Relationship between receptors for epidermal growth factor and steroid hormones in normal, dysplastic and neoplastic canine mammary tissues

The concentrations of receptors for epidermal growth factor (EGF-R), oestrogen (ER) and progesterone (PR) were measured in 108 samples from canine mammary tumours and 132 samples of normal mammary tissue removed surgically from 84 bitches. The history and clinical signs were also recorded. Binding sites of high affinity were detected in 70 per cent of both types of tissue and no significant variations in EGF-R concentrations or positivity were observed with the histology, location, size or number of mammary tumours or the age of the animal. A significant direct correlation (P=0002) was observed between the concentrations of ER and EGF-R only in malignant tumours. The concentrations of EGF-R were significantly correlated (P=0.04) in normal mammary tissues adjacent to and distant from the lesions, but not between normal tissue and tumour tissue. No significant differences were observed in the expression of EGF-R in normal and neoplastic tissues from the same bitches. The direct correlation between the concentrations of EGF-R and ER in malignant tumours could be related to an oestrogen-dependent expression of EGF-R or to a similar pattern of regulation of the receptors.     

Oxidative stress parameters in bitches with mammary gland tumours

The aim of the present study was to describe some of the oxidative stress parameters in bitches suffering from spontaneously occurring mammary gland tumours. The experiment involved 28 bitches which had mammary gland tumours removed surgically (15 bitches with malignant tumour and 13 with benign tumour) as well as 10 clinically healthy bitches. The activities of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were determined in haemolysates of erythrocytes derived from the animals. The concentrations of thiobarbituric acid reactive substances (TBARS), as well as -SH groups, were determined in blood plasma. GSH-Px activity was significantly higher in the malignant tumour group than in healthy animals. SOD activity was significantly higher in animals with tumours compared with the control group. Activities of both enzymes were higher in animals with malignant tumours than in benign groups, but the differences were not statistically significant. The concentrations of TBARS and -SH groups were similar in all examined groups. The increase of antioxidative enzyme activities in these animals may suggest the activation of antioxidative defence mechanisms in mammary gland carcinogenesis. Moreover, it might indicate the participation of oxidative stress in malignancies. Further experiments involving more animals, with more frequent sample collection and the use of other oxidative stress markers are necessary.     

Quantification of epidermal growth factor receptor (EGFR) in canine mammary tumours by ELISA assay: clinical and prognostic implications

The involvement of epidermal growth factor receptor (EGFR) is well established in human breast cancer, however, in canine mammary tumours (CMT), including inflammatory mammary carcinomas (IMC), still needs to be clarified. Enzyme immune assay techniques were used for EGFR determinations in tumour tissue from 45 bitches with CMT and in normal mammary glands from eight control dogs. Higher tissue EGFR levels were found in CMT compared with controls (P < 0.05). In malignant CMT, tissue EGFR elevated concentrations were statistically significantly associated with tumour relapse and/or distant metastasis during follow‐up and with reduced disease‐free and overall survival times. The IMC cases had the highest tissue EGFR levels compared with other malignant non‐IMC tumours (P < 0.001). The results support the hypothesis that EGFR levels influence prognosis in malignant CMT, suggesting that EGFR may represent a therapeutic target in cases of high histological aggressiveness and especially in cases of metastatic phenotype and poor prognosis.     

Influence of ovariectomy at the time of mastectomy on the prognosis for canine malignant mammary tumours

The two-year prognosis for malignant mammary tumours seen in 175 bitches in the Tokyo metropolitan area was assessed based on the extent of mastectomy and on whether an ovariectomy was carried out at the time of mastectomy. The prognosis for the bitches was not influenced by the excision size of the affected mammary glands. Ovariectomy had no effect on the two-year survival rate of the dogs that underwent the tumour excision. There was no significant difference in the two-year survival rate between the dogs ovariectomised prior to mastectomy and those ovariectomised at the time of mastectomy. These results suggest that ovariectomy at the time of mastectomy has no effect on the prognosis in dogs with established neoplasms of the mammary gland.     

Clinical value of carcinoembryonic antigen in mammary neoplasms of bitches

This study aimed at evaluating the behaviour and understanding the diagnostic value of the carcinoembryonic antigen (CEA) in bitches with mammary carcinoma as a tool for monitoring and prognosis of canine cancer patients. Serum samples from 77 bitches were divided into four groups, G1 (n = 21), control group (healthy/neoplasia free bitches); G2 (n = 31), bitches with non‐metastatic mammary carcinoma less than 3 cm; G3 (n = 12), bitches with non‐metastatic mammary carcinoma greater than 3 cm; and, G4 (n = 13) bitches with mammary carcinoma and lymph node metastasis. The marker was dosed once in G1, whereas in G2, G3 and G4, CEA levels were determined before (M0) and 15 days after (M1) mastectomy, using the ELISA kit for humans while reading used ELISYS ONE human. A group of 11 bitches was followed up 45 days after mastectomy (M2). The results for the concentration of markers in blood serum samples at the evaluated times and their relationship with neoplasia biological behaviour and observed clinicopathological changes were evaluated by the Tukey test at 5% significance. The ROC curve was established to find the cut‐off value and calculate the test sensitivity and specificity, the multivariate matching analysis was performed to confirm the association between CEA values and clinicopathological variables. CEA values increased significantly in bitches with mammary carcinoma, metastatic tumours with a diameter larger than 3.0 cm and high grade, compared with healthy ones. In addition, mastectomy reduced the CEA concentration in the blood (P < .05) whereas high CEA levels were associated with unfavourable prognostic factors (P < .05). The biomarker presented good diagnostic value, especially for more aggressive tumours. In conclusion, CEA serum concentrations allowed to follow efficiently the evolution of mammary tumours in bitches, since CEA values increased in bitches with mammary gland tumour and decreased after mastectomy while correlating with prognostic factors such as tumour size, nodal metastasis and histological grade. Further studies are still needed to confirm its diagnostic value for follow‐up of relapse and early metastasis.     

Hysterectomy leads to fast improvement of haematological and immunological parameters in bitches with pyometra

OBJECTIVES: To investigate whether a combination of hysterectomy and antibiotic treatment leads to an improvement of altered haematological and immunological parameters in bitches affected by pyometra. METHODS: Blood samples obtained from 13 bitches affected by pyometra were investigated before hysterectomy and seven days later for a total and differential leucocyte count, activity of neutrophils and lymphocytes, and quantification of total serum immunoglobulins, lysozyme and circulating immune complexes. RESULTS: The parameters most affected included changes in blood profile (leucocytosis because of neutrophilia or leucopenia) and inhibition of lymphocyte activity. Seven days after hysterectomy, all affected parameters returned to normal levels comparable to clinically healthy dogs. CLINICAL SIGNIFICANCE: Application of immunostimulating therapy is not necessary to manage changes in blood cell counts and suppression of lymphocyte activity in bitches with pyometra. Removal of the infected uterus, being the source of infection, leads to improvement.     

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.     

Studies on canine mammary tumours. I. Age, seasonal and breed distribution.

The incidence as well as age, seasonal and breed distribution of canine mammary tumours (n = 521) were studied at the Clinic of Obstetrics and Gynaecology of the University of Veterinary Science, Budapest, between 1985 and 1989. In 39 cases of mammary tumour, blood plasma oestradiol (E2) and progesterone (P) concentrations were also determined. Of all dogs referred to the clinics of the University in 1985, 0.7% had mammary tumour. On the average, 104 +/- 9.3 cases of mammary tumour were recorded at the Clinic of Obstetrics per year. This number did not increase after the Chernobyl atomic reactor catastrophe of 1986. The age distribution of canine mammary tumour found in this study shows good agreement with earlier data of the literature: mammary tumour showed the highest incidence in 10 years old dogs. The incidence of mammary tumour kept increasing with age until the 14th year of life (as expressed in per cent of animals of identical age). The number of mammary tumours was markedly higher in the spring (April-May) and autumn (September). This seasonality was demonstrable in 11 to 16 years old bitches, too. On the basis of the blood plasma E2 and P profiles, 61.5% of the clinically anoestrous animals were found to be cycling. The strikingly high ratio of pulis among dogs with mammary cancer was suggestive of a breed disposition.     

Ultrastrukturelle und ultrahistochemische Untersuchungen an der Milchdrüse des Hundes: II. Ultrahistochemische Untersuchungen

Ultrastructural and ultrahistochemical examinations on the canine mammary gland II. Ultrahistochemical examinations Mammary glands of mostly oldder bitches were examined ultrahistochemically for the distribution of ATPase, alkaline phosphatase, and acid phosphatase. The reactions for ATPase were found regularly in the walls of the blood vessels and often on the plasma membranes of the myoepithelial and epithelial cells. However, plasma membranes adjacent to lumen and basement membrance did not show the reaction. Alkaline phosphatase was found regularly only on the plasma membranes of the myoepithelial cells. Acid phosphatase was found in the lysosomes of myoepithelial and epithelial cells, and increased in amount during the regression of the gland.     

The Effect of the Bacteriological Status of Feed on Some Haematological and Blood Chemical Data on Mink

Blood samples from 100 minks from a research farm using bacteriologically high quality feed and from 55 minks from another farm supplying bacteriologically inferior feed, as well as from nine minks from Denmark from two farms providing still better quality feed than both Finnish farms – all minks apparently clinically healthy – were analyzed for some haematological and chemical data: total leucocyte count, haemoglobin, ornithine carba-moyltransferase (OCT), alkaline phosphatase (AP), alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), γ-glutamyl-transferase (Γ-GT), total bilirubin and creatinine. The Finnish minks supplied with high quality feed had more optimal values of total leucocytes, haemoglobin, OCT, AP and creatinine than minks receiving feed with higher bacterial contamination. The Danish minks had better blood values in the investigated parameters except for lower haemoglobin and total bilirubin, which showed no significant difference.     

Prognostic significance of serum alkaline phosphatase activity in canine appendicular osteosarcoma

Sixty-one dogs with appendicular osteosarcoma were treated with amputation and chemotherapy of cisplatin and doxorubicin. Serum samples were obtained before and after treatment for determination of total alkaline phosphatase (TALP) activity as well as the activities of the constituent bone (BALP), liver (LALP), and corticosteroid-induced (CALP) isoenzymes. The relationship between alkaline phosphatase activities and survival was examined by Cox proportional hazards regression analysis and Kaplan-Meier log rank analysis. Mean activity of TALP, BALP, and LALP decreased significantly after treatment (P < .001). TALP and LALP activities before treatment were significantly correlated with survival (P = .006 and .001, respectively). The correlation between BALP activity before treatment and survival approached significance (P = .054). CALP activity and TALP, BALP, and LALP activities after treatment were not significantly correlated with survival. Dogs with normal pretreatment TALP and BALP activities survived significantly longer than dogs with increased pretreatment activities (P = .001 and .003, respectively). Median survival times for dogs with normal or increased TALP activities before treatment were 12.5 and 5.5 months, respectively; and median survival times for dogs with normal or increased BALP activities before treatment were 16.6 and 9.5 months, respectively. In the design of future clinical trials involving dogs with osteosarcoma, consideration should be given to stratifying the randomization according to alkaline phosphatase activity. In addition, alkaline phosphatase activity should be a factor considered by clinicians attempting to tailor the aggressiveness of adjuvant chemotherapy to the needs of individual patients or owners.     

Correlation between histologic diagnosis mean nucleolar organizer region count and prognosis in canine mammary tumors.

In this study, surgically excised mammary tumors from 98 bitches were graded histologically, and the grade was compared with the mean nucleolar organizer region (NOR) count in silver-stained paraffin-embedded sections. Histologically benign tumors, papillary adenocarcinomas, and intraductal carcinomas showed relatively little variation; the mean count for each category was between three and four NOR per nucleus. There was, however, a significant increase in the NOR counts in tubular and solid carcinomas. This increase was most pronounced for tumors that showed evidence of infiltration into the surrounding connective tissues. The mean NOR count for noninfiltrative carcinomas was 5.1, and that for invasive carcinomas was 7.3 (P less than 0.03). The mean NOR count for individual carcinomas ranged from 2.0 to 12.3, and a significant correlation was found between an increased NOR count and tumor-related death during the first post-surgical year. The 39 bitches in which the tumor had an NOR count less than 8.0 had a generally favorable prognosis; only six (15%) died as a result of the original neoplasm. In contrast, 18/21 dogs (85%) with a carcinoma having an NOR count greater than 8.0 died from the tumor during the first post-surgical year. A similar, although less pronounced result was obtained specifically for invasive carcinomas, in which 3/12 (25%) tumors with an NOR count less than 6.0 resulted in the death of the host, compared with 17/20 (85%) that had an NOR count greater than 6. By using this technique, it is possible to identify a subgroup of bitches with invasive mammary carcinomas that have a very poor prognosis following apparently adequate surgical ablation of the primary tumor.     

Changes in blood cellular components, serum proteins and serum enzyme activities in pigs naturally infected with Cysticercus tenuicollis

A study was made of blood cellular components, serum proteins and serum enzymes in 48 pigs naturally infected with Cysticercus tenuicollis. Twenty-five healthy pigs were studied for comparison. Affected animals showed a reduction in total erythrocyte count, packed cell volume and haemoglobin content and an increase in mean corpuscular volume and total leucocyte count. Significantly higher activities of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and isocitric dehydrogenase serum enzymes were recorded in all affected pigs. Total protein, albumin and globulin values of affected pigs remained unchanged when compared with healthy controls.     

Perioperative desmopressin prolongs survival in surgically treated bitches with mammary gland tumours: a pilot study

Desmopressin (1-deamino-8-d-arginine vasopressin, also known as DDAVP) is a safe haemostatic compound capable of inhibiting lymph node and lung metastasis in a mouse model of mammary tumour manipulation and surgical excision. The aim of this study was to test the efficacy and safety of perioperative DDAVP (1microg/kg) in surgically treated bitches with mammary gland tumours (MGT). Twenty-one, otherwise healthy, intact bitches, with malignant MGT stage III or IV were randomly allocated to DDAVP (n=11) or placebo (n=10) groups. En bloc mastectomy of the affected gland/s was performed. DDAVP had a significant beneficial effect on disease-free period (P<0.01) and overall survival time (P<0.05). No side effects were seen in any of the cases. Whatever the mechanism of action, it seems that DDAVP may have a novel use in cancer surgery to minimise spread or survival of residual malignant cells. Additional, large scale controlled trials are required to fully evaluate this adjuvant pharmacological protocol.     

Immunohistologic detection of estrogen receptor alpha in canine mammary tumors: clinical and pathologic associations and prognostic significance

Eighty-nine canine mammary tumors and dysplasias of 66 bitches were investigated to determine the immunohistochemical expression of classical estrogen receptor (ER-alpha) and its clinical and pathologic associations and prognostic value. A complete clinical examination was performed and reproductive history was evaluated. After surgery, all animals were followed-up for 18 months, with clinical examinations every 3-4 months. ER-alpha expression was higher in tumors of genitally intact and young bitches (P < 0.01, P < 0.01) and in animals with regular estrous periods (P = 0.03). Malignant tumors of the bitches with a previous clinical history of pseudopregnancy expressed significantly more ER-alpha (P = 0.04). Immunoexpression of ER-alpha decreased significantly with tumor size (P = 0.05) and skin ulceration (P = 0.01). Low levels of ER-alpha were significantly associated with lymph node involvement (P < 0.01). Malignant tumors had lower ER-alpha expression than did benign tumors (P < 0.01). Proliferation index measured by proliferating cell nuclear antigen immunostaining was inversely correlated with ER-alpha scores (P = 0.05) in all tumors. Low ER-alpha levels in primary malignant tumors were significantly associated with the occurrence of metastases in the follow-up (P = 0.03). Multivariate analyses were performed to determine the prognostic significance of some follow-up variables. ER-alpha value, Ki-67 index, and age were independent factors that could predict disease-free survival. Lymph node status, age, and ER-alpha index were independent prognostic factors for the overall survival. The immunohistochemical detection of ER-alpha in canine mammary tumors is a simple technique with prognostic value that could be useful in selecting appropriate hormonal therapy.     

ULTRASONOGRAPHIC DESCRIPTION OF CANINE MASTITIS

Ultrasonographic images were acquired of the mammary glands of 40 bitches with physiologically lactating (n = 20) or inflamed glands (n = 20). Echogenicity, structure, homogeneity, thickness, and distinguishability of each tissue layer were assessed. Additionally, overall echogenicity was noted. In the normal lactating gland, different tissues could be differentiated easily. The parenchyma was, without exception, separated from adjacent tissues and was visible as medium echogenic tissue with a coarse-grained structure. The tissue always had some echogenic lines and anechoic areas and was slightly heterogeneous. The loss of distinct layering of the tissue was characteristic of an inflamed mammary gland and inflamed regions had reduced echogenicity. Additionally in five bitches with mastitis, the ultrasound examination was repeated five times for documentation of the progress of the illness and associated changes, supplemented with a color Doppler sonogram to assess changes in blood vessel density. Information from the examinations carried out via B-mode did not allow treatment success to be predicted. Two bitches with reduced blood vessel density centrally had a poor outcome whereas three bitches with increased blood vessel density had a good outcome. Thus, Doppler sonography might be a useful tool to obtain information of the prognosis in acute canine mastitis.     

Age‐Related Pregnancy Results and Further Examination of Bitches after Aglepristone Treatment of Pyometra

The cystic endometrial hyperplasia and pyometra complex is one of the most common uterine diseases in bitches. The appearance of pharmacological preparations containing anti‐progestagens created new possibilities for pyometra treatment. The aim of this study was to evaluate the curative effect of the anti‐progestagen aglepristone treatment of pyometra in bitches of different ages. Twenty four bitches of different breeds, aged from 0.8 to 9.5 years (21–48 kg) exhibiting clinical pyometra symptoms (two groups – I ≤ 5 years, n = 14 and II >5 years, n = 10) were evaluated. Information about the general reproductive health was collected up to 54 months after anti‐progestagen treatment. Remission of clinical symptoms and return of blood chemistry results and total leucocyte count to referential values were achieved in all cases within 14 days of treatment. Bitches were naturally mated at the first, and when unsuccessful, the second oestrus after treatment. In group I, no recurrence of pyometra symptoms was observed during following cycle(s). Eight bitches (57.1%) had a full‐term pregnancy and the number of newborn pups ranged from 1 to 12. None of the bitches from the group II became pregnant. In conclusion, the basic indication for conservative pharmacological treatment of pyometra is preserving female fertility and obtaining offspring. The important conditions for successful aglepristone treatment are: the young age (up to 5 years) and the lack of detectible ovarian cysts. It seems necessary to mate bitches in the first or second oestrus after finishing treatment. The efficacy of treatment can be measured by the after‐treatment pregnancy rate.     

Immunohistochemical identification method of tumour cells in the S phase of mitotic cycle and its usefulness in diagnostics of mammary gland adenocarcinomas in bitches

The studies aimed at identification of neoplastic cells at the S phase of mitotic cycle in mammary gland adenocarcinomas of bitches. The material was sampled from bitches of various races, aging 6 to 12 years, in which the mammary gland tumours developed spontaneously. The tumours were verified histopathologically and, then, immunohistochemical reactions were performed in order to detect cells which had incorporated BrdU (bromodeoxyuridine), contained Ki-67 or PCNA antigen. The histological preparations were photographed and obtained pictures were subjected to computer-assisted image analysis using Axiophot microscope (Carl Zeiss) coupled to a computer and the Multi-ScaneBase V 8.08 software, working under Windows. Fifty percent of sections from mammary gland adenocarcinomas demonstrated BrdU labelling index of 4-5%, 40% of 1-3%, while in the remaining 10% of examined tumours no BrdU incorporation could be demonstrated. No evident relationship could be detected between the presence of BrdU incorporation and Ki-67 or PCNA antigen presence but a significant correlation was demonstrated between the expression of Ki-67 and PCNA.     

Expression and significance of PTEN in canine mammary gland tumours

To explore the expression and clinical importance of the anti-oncogene phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in canine mammary gland tumours, PTEN expression was compared in 50 cases of canine mammary tumour and four examples of normal mammary tissue using real-time quantitative PCR. PTEN expression was similar in benign mammary tumours and normal mammary tissues (P>0.05), but was lower in malignant tumours than in normal mammary tissues or benign mammary tumours (P<0.001). PTEN expression was also low in the lymph node metastases of malignant mammary tumours. The expression profile of PTEN in malignant mammary tumours compared to those without lymph node metastasis varied significantly. Low-level PETN expression might play an important role in carcinogenesis and the progression of canine mammary tumours, and PTEN protein detection might be useful in evaluating tumour development and prognosis.     

Of humans and canines: Immunohistochemical analysis of PCNA, Bcl-2, p53, cytokeratin and ER in mammary tumours

Mammary tumours are the most common neoplasms in humans and canines. Human and canine mammary tumours share several important epidemiological, clinicopathological and biochemical features. Development of mammary tumours involves accumulation of mutant cells caused by excessive proliferation and insufficient apoptosis or dysregulation of cellular differentiation. The present study was therefore designed to investigate the expression of proliferation, differentiation, and apoptosis associated proteins together with expression of estrogen receptors (ER) in both human and canine mammary tumours. Thirty breast cancer patients categorized as pre- and postmenopausal, and 30 mammary gland tumours obtained from bitches were included in this study. The expression of proliferating cell nuclear antigen (PCNA), Bcl-2, p53, cytokeratin and ER in tumour tissues and adjacent tissues were investigated using immunohistochemical staining. While the expression of PCNA, Bcl-2, p53 and ER was significantly increased, expression of cytokeratin was significantly lower in both human as well as canine mammary tumours compared to corresponding adjacent tissues. The magnitude of the changes was however more pronounced in premenopausal patients compared to postmenopausal patients. The changes in proliferation, apoptosis and differentiation associated proteins in human and canine mammary tumours validate use of the canine model to understand the molecular mechanisms of mammary carcinogenesis.