Ki-67 and PCNA Expression in Canine Mammary Tumors and Adjacent Nonneoplastic Mammary Glands: Prognostic Impact by a Multivariate Survival Analysis.

The assessment of tumor proliferation has been considered a determining prognostic factor in canine mammary tumors (CMTs). However, no studies have assessed the prognostic importance of proliferation in adjacent nonneoplastic mammary glands. We included 64 CMTs (21 benign and 43 malignant) and studied the proliferation index (PI) of Ki-67 and proliferating cell nuclear antigen (PCNA) together with several clinicopathological characteristics. A positive and statistically significant correlation between the PI of Ki-67 and PCNA in tumors and adjacent nonneoplastic mammary glands was observed in benign and malignant tumors. Tumor size, skin ulceration, histological type, mitotic index, nuclear grade, differentiation grade, histological grade of malignancy, lymph node metastasis, Ki-67, and PCNA expression in tumors and adjacent nonneoplastic mammary glands were statistically associated with overall survival by univariate analysis in malignant cases (n = 43). Histological grade of malignancy and high intratumoral PCNA retained their significance by multivariate analysis arising as independent predictors of overall survival. Interestingly, the PI of Ki-67 and PCNA of adjacent nontumoral mammary glands were associated with clinicopathological features of tumor aggressiveness and shorter overall survival, demonstrating the need to better explore this adjacent non-neoplastic tissue.

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), 17ß-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.

Validation of a hematology analyzer in donkey medicine.

Asinina de Miranda is a protected donkey sub-species from the Mirandês plateau in northeastern of Portugal. Donkeys are animals that have substantially lost their place as working animals in modern society, this had led to a decrease in their population numbers. A need to preserve native species has led to the foundation of organizations like Associação para o Estudo e Proteção do Gado Asinino (AEPGA) and the development of studies regarding breed welfare, such as hematology. The IDEXX ProCyte Dx is a veterinary hematology analyzer validated for several species, but not for donkeys. The aim of this study was to validate the ProCyte Dx for Asinina de Miranda donkeys. The validation requires a controlled study of precision, carryover, linearity and comparison between the equipment and the manually obtained values for the leukocyte differential count and hematocrit. Results indicated coefficient of variation was good (below 5 %) for both the intra-assay and the inter-assay precision, except for basophils. Carryover was 0 % for all the parameters except platelets (5.88 %). Linearity showed a very high Pearson correlation coefficient, above 0.99, for erythrocytes, hematocrit, hemoglobin, leucocytes, neutrophils, lymphocytes, monocytes, eosinophils, platelets and plateletcrit. Comparison demonstrated excellent agreement for hematocrit (rs=0.96) and good Spearman rank correlation for neutrophils (rs=0.84) and lymphocytes (rs=0.90). Accuracy for total leukocyte count and platelets could not be determined. In conclusion, the ProCyte Dx seems appropriate to be used in Asinina de Miranda hematology.

Assessing domain match and feasibility of candidate instruments matching with OMERACT endorsed domains to measure flare in knee and hip osteoarthritis.

PURPOSE: To evaluate the domain match (truth) and feasibility of candidate instruments assessing flare in knee and hip osteoarthritis (OA) according to the identified domains. MATERIAL AND METHODS: From a literature review (575 papers), instruments were selected and evaluated using the truth and feasibility elements of the OMERACT Filter 2.2. These were evaluated by 26 experts, including patients, in two Delphi survey rounds. The final selection was obtained by a vote. RESULTS: 44 instruments were identified. In Delphi Round 1, five instruments were selected. In Round 2, all instruments obtained at least 75 % in terms of content match with the endorsed domains and feasibility. In the final selection, the Flare-OA questionnaire obtained 100 % favorable votes. CONCLUSION: Through consensus of the working group, the Flare-OA questionnaire was selected as the best candidate instrument to move into a full assessment of its measurement properties using the OMERACT Filter 2.2.

CPAP and survival in moderate-to-severe obstructive sleep apnoea syndrome and hypoxaemic COPD.

Obstructive sleep apnoea syndrome (OSAS) often coexists in patients with chronic obstructive pulmonary disease (COPD). The present prospective cohort study tested the effect of OSAS treatment with continuous positive airway pressure (CPAP) on the survival of hypoxaemic COPD patients. It was hypothesised that CPAP treatment would be associated with higher survival in patients with moderate-to-severe OSAS and hypoxaemic COPD receiving long-term oxygen therapy (LTOT). Prospective study participants attended two outpatient advanced lung disease LTOT clinics in São Paulo, Brazil, between January 1996 and July 2006. Of 603 hypoxaemic COPD patients receiving LTOT, 95 were diagnosed with moderate-to-severe OSAS. Of this OSAS group, 61 (64%) patients accepted and were adherent to CPAP treatment, and 34 did not accept or were not adherent and were considered not treated. The 5-yr survival estimate was 71% (95% confidence interval 53-83%) and 26% (12-43%) in the CPAP-treated and nontreated groups, respectively (p<0.01). After adjusting for several confounders, patients treated with CPAP showed a significantly lower risk of death (hazard ratio of death versus nontreated 0.19 (0.08-0.48)). The present study found that CPAP treatment was associated with higher survival in patients with moderate-to-severe OSAS and hypoxaemic COPD receiving LTOT.

Respiratory muscle unloading improves leg muscle oxygenation during exercise in patients with COPD.

BACKGROUND: Respiratory muscle unloading during exercise could improve locomotor muscle oxygenation by increasing oxygen delivery (higher cardiac output and/or arterial oxygen content) in patients with chronic obstructive pulmonary disease (COPD). METHODS: Sixteen non-hypoxaemic men (forced expiratory volume in 1 s 42.2 (13.9)% predicted) undertook, on different days, two constant work rate (70-80% peak) exercise tests receiving proportional assisted ventilation (PAV) or sham ventilation. Relative changes (Delta%) in deoxyhaemoglobin (HHb), oxyhaemoglobin (O(2)Hb), tissue oxygenation index (TOI) and total haemoglobin (Hb(tot)) in the vastus lateralis muscle were measured by near-infrared spectroscopy. In order to estimate oxygen delivery (Do(2)est, l/min), cardiac output and oxygen saturation (Spo(2)) were continuously monitored by impedance cardiography and pulse oximetry, respectively. RESULTS: Exercise tolerance (Tlim) and oxygen uptake were increased with PAV compared with sham ventilation. In contrast, end-exercise blood lactate/Tlim and leg effort/Tlim ratios were lower with PAV (p<0.05). There were no between-treatment differences in cardiac output and Spo(2) either at submaximal exercise or at Tlim (ie, Do(2)est remained unchanged with PAV; p>0.05). Leg muscle oxygenation, however, was significantly enhanced with PAV as the exercise-related decrease in Delta(O(2)Hb)% was lessened and TOI was improved; moreover, Delta(Hb(tot))%, an index of local blood volume, was increased compared with sham ventilation (p<0.01). CONCLUSIONS: Respiratory muscle unloading during high-intensity exercise can improve peripheral muscle oxygenation despite unaltered systemic Do(2 )in patients with advanced COPD. These findings might indicate that a fraction of the available cardiac output had been redirected from ventilatory to appendicular muscles as a consequence of respiratory muscle unloading.

The use of low-dose metronomic chemotherapy in dogs-insight into a modern cancer field.

The era of chemotherapy, which started in the middle of the last century, has been ruled by the routine use of dose-intense protocols, based on the "maximum-tolerated dose" concept. By promoting a balance between patient's quality of life and the goal of rapidly killing as many tumour cells as possible, these protocols still play a prominent role in veterinary oncology. However, with the opening of a new millennium, metronomic chemotherapy (MC) started to be considered a possible alternative to traditional dose-intense chemotherapy. Characterized by a long-term daily administration of lower doses of cytotoxic drugs, this new modality stands out for its unique combination of effects, namely on neovascularization, immune response and tumour dormancy. This article reviews the rationale for treatment with MC, its mechanism of action and the main studies conducted in veterinary medicine, and discusses the key challenges yet to be solved.

Expression of Cox-1 and Cox-2 in canine mammary tumours.

The aim of this study was to investigate immunohistochemically the expression of cyclooxygenase-1 (Cox-1) and cyclooxygenase-2 (Cox-2) in canine mammary tumours of different histological types. Cox-1 and Cox-2 enzyme expression was evaluated in 70 mammary samples (four normal, six hyperplastic, 60 neoplastic [21 benign and 39 malignant]). Cox-1 expression was identified in all the samples, and Cox-2 in all the mammary lesions except ductal hyperplasia. Two of the four normal mammary gland samples showed focal immunoreactivity for Cox-2. Cox-1 immunoexpression did not differ significantly between benign and malignant lesions (P=0.272). Cox-2 immunoexpression was higher in malignant tumours than in benign counterparts (P<0.001). Of the malignant tumours, carcinosarcomas and tubulopapillary and squamous cell carcinomas had the highest Cox-2 scores. The study showed that malignant tumours had the highest values of Cox-2 expression, and Cox-2 immunolabelling was particularly intense in histological types classically associated with high malignancy. This suggests that nonsteroidal anti-inflammatory drugs (NSAIDs), particularly Cox-2 inhibitors, may have a useful role to play in the treatment of canine malignant mammary tumours.

High COX-2 expression in canine mast cell tumours is associated with proliferation, angiogenesis and decreased overall survival.

COX-2 overexpression is associated with several hallmarks of carcinogenesis such as proliferation, angiogenesis, invasion and metastasis. Fifty cases of canine mast cell tumours (MCT) were retrospectively evaluated and submitted to immunohistochemistry for COX-2, CD31, Ki-67, MAC-387 and CD3. Furthermore its relationship with clinicopathological variables and overall survival (OS) was analysed. COX-2 intensity (P = 0.016), but not COX-2 extension nor score was associated with decreased OS and higher grades of malignancy according to Patnaik (P = 0.002) and Kiupel (P < 0.001) grading systems. Cox-2 intensity was also associated with higher Ki-67 scores (P = 0.009), higher mitotic index (P = 0.022) and higher microvascularization density (P = 0.045). No association was observed for COX-2 intensity and CD3-T lymphocyte (P = 0.377) and macrophage infiltration (P = 0.261) by MAC-387 immunollabelling, suggesting an active role of COX-2 in MCT oncogenesis mainly through proliferation and angiogenesis stimulation making it a potentially clinical relevant prognosis marker and therapeutic target.

High COX-2 expression is associated with increased angiogenesis, proliferation and tumoural inflammatory infiltrate in canine malignant mammary tumours: a multivariate survival study.

COX-2 expression affects mammary tumourigenesis by promoting angiogenesis and cell proliferation, encouraging metastatic spread and tumour-associated inflammation. Samples of canine mammary tumours (n = 109) were submitted to immunohistochemistry to detect COX-2, CD31, VEGF, Ki-67, CD3 and MAC387 expression. Concurrent high expression of COX-2/CD31, COX-2/VEGF, COX-2/Ki-67, COX-2/CD3 and COX-2/MAC was associated with elevated grade of malignancy, presence of intravascular emboli and presence of lymph node metastasis. Tumours with high COX-2 (P < 0.001) and tumours with concurrent expression of high COX-2 and high CD31 (P = 0.008); high VEGF (P < 0.001); high Ki-67 (P < 0.001); high CD3+ T-lymphocytes (P = 0.002) and elevated MAC387 macrophages (P = 0.024) were associated with shorter overall survival (OS) time. Interestingly the groups with high COX-2/CD31 and high COX-2/VEGF retained their significance after multivariate analysis arising as independent predictors of OS. Present data highlight the importance of COX-2 in canine mammary tumourigenesis.

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.

Exploring new biomarkers in the tumour microenvironment of canine inflammatory mammary tumours.

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (CIMC) are the most aggressive forms of mammary cancer. Current research aims to identify new therapeutic targets. Here, we investigated gene expression levels of biomarkers associated with the inflammatory microenvironment. A total of 32 formalin-fixed paraffin-embedded samples of canine mammary carcinoma (CIMC = 26; non-CIMC = 6) were used and their cDNA subjected to quantitative polymerase chain reaction (qPCR) to establish gene expression levels for mediators commonly implicated in linking carcinogenesis with inflammation. Gene expression differences between CIMC and non-CIMC types were obtained for cyclooxygenase 2 (COX-2) (P = 0.004), synuclein gamma (SNCG) (P = 0.006), tribbles 1 (P = 0.025), vascular endothelial growth factor (VEGF) (P = 0.017) and CSF1R (P = 0.045). Among these biomarkers correlations were found, particularly between SNCG and tribbles 1 (r = 0.512, P = 0.001). The efficient metastasis of CIMC is intimately linked to components in the tumour microenvironment. This study suggests that upregulation and correlation of SNCG and tribbles 1 deserves to be further explored.

Primary and secondary tumours occurring simultaneously in the brain of a dog.

Primary brain tumours of a single histological type and metastatic brain tumours are well described in dogs in the current veterinary literature. However, the concurrent presence of a primary and secondary tumour in the brain of a dog has never, to the authors' knowledge, been previously reported. The clinical and pathological features of a nine-year-old, female boxer with an oligodendroglioma and metastases from a mammary gland adenocarcinoma occurring simultaneously in the brain are described in this case report. Information in the veterinary literature on multiple malignancies affecting the central nervous system is very limited; therefore, a discussion about comparative situations in human medicine has been included.

Immunohistochemical Labelling for Cyclo-oxygenase-2: Does the Positive Control Guarantee Standardized Results?

Since the identification of cyclo-oxygenase-2 as a potentially important therapeutic target in veterinary oncology, numerous studies on its expression have been conducted. Unfortunately, results have been heterogeneous and conclusions are difficult to draw. We tested the ability of a defined positive control to guarantee reproducibility of results among different laboratories. Valid positive controls were defined by positivity of the renal macula densa without background labelling. Fifteen colorectal tumours and 15 oral squamous cell carcinomas were labelled immunohistochemically by six European laboratories. Slides were evaluated in blinded fashion for percentage of positive cells and labelling intensity by three pathologists, and results were analyzed statistically for reproducibility and inter-reader variability. Macula densa positivity was an insufficiently sensitive control to guarantee reproducible results for percentage of positive cells and labelling intensity. Inter-reader variability was proven statistically, making the case for image analysis or other automated quantitative evaluation techniques.

Role of steroid hormones and prolactin in canine mammary cancer.

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.

Inflammation and cancer: till death tears them apart.

Advances in biotechnology have enabled the collection of an immeasurable amount of information from genomic, transcriptomic, metabolomic and proteomic studies of tumours within their microenvironments. The dissection of cytokine and chemokine networks has provided new clues to the interactions between cancer cells and their surrounding inflammatory landscape. To bridge the gap between chronic inflammation and cancer, dynamic participants in the tumour microenvironment have been identified, including tumour-associated macrophages (TAMs) and regulatory T cells (Tregs). Both of these cell types are notable for their ability to cause immunosuppressive conditions and support the evasion of tumour immune surveillance. It is clear now that the tumour-promoting inflammatory environment has to be included as one of the major cancer hallmarks. This review explores the recent advances in the understanding of cancer-related inflammation and how this is being applied to comparative oncology studies in humans and domestic species, such as the dog.

Tumour-associated macrophages are associated with vascular endothelial growth factor expression in canine mammary tumours.

Tumour-associated macrophages (TAMs) have been implicated in carcinogenesis including an important role in angiogenesis. In this study, we describe the relationship between TAMs and angiogenesis in canine mammary tumours (CMT). Formalin-fixed paraffin-embedded CMT samples [(n = 128: malignant (n = 97) and benign (n = 31)] were submitted to immunohistochemical staining to detect MAC387, vascular endothelial growth factor VEGF and CD31 expression. A statistical analysis was carried out to assess possible associations with clinicopathological variables and biological markers of tumour angiogenesis. TAMs, detected by MAC387 expression, were significantly associated with malignant CMT (P < 0.001) and VEGF positive tumours (P = 0.002) and also associated with VEGF expression within malignant CMT (P = 0.043). Associations with clinicopathological variables were found between TAMs and the presence of infiltrative growth (P = 0.031), low tubule formation (P = 0.040) and lymph node metastasis (P = 0.016). The results support the hypothesis that TAMs influence angiogenesis in CMT suggesting TAMs may represent a therapeutic target in this disease.

Prognostic value of tumour-associated macrophages in canine mammary tumours.

Tumour-associated macrophages (TAMs) have already been associated in human breast cancer to a poor prognosis. As a part of a tumoural microenvironment, TAMs have an important contribution influencing neoplastic progression. Hitherto, in canine mammary tumours (CMT) the prognostic value of TAMs has not been reported. In this study, MAC387 immunohistochemical expression was evaluated in 59 CMTs (20 benign and 39 malignant). The TAM value was significantly higher in malignant than benign CMT (P = 0.011). In malignant CMT, TAMs were associated with skin ulceration (P = 0.022), histological type (P = 0.044), nuclear grade (P = 0.031) and tubular differentiation (P = 0.042). The survival analysis revealed a significant association between tumours with higher levels of TAMs and the decrease in overall survival (P = 0.030). TAMs have proven to have a prognostic value. These findings suggest the future possibility of using TAMs as a novel therapeutic target in CMT.

Concurrent expression of cyclo-oxygenase-2 and epidermal growth factor receptor in canine malignant mammary tumours.

Canine mammary tumours (CMTs) are reported to express cyclo-oxygenase (COX)-2 and epidermal growth factor receptor (EGFR); however, no studies have evaluated concurrent expression of these proteins. In this study, 43 malignant CMTs were evaluated immunohistochemically for concurrent expression of COX-2 and EGFR and expression was correlated with malignancy. High COX-2 expression was associated with tumour size (P = 0.033), mitotic index (P = 0.040), nuclear grade (P = 0.021), histological grade of malignancy (P = 0.020) and lymph node metastasis (P = 0.029). High EGFR immunoreactivity was associated with tumour size (P = 0.001), necrosis (P = 0.001), mitotic index (P = 0.022), histological grade of malignancy (P = 0.041) and lymph node metastasis (P = 0.005). Simultaneous high-expression of COX-2 and EGFR was associated with high-nuclear grade (P = 0.049), high-histological grade of malignancy (P = 0.031) and the presence of lymph node metastasis (P = 0.025). A positive correlation between COX-2 and EGFR expression (r = 0.474; P = 0.001) was also observed. These results suggest that combined use of selective inhibitors of COX-2 and EGFR may be a useful approach to the treatment of malignant CMTs.