Serum neopterin, sialic acid and nitric oxide levels in dogs with malignant mammary tumours

Mammary cancer is one of the leading causes of death in pet population. Early diagnosis and malignancy detection is important for prognosis. The levels of neopterin, sialic acid and nitric oxide in serum of dogs with malignant mammary tumours were evaluated to investigate the importance of these biochemical parameters for malign mammary tumour. Twelve healthy dogs and twenty dogs with malignant mammary tumours were used as research materials. Blood samples were collected from both groups for neopterin analysis by enzyme-linked immunosorbent assay, whereas nitric oxide and sialic acid were measured by modified nitrate reductase method and spectrophotometry, respectively. Tissue specimens were evaluated and defined as malignant tumours. Serum nitric oxide and sialic acid levels in dogs with mammary tumours were significantly higher than those in the healthy dogs. Serum neopterin levels were not found significantly different in dogs with mammary tumours compared to healthy dogs. Malignancy of canine mammary tumours are accompained by an elevation of nitric oxide and sialic acid levels.     

A Statistical Study on the Tumours of Dogs

The author presents a statistical investigation of tumours from 164 dogs examined at the Department of Pathology of the College of Veterinary Medicine, Helsinki, Finland.It was found that Boxers and Airedale terriers were predisposed to tumours and especially to cutaneous neoplasms. The dominance of mammary tumours in Dachshunds was statistically highly significant. A statistically highly significant under-representation of Finnish harriers and Finnish spitzes was noted.Sex distribution was female-dominated, obviously owing to high incidence of mammary tumours.It was shown that tumours, especially malignant neoplasms, were primarily found in the high age groups.No difference was observed in the frequency of epithelial and mesenchymal tumours. Malignant tumours were strongly dominant, obviously due to common incidence of carcinoma. The carcinoma/sarcoma ratio was 1.5:1.Analysis of the localization showed that cutaneous tumours (37.0 %) and mammary tumours of females (19.1 %) were most common.Nine dogs showed 18 multiple primary tumours. The high frequency of these tumours in older dogs was obvious. Their mean age was 10 ± 0.9 year.     

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.     

Plasma free amino acid profiles of canine mammary gland tumors

The purpose of this study was to elucidate the relationship between plasma free amino acid (PFAA) levels and the clinical stages of mammary gland tumors (MGT) in dogs. PFAA levels in canines with malignant mammary tumors were decreased compared to those of healthy animals. The levels of aspartate and ornithine, in the dogs with tumor metastasis were significantly decreased when compared to those of dogs that did not have metastases. Results of this study indicate that PFAA levels could be a risk factor or biomarker for canine MGT metastasis.     

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.     

Ovarian function suppression with a GnRH analogue: D-ser(But[t])[6]-Arzgly[10]-LHRH (Goserelin) in hormone dependent canine mammary cancer

Hormones and hormone level modifying substances have long been used to treat hormone-dependent tumours in humans. Recently, attempts have been made to use hormone manipulation regimens for the treatment of these tumours in veterinary medicine. The aim of the present study was to evaluate the activity of the luteinizing hormone-releasing hormone (LHRH)-agonist, D-ser(But[t])[6]-Azgly[10]-LHRH (Goserelin) in hormone-dependent mammary cancer in dogs. Eighteen female dogs with hormone-dependent mammary cancer (T2-T4, N0, M0 according to TNM clinical staging classification) were selected and allocated into two groups: nine dogs not treated with Goserelin (Group 1) referred to as control; and nine dogs treated with 60 microg/kg depot Goserelin every 21 days for 12 months (Group 2). Goserelin treatment decreased circulating levels of oestradiol and progesterone and reduced the size of mammary tumours; all the animals showed objective response (OR) to treatment after 3 months, and the relapse-free survival after 2 years was 88%. Haematology and blood chemistry parameters, measured every month from the beginning of treatment, as well as physical examination, showed that the drug was without toxic effects. This suggests that, at the dose administered, Goserelin blocks the hypothalamus-pituitary-ovary axis, and consequently can be useful to treat hormone-dependent mammary tumours in female dogs.     

The relationship between tumour size and expression of prognostic markers in benign and malignant canine mammary tumours

Tumour size is considered one of the most important determinants of clinical staging in cancer patients. The aim of this study was to assess the value of tumour size as an indicator of the differentiation of mammary neoplasias in female dogs. The tumour, nodes metastates (TNM) system, based on primary lesion size, the extent of its dissemination to regional lymph nodes and the presence or absence of distant metastases, was applied to 120 female dogs diagnosed with mammary neoplasias. Paraffin blocks from 38 cases were selected and studied by immunohistochemical staining for prognostic and predictive markers of breast cancer. The Kaplan–Meier survival curve was estimated for 110 female dogs. Larger tumours (T3) were mostly malignant and showed lower expression of progesterone receptor and higher expression of cellular proliferation markers. Global survival time was shorter in female dogs with large tumour masses. This study highlights the importance of tumour size as a prognostic indicator of mammary neoplasias in female dogs.     

Canine mammary gland tumours; a histological continuum from benign to malignant; clinical and histopathological evidence*

This study describes the clinical and histopathological findings in dogs with mammary gland tumours, and compares the histopathological and clinical evidence consistent with progression from benign to malignant to human breast cancer epidemiology. Clinical and histopathological data on 90 female dogs with 236 tumours was included. Dogs with malignant tumours were significantly older than dogs with benign tumours (9.5 versus 8.5 years), P = 0.009. Malignant tumours were significantly larger than benign tumours (4.7 versus 2.1 cm), P = 0.0002. Sixty‐six percent had more than one tumour, and evidence of histological progression was noted with increasing tumour size. Dogs with malignant tumours were significantly more likely to develop new primary tumours than dogs with benign tumours, P = 0.015. These findings suggest that canine mammary tumours progress from benign to malignant; malignant tumours may be the end stage of a histological continuum with clinical and histopathological similarities to human breast carcinogenesis.     

A statistical assessment of the biological relationship between simultaneous canine mammary tumours

Simultaneous canine mammary tumours (CMTs) are frequently reported in the literature, but few studies have addressed their biological relationship in detail or performed statistical assessments. In this study, 269 canine mammary gland tumours from 216 dogs were categorized using an extended histopathological classification, where semiquantitative and binomial scales enumerated morphological parameters of the tumours. The classification facilitated a statistical study of the biological relationship between simultaneous within‐dog tumours. Seventy‐seven percent of the dogs had single tumours and 23% had simultaneous tumours. Sixty‐one percent of the neoplasias were benign, with complex adenoma as the most frequent diagnosis and 39% were malignant, with complex carcinoma as the most common malignancy. Simultaneous tumours within dogs more often had equal diagnoses and neoplastic level (benign or malignant) than would be expected by chance alone, as compared with random pairs of single tumours from different dogs. This statistically supported finding indicated the presence of a biological relationship between simultaneous tumours.     

Expression of TopBP1 in canine mammary neoplasia in relation to histological type, Ki67, ERalpha and p53

TopBP1 is aberrantly expressed in human and feline mammary carcinomas, but expression of this BRCA1-related protein has not been investigated in canine mammary carcinomas. In this study, 132 canine mammary tumours (46 benign, 86 carcinomas) were examined immunohistochemically for expression of TopBP1, oestrogen receptor α (ERα), Ki67 and p53. Positive staining for TopBP1 was evident in all canine mammary lesions, although five samples had <20% positive cells. The number of samples with high levels of staining increased in different categories from benign mixed tumour to adenoma to carcinoma. Most TopBP1 staining was nuclear, but both nuclear and cytoplasmic staining were observed as the degree of malignancy increased, similar to human and feline mammary carcinomas. Benign mixed tumours, however, had more cytoplasmic staining than adenomas. Expression of p53 and the proliferation marker Ki67 increased from benign mixed tumour to adenoma to carcinoma, but the differences between benign and malignant tumours were more distinct than for TopBP1 expression. ERα expression decreased from malignant to benign tumours, although over half of the benign mixed tumours were negative. TopBP1 was expressed in canine mammary tumours at higher levels than has been reported previously for cats, although the shift in cellular localisation with malignancy was similar.     

Effects of different liver diseases on the platelet count in dogs

Changes of the platelet count in liver diseases are described in humans. Thrombocytopenia was observed more frequently than thrombocytosis. There are only a few investigations on platelet counts in liver diseases in dogs. The goal of the present study was to investigate the influence of different liver diseases including degeneration, hepatitis and liver tumours, on the platelet count. Platelet counts of 52 dogs with different liver diseases were measured and compared with 52 healthy dogs. The results showed, that dogs with liver degeneration have thrombocytosis in 41% of the cases and a group of dogs with liver tumours (malignant histiocytosis, hepatoma, malignant lymphoma anaplastic sarcoma, cholangiocarcinoma, hepatocellular carcinoma) had thrombocytopenia in 50% of the cases. The dogs with hepatitis showed no specific changes in the platelet count. The statistical comparison of our patients with liver disease and a control group of healthy dogs showed significantly higher platelet counts in cases of liver degeneration (p < 0.0001) and significantly lower platelet counts in cases of liver tumour (p < 0.001). The comparison between the dogs with different liver diseases showed significantly lower platelet counts in dogs with liver tumours when compared to dogs with liver degeneration (p < 0.0001). There was no significant difference between dogs with liver tumours and dogs with hepatitis and between dogs with liver degeneration and dogs with hepatitis. Based on the results of this study the author recommends to assess platelet counts in all dogs with liver disease, especially if liver biopsy is planed.     

Routine hematology and clinical chemistry in female dogs with benign and malignant mammary tumors

Routine hematological and serum biochemical screening was done in 61 female dogs with benign mammary tumors and 51 female dogs with malignant mammary tumors. Most parameters were not significantly different from age-matched female controls; moreover, no significant difference could be observed between animals with benign and malignant tumors. It is concluded that routine hematology and biochemistry offer little diagnostic or prognostic benefit in female dogs with mammary tumors.     

Conventional and Doppler ultrasound for the differentiation of benign and malignant canine mammary tumours

Objectives : The aim of this study was to evaluate the sensitivity of conventional and Doppler ultrasound for differentiation of benign and malignant mammary tumours in female dogs. Methods : Mammary tumours were evaluated from 60 animals and divided into two distinct groups, group 1 (benign tumours) and group 2 (malignant tumours). The tumours were assessed by conventional ultrasound, Doppler ultrasound mode, histopathology and immunohistochemical detection of vascular endothelial growth factor. Results : Conventional ultrasound examination was found to be ineffective in separating tumours into the two experimental groups. Similarly, using colour‐flow Doppler ultrasound, no correlation was found between the presence of vascularisation and its characteristics between the two groups. Triplex Doppler ultrasound yielded average maximum velocities of 28·71 cm/s for malignant and 19·91 cm/s for benign tumours, which were significantly different (P=0·01). For vascular endothelial growth factor, an average score of 2·22 was found for group 2 and 1·66 for group 1 (P=0·03). Positive correlations were found between vascular endothelial growth factor and presence of vascularisation (P=0·04 and r=0·3658) and between vascular endothelial growth factor and maximum velocity (P=0·03 and r=0·3913). Clinical Significance : Doppler evaluation may be used to predict malignancy of mammary tumours in bitches.     

Mammary tumors in a colony of beagle dogs

In a lifetime study, female beagle dogs in a closed colony were administered 226radium and 90strontium. An unirradiated control group was included in the study. A total of 223 of 356 dogs at risk developed 1,112 mammary proliferative growths (hyperplastic nodules and neoplasms). There was no correlation between occurrence and types of lesions in radiation and control groups. The age range for first occurrence of lesions was 10.4 to 13.9 years; hyperplastic nodule and benign mixed tumor occurred 1 to 2 years earlier than other lesions. A multiplicity of growths of similar or different morphological type were common throughout the lifetime of the dog. The female beagles, collectively, developed 244 hyperplastic nodules, 78 adenomas, 694 benign mixed tumors, 78 carcinomas, 14 malignant mixed tumors, and four myoepitheliomas. Proliferations occurred with increasing frequency from the cranial to caudal mammary glands. Metastasis was found in 77% of the dogs with carcinoma. The median time from diagnosis to metastasis was 10 months, but was shorter in dogs with infiltrative carcinoma.     

Expression and the molecular forms of neutrophil gelatinase‐associated lipocalin and matrix metalloproteinase 9 in canine mammary tumours

Neutrophil gelatinase‐associated lipocalin (NGAL) is a new biomarker for renal injury. It is also involved in tumorigenesis of different human cancer types. The oncogenic role of NGAL is related to its molecular forms, and heterodimer formation with matrix metalloproteinase 9 (MMP9) promotes human breast cancer (HBC) invasion and metastasis. To date, the levels of NGAL and NGAL/MMP9 complex have not yet been explored in canine mammary tumours (CMTs). Hence, this study aimed to investigate whether NGAL and its molecular forms could be the biomarker for CMT diagnosis. To this end, expression profile of NGAL and MMP9 in mammary epithelial cells as well as in urine samples were detected. By immunohistochemistry staining, NGAL was expressed at variable levels. Unlike HBC, a significant reduction in NGAL expression was demonstrated in benign and malignant CMTs as compared with normal controls. Additionally, NGAL expression was significantly reduced in dogs with metastatic CMTs. By contrast, the mean score of MMP9 expression in ascending order was normal groups, benign, and malignant CMTs. Interestingly, analysis of the molecular form revealed the NGAL/MMP9 complex presents in most mammary tissues and urine of dogs with benign or malignant CMTs, whereas the complex was absent in samples from dogs without CMTs. In conclusion, NGAL and MMP9 are ubiquitously expressed in canine mammary epithelial cells in normal and cancerous status. However, the NGAL/MMP9 complex exclusively presents in mammary tissues and urine of dogs with tumours.     

Phase II evaluation of doxorubicin for treatment of various canine neoplasms

One hundred eighty-five dogs with histologically confirmed, measurable malignant tumors were used in a prospective study to determine the response to 2 doses of the anthracycline antitumor antibiotic, doxorubicin. Eighty-three dogs had been refractory to one or more previous treatment modalities (surgery, n = 54; chemotherapy, n = 22; radiation, n = 10; hyperthermia, n = 1; biological response modifier, n = 1). The extent of neoplastic disease was determined immediately prior to and 3 weeks after 2 doses of doxorubicin were administered (30 mg/m2 of body surface area, iv) 21 days apart. Eighty-four percent (n = 157) of the dogs received 2 doses of doxorubicin and were evaluated. Of the 28 dogs ruled ineligible, 4 had serious side effects to the first dose of doxorubicin, and 24 others acquired complications resulting from their malignant tumors. A partial or complete remission was obtained in 41% (64/157) of all evaluable dogs: 26% (11/43) of the dogs with carcinoma, 67% (42/63) of the dogs with lymphoma, and 22% (11/51) of the dogs with sarcoma. Tumors in which there was at least a 50% volume reduction (partial or complete remission) included malignant lymphoma (42/63), fibrosarcoma (1/14), solid follicular thyroid carcinoma (3/13), mammary adenocarcinoma (2/8), hemangiosarcoma (2/8), osteosarcoma (1/6), circumanal carcinoma (3/5), synovial cell sarcoma (2/3), undifferentiated sarcoma (2/3), nasal adenocarcinoma (1/2), liposarcoma (1/2), infiltrating lipoma (1/1), malignant melanoma (1/1), sclerosing mesothelioma (1/1), and neurofibrosarcoma (1/2).(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum alkaline phosphatase isoenzyme activities in canine malignant mammary neoplasms with and without osseous transformation

BACKGROUND: Increased serum activity of total alkaline phosphatase (TALP) has been found in dogs with mammary neoplasms, especially malignant mixed tumors. We hypothesized that the bone isoenzyme of alkaline phosphatase (BALP), a specific indicator of osteoblastic activity and bone formation, may contribute to increased TALP in dogs with mammary neoplasms with osseous transformation. OBJECTIVE: The purpose of this study was to compare serum TALP, BALP, and other ALP isoenzyme activities in dogs with mammary malignant neoplasms with and without osseous transformation. METHODS: Twenty-one female dogs with malignant mammary neoplasms were compared with 21 clinically healthy, age-matched female control dogs. Physical, clinicopathologic (including preprandial and postprandial serum bile acids, ACTH stimulation, and low-dose dexamethasone suppression tests), radiographic, and ultrasonographic examinations were performed on all dogs with tumors to assess coexisting conditions. On the basis of histologic examination of excised tumors, dogs were further classified as having epithelial (n = 11) or mesenchymal/mixed (epithelial-mesenchymal) (n = 10) neoplasms, the latter of which had histologic and radiologic evidence of bone formation. Serum TALP, BALP, liver alkaline phosphatase (LALP), and corticosteroid-induced alkaline phosphatase (CALP) activities were measured using biochemical methods. RESULTS: Dogs with malignant mammary tumors had significantly higher (P < .05) median serum TALP (170 U/L), BALP (59 U/L), LALP (49 U/L), and CALP (24 U/L) activities, compared with control dogs (81, 32, 37, and 5 U/L, respectively). Significantly higher activities of BALP and LALP were found in dogs with epithelial neoplasms; whereas, only CALP activity was higher in dogs with mesenchymal/mixed neoplasms. There was no significant difference in TALP or isoenzyme activitities between epithelial and mesenchymal/mixed groups. CONCLUSION: BALP activity is increased in some dogs with malignant mammary tumors but does not account for the increase in TALP in dogs with neoplasms that have osseous transformation.     

Immunohistochemical detection of P-glycoprotein (clone C494) in canine mammary gland tumours

Elevated levels of P-glycoprotein have been reported in multidrug-resistant tumours in both humans and dogs. In the present study, we investigated the expression of P-glycoprotein in 57 canine mammary gland tumours, 10 mammary gland hyperplasia and seven normal mammary glands by immunohistochemistry. Tissue sections were incubated with an anti-Pgp monoclonal antibody and visualized with En Vision-DAB polymer. Normal and hyperplastic mammary tissues were negative or showed slight cytoplasmic immunoreactivity. Neoplastic cells in benign mammary tumours showed diffuse cytoplasmic staining, in contrast to malignant tumours that showed mainly a membranous staining pattern for Pgp (C494). We observed statistically significant differences among all the different groups of tissues analysed except for benign tumours versus hyperplasia (P = 0.221). Receiver-operating characteristic analysis showed that the best cut-off point to differentiate the threshold to differentiate negative from positive tissue samples was 18.40% of immunostained cells. These results provide a first indication that routine evaluation of Pgp expression in canine mammary gland tumours, taking into consideration a cut-off point for positivity, may be useful for selecting cases for chemotherapy.     

Correlation of haemostatic abnormalities with tumour stage and characteristics in dogs with mammary carcinoma

Sixty female dogs with untreated mammary carcinoma, comprising equal numbers of dogs in tumour stages I to IV, were evaluated for haemostatic abnormalities using the following tests: platelet count, prothrombin time, activated partial thromboplastin time, thrombin time, plasma activity of factor V, VIII and X, plasma concentration of fibrinogen, fibrin monomers and fibrinogen degradation products, and plasma antithrombin III activity. Two-thirds of all dogs had one or more haemostatic test abnormality of which the likelihood and frequency was increased in those with stage III and IV neoplasia. Haemostatic abnormalities were more frequently observed in dogs which had mammary tumours with distant metastases, extended tumour necrosis, inflammatory carcinomas, tumours fixed to underlying structures, or tumours in which there was penetration of the tumour capsule by tumour cells. As in humans with mammary carcinoma, these haemostatic abnormalities might be used as prognostic indicators, but their clinical importance remains unknown.     

Influence of different tumour types on natural cytotoxicity (NK cell activity) and mitogen-induced lymphocyte proliferation in isolated blood lymphocytes from 110 dogs with tumours

The cell-mediated immune response of blood lymphocytes from 110 untreated dogs with different tumours was evaluated. The influence of different tumour types on the cellular immune system was examined by assessing the percentage of isolated large granular lymphocytes (LGL), in vitro natural cytotoxicity and mitogen-induced lymphocyte proliferation. Although the overall natural cytotoxicity of dogs with different tumours was decreased, the overall difference from control values was not statistically significant. However, mitogen-induced lymphocyte proliferation was significantly depressed in dogs with tumours in comparison with the controls. Dogs with mammary carcinomas showed significantly lower natural cytotoxicity than controls and dogs with myeloid neoplasms showed significantly lower mitogen-induced lymphocyte proliferation. Abnormalities exist not only in natural cytotoxicity but also in mitogen-induced lymphocyte proliferation. For the dog, this is the first study to assess the influence of different tumours using a combined evaluation of natural cytotoxicity and mitogen-induced lymphocyte proliferation in such a large number of animals.