs and in 11 NAs with quantitative RT‐PCR. The genes' expression levels were statistically compared between NAs, LRTs and MHRTs. Univariate and multivariate analyses were performed to determine the association of the genes' expression levels with survival. Seven genes were differentially expressed between NAs and ACTs, of which pituitary tumour‐transforming gene‐1 (PTTG1) and topoisomerase II alpha (TOP2A) were also differentially expressed between LRTs and MHRTs. In survival analyses, high expression levels of Steroidogenic factor‐1 (SF‐1), PTTG1 and TOP2A were significantly associated with poor survival. In conclusion, we have identified several genes that are part of the molecular signature of malignancy in canine ACTs. These findings can be used to refine prognostic prediction, but also offer insights for future studies on druggable targets.     

PCNA and grade in 13 canine oral squamous cell carcinomas: association with prognosis

This study evaluated the prognosis factors of age, tumour size, anatomic location, histological grade and proliferating cell nuclear antigen (PCNA) expression in 13 dogs with oral squamous cell carcinoma (OSCC) with bone invasion and without signs of lymph node or distant metastasis. All animals were treated with radical excision performed with at least 1 cm margin, based on computed tomography images. In the 2‐year follow‐up, median disease‐free survival was 138 days for dogs with grade 3 tumours and was not reached for those with grade 2 tumours. Grade 3 tumours and PCNA labelling index ≥65% were related with a shorter disease‐free survival time and consequently poor prognosis (p = 0.003 and p = 0.034, respectively). Mean PCNA labelling index was significantly higher in recurrent cases (p = 0.011). Histological grade and PCNA expression may be important prognosis factors in canine OSCC.     

Immunohistochemical evaluation of MMP-2 and TIMP-2 in canine mammary tumours: a survival study

Canine mammary tumours (CMTs) are a very heterogeneous group of neoplasms with variable prognosis. Their aggressiveness is mainly due to their ability to invade locally and to metastasize. The degradation of extracellular matrix components is an important determinant of the invasive phenotype. The aims of this study were to analyse by immunohistochemistry and double immunofluorescence the expression of metalloproteinase 2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in eight normal canine mammary glands and 118 CMTs (24 benign, 94 malignant) and to investigate relationships with metastatic disease and survival.MMP-2 and TIMP-2 expression was higher in malignant tumours than in normal canine mammary tissue and benign tumours. The main difference between benign and malignant CMTs was the pattern of expression of both molecules: benign tumours presented TIMP-2 and MMP-2 immunoreactivity in the myoepithelial cells lining the basement membrane of tubuloalveolar structures, while malignant tumours showed mainly diffuse expression in neoplastic cells. In malignant tumours, increased TIMP-2 expression was significantly associated with the development of distant metastases, lower overall survival and lower disease-free survival. MMP-2 expression was not significantly associated to any of these parameters. These results suggest that the immunohistochemical expression of TIMP-2 is a useful prognostic factor in CMTs.     

Apoptotic intrinsic pathway proteins predict survival in canine cutaneous mast cell tumours

Mast cell tumours (MCTs) are the most frequent canine round cell neoplasms and show variable biological behaviours with high metastatic and recurrence rates. The disease is treated surgically and wide margins are recommended. Adjuvant chemotherapy and radiotherapy used in this disease cause DNA damage in neoplastic cells, which is aimed to induce apoptotic cell death. Resisting cell death is a hallmark of cancer, which contributes to the development and progression of tumours. The aim of this study was to investigate the expression of the proteins involved in the apoptotic intrinsic pathway and to evaluate their potential use as prognostic markers for canine cutaneous MCTs. Immunohistochemistry for BAX, BCL2, APAF1, Caspase‐9, and Caspase‐3 was performed in 50 canine cases of MCTs. High BAX expression was associated with higher mortality rate and shorter survival. BCL2 and APAF1 expressions offered additional prognostic information to the histopathological grading systems. The present results indicate that variations in the expression of apoptotic proteins are related to malignancy of cutaneous MCTs in dogs.     

Expression of oestrogen and progesterone receptors in canine sebaceous gland tumours

Sebaceous gland oestrogen α (ERα) and progesterone (PR) receptor expression was examined immunohistochemically in 26 and 32 dogs respectively with sebaceous gland hyperplasia/adenomas, epitheliomas and carcinomas, and in the glands of 10 healthy controls. The mean percentage of ERα positive nuclei in control sebaceous glands was 21.31% compared with 11.5% in hyperplasia/adenoma‐type lesions, although these values were not statistically different. In sebaceous gland epitheliomas and carcinomas, positive basal cells represented 7.86% and 3.53% of neoplastic cells respectively and these mean percentages were significantly lower in epitheliomas (P < 0.024) and carcinomas (P < 0.015) than in controls. The mean percentage of PR‐positive nuclei in control sebaceous glands was 23.96%, similar to the 22.07% found in hyperplasia/adenoma‐type lesions. In sebaceous gland epitheliomas and carcinomas, positive cells were scarce and represented 13.5% and 4.06% of neoplastic cells respectively. Differences in the percentage of positive cells between normal and pathological glands reached statistical significance for carcinomas (P < 0.043). In the control group there was greater PR (P < 0.001) and ERα expression (P < 0.014) in sebaceous glands in female dogs. The PR and ERα immunoreactivity in each category of neoplastic lesions could not be analysed because sample size was too small but when all the sebaceous gland tumours were grouped and analysed, no sex difference was found. The results suggest that oestrogen and progesterone receptor expression is reduced in some canine sebaceous gland tumours. These changes may represent a contributing factor for tumour growth or simply be a consequence of tumour progression.     

Expression of epidermal growth factor receptor and vascular endothelial growth factor in malignant canine epithelial nasal tumours*

Epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) signalling pathways play a role in carcinogenesis. Inhibition of EGF receptor (EGFR) and of VEGF is effective in increasing the radiation responsiveness of neoplastic cells both in vitro and in human trials. In this study, immunohistochemical evaluation was employed to determine and characterize the potential protein expression levels and patterns of EGFR and VEGF in a variety of canine malignant epithelial nasal tumours. Of 24 malignant canine nasal tumours, 13 (54.2%) were positive for EGFR staining and 22 (91.7%) were positive for VEGF staining. The intensity and percentage of immunohistochemically positive neoplastic cells for EGFR varied. These findings indicate that EGFR and VEGF proteins were present in some malignant epithelial nasal tumours in the dogs, and therefore, it may be beneficial to treat canine patients with tumours that overexpress EGFR and VEGF with specific inhibitors in conjunction with radiation.