Comparison of surgical resection of Axillary Lymph Nodes in Dogs with Mammary Gland Tumors with or without sentinel lymph node visualization with patent blue dye.

Introduction: Dogs' axillary lymph node (ALN) is often difficult to locate before surgical resection. The anatomical location of ALN often discourages Veterinarians from surgical lymphadenectomy. Considering the limited literature available, the actual incidence of metastases and the prognostic relevance are poorly understood. Methods: A non-randomized, prospective clinical study was conducted with female dogs (n = 41) with mammary gland tumor (MGT) in the thoracic or cranial abdominal mammary glands. The study investigated the risks of ALN metastasis based on tumors clinical findings, tumor size, histopathological diagnosis and grade. The main aim of this study was to compare ALN resection with or without patent blue 2.5% (PB) dye injection for sentinel lymph node visualization. A total of 46 mastectomies were performed and five animals underwent two mastectomies. In the first group, 17 patients underwent a mastectomy and lymphadenectomy without PB injection (G1). In contrast, in the second group, 24 patients also received PB injections for sentinel lymph node mapping (G2). The ALN was identified in 38/46 cases (82%). The ALN was identified and excised in only 58% of surgeries in G1(19/46), while in group 2, the lymph node was identified in 92% of the cases and resected in 100% of the cases. The use of PB improves ALN's identification and reduces the surgical resection time in dogs with MGT. Results and discussion: Surgical time differed between the two groups, as it was significantly shorter in the PB injection group compared to group 1 (80 vs. 45 min) (p < 0.0001). The overall frequency of ALN metastasis was 32%. Macroscopic abnormalities in the lymph nodes, tumor size (>3 cm), and diagnosis of anaplastic carcinoma or grade II/III mammary gland tumors were associated with a higher probability of ALN metastasis. Metastases in the ALNs are more common, in dogs presenting with tumors larger than 3 cm and diagnosed with aggressive histological subtypes. The ALNs should be removed for correct staging, prognostic evaluation, and decision for adjuvant therapy.

Transcriptomic Profile of Canine Mammary Ductal Carcinoma.

Dogs can be excellent models for spontaneous studies about breast cancers, presenting similarities in clinical behavior and molecular pathways of the disease. Thus, analyses of the canine transcriptome can identify deregulated genes and pathways, contributing to the identification of biomarkers and new therapeutic targets, benefiting humans and animals. In this context, this study aimed to determine the transcriptional profile of canine mammary ductal carcinoma and contribute to the clarification of the importance of deregulated molecules in the molecular pathways involved in the disease. Therefore, we used mammary ductal carcinoma tissue samples and non-tumor mammary tissue from the radical mastectomy of six female dogs. Sequencing was performed on the NextSeq-500 System platform. A comparison of carcinoma tissue and normal tissue revealed 633 downregulated and 573 upregulated genes, which were able to differentiate the groups by principal component analysis. Gene ontology analysis indicated that inflammatory, cell differentiation and adhesion, and extracellular matrix maintenance pathways were mainly deregulated in this series. The main differentially expressed genes observed in this research can indicate greater disease aggressiveness and worse prognosis. Finally, the study of the canine transcriptome indicates that it is an excellent model to generate information relevant to oncology in both species.

A pilot study of chemotherapy combinations in rats: Focus on mammary cancer treatment in female dogs.

The use of combined chemotherapy is an essential alternative in treating breast cancer. However, knowledge of the pharmacokinetics of drugs is necessary to obtain maximum efficiency of the protocol and reduce adverse reactions. This study suggests for the first time the effect of the association of carboplatin with ivermectin and carboplatin with cyclophosphamide. This investigation was performed with 36 healthy Wistar rats, divided into four groups: group control, carboplatin (C), carboplatin preceded by ivermectin (C + IV), and carboplatin associated with cyclophosphamide (C + CI). Plasma concentrations quantification was performed using the High-Performance Liquid Chromatographic (HPLC) equipment with an Ultraviolet (UV) detector at eight different time points. Then, the animal was euthanized and necropsied. The bioanalytical method was validated for the two matrices (dogs and rats' plasma), with full validation in female dogs and partial validation in rats, as recommended by the EMA. In both matrices, the method was linear and reproducible. Here, we show the results in female rats' plasma. When comparing the experimental rats' groups (C; C + IV, and C + CI), there is a tendency to increase the bioavailability of carboplatin when used in association, a slight increase for C + IV and more evident to the C + CI group with an AUC rise higher than 2-fold (AUC0-∞ = 2983.61 for C; 4459.06 for C + CI; 7064.68 for C + CI min·mg·mL-1). The blood count, biochemistry profile, and histopathology of the organs revealed only alterations inherent to the metabolic effects of the drugs used. The carboplatin association with ivermectin appeared safe for this pilot group. We believe the carboplatin dose can be maintained without risk to the patient. However, in the carboplatin association with cyclophosphamide, a slight reduction in carboplatin's amount is suggested, seeking to avoid increased effects due to cyclophosphamide. Thus, studies with a more significant number per group must confirm the relevance of this pilot study.

Activity of the glycosidases ß-galactosidase, α-l-fucosidase, ß-N-acetyl-hexosaminidase, and sialidase in uterine tissues from female dogs in diestrus with and without pyometra.

This study aimed to compare the activity of selected glycosidases (ß-galactosidase, α-l-fucosidase, ß-N-acetyl-hexosaminidase, and sialidase) in homogenates of uterine tissues obtained from female dogs with and without pyometra. In addition, it examined the availability of substrates for these glycosidases in the homogenates. The study was carried out on female dogs undergoing ovariohysterectomy for pyometra (n = 10) and clinically healthy dogs (n = 10) undergoing elective spaying. The activity of ß-galactosidase, α-l-fucosidase, and ß-N-acetyl-hexosaminidase was analyzed using a spectrofluorometer and that of sialidase using a colorimetric method. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis with Alcian Blue (AB) and Periodic Acid-Schiff (PAS) staining was performed to determine the presence of substrates for these glycosidases in the homogenates of uterine tissues. The results revealed that the activity of all the examined glycosidases was significantly higher (P < 0.05) in the uterine tissues isolated from dogs with pyometra in comparison to healthy dogs. The electrophoretic patterns of the selected samples showed several proteins, which contained different sugar moieties stained by AB and PAS and the profiles differed significantly between the pyometra group and the healthy group. Densitometric analysis of AB staining showed patterns between 233 and 148, 86 and 55, and 43 and 20 kDa, which differed markedly in sugar content between the examined groups of animals. Similarly, PAS staining analysis revealed patterns of different molecular weights, between 233 and 117 and between 55 and 32 kDa, which also differed in sugar content. These findings suggest that canine pyometra is accompanied by the increase in the activity of selected glycosidases in the uterus. This could potentially modify the glycan structures of uterine glycoproteins and in result their biological functions. Further studies are needed to elucidate the potential role of the increased activity of glycosidases in the pathogenesis of this disease.

Evaluation of immunohistopathological profile of tubular and solid canine mammary carcinomas.

Breast cancer is the most common cancer in women, but the incidence of mammary carcinoma in female dogs is even higher than in humans. These two tumors have similarities that can be seen by its biological behavior, molecular genetic alterations, and histology. This suggest that female dogs can be an excellent model for preclinical oncological studies. And the mammary carcinoma most frequently found in this species is the tubular and solid carcinomas. The extracellular matrix (ECM) has an important role in the progression of these tumors. Because of that we proposed to evaluate the ECM components of these carcinomas through histology with specific stains such as Masson's Trichrome, Picrosirius Red and the technique of scanning electron microscopy. With that, we found the presence of collagen fibers in the tubular carcinoma and around its parenchyma. On the other hand, the solid carcinoma presented collagen fibers throughout the parenchyma and around each tumor cell. With the transmission electron microscopy, we observed the presence of mitochondrias and rough endoplasmic reticulum in both tumors. And finally, we evaluated the expression of proteins through the immunohistochemistry, in which we found a high expression of VEGF, PCNA, CK-18 and vimentin in solid carcinoma, and a positive mark in the tubular and solid carcinoma for collagen I, III and fibronectin. Thus, we demonstrated some differences in the ECM of these mammary carcinomas, allowing a better understanding of its histological characteristics, and these data may contribute to future studies about therapies focused on tumors ECM.

Oxidative stress in female dogs with mammary neoplasms

ABSTRACT: The result of the reaction of free radicals with biomolecules is the formation of substances with the potential of inducing oxidative damage, a condition known as oxidative stress. There are voluminous literature data reporting the association, both as a cause and as a consequence, between different diseases and oxidative stress. In this study, 144 female dogs with mammary neoplasia were analyzed. The animals were submitted to clinical evaluation for disease staging, hematological evaluation, serum biochemistry (renal and hepatic function tests), and dosage of the oxidative damage biomarker, malondialdehyde (MDA), at the time of its approach and 30 days after treatment. A control group of 100 healthy animals was also submitted to determination of serum MDA levels. The mean age of the animals affected by mammary neoplasms was 9.88±2.95 (4 to 14) years, while in healthy animals it was 2.31±1.90 years (1 to 6). Of the 144 animals, 113 (78.9%) had malignant neoplasms, and 15, 21, 46, 17 and 14 animals were in clinical stage I, II, III, IV and V respectively and the carcinoma in a mixed tumor was the most frequent histological pattern in this group (26%). Thirty-one animals were diagnosed with benign neoplasms and mammary adenoma was the most frequent histological pattern in 15 animals (51.61%). Hematological changes in the preoperative period were observed in 44 (38.9%) and 12 (38.7%) animals with malignant and benign neoplasias, respectively, and there was a positive correlation between anemia and higher levels of MDA (P=0.0008) for animals with malignant tumors. Regarding serum biochemical parameters, the most frequent alterations in animals with malignant neoplasms were elevated ALT levels in 12 animals (10.6%), creatinine in 10 animals (8.84%) and urea in eight animals (7.07%). Females with benign neoplasms presented less occurrence of changes in these parameters. In the group of healthy animals (control), the mean serum MDA values were 12.08±4.18, whereas in the pre-treatment group, mean MDA was 24.80±5.74 for bitches with benign neoplasms and 32.27±10.24 for bitches with malignant tumors. A significant increase (P 0.001) in MDA levels was observed in animals with malignant mammary neoplasms when compared to healthy animals and with benign tumors. In addition, a significant reduction (P 0.001) was observed 30 days after treatment in MDA levels (27.37±7.86) in animals with malignant tumors. In conclusion, our results indicate an association between MDA seric levels and mammary neoplasms in dogs. The results suggest that this factor can be used as a biomarker of oxidative stress with a potential impact in the prognostic of mammary tumors, since significantly higher levels of MDA were detected especially in dogs carrying malignant tumors and presenting anemia.

RESUMO: O resultado da reação de radicais livres com biomoléculas é a formação de substâncias que podem ser utilizadas como marcadores de dano oxidativo, condição mais conhecida como estresse oxidativo. Evidências científicas comprovam a relação, quer como causa, quer como consequência, entre muitas doenças e o estresse oxidativo. Neste estudo, 144 cadelas portadoras de neoplasia de mama, foram submetidas à avaliação clínica para estadiamento da doença, avaliação hematológica, testes de função renal e hepática e dosagem do biomarcador de dano oxidativo, malondialdeído (MDA), no momento de sua abordagem e 30 dias após realização de tratamento. Um grupo controle de 100 cadelas saudáveis foi submetido também à determinação dos níveis séricos de MDA. A idade média dos animais acometidos por neoplasias mamárias foi de 9,88±2,95 (4 a 14) anos, enquanto que nos animais saudáveis foi de 2,31±1,90 anos (1 a 6). Dos 144 animais, 113 (78, 9%) apresentavam neoplasias malignas, sendo que 15, 21, 46, 17 e 14 animais encontravam-se em estadiamento clínico I, II, III, IV e V respectivamente, e o carcinoma em tumor misto foi o padrão histológico mais frequente neste grupo (26%). Trinta e um animais tiveram diagnóstico de neoplasias benignas, sendo que 7 estavam no estádio I, 16 no estádio II e 8 no estádio III e o adenoma mamário foi o padrão histológico mais frequente em 15 animais (51,61%). Alterações hematológicas no período pré-operatório foram observadas em 44 (38,9%) e 12 (38,7%) animais portadores de neoplasias malignas e benignas, respectivamente, sendo que houve correlação positiva entre anemia e níveis mais elevados de MDA (P=0,0008), para os animais com tumores malignos. Em relação aos parâmetros bioquímicos séricos, as alterações mais frequentes nos animais com neoplasias malignas foram a elevação dos níveis de ALT em 12 animais (10,6%), de creatinina em 10 animais (8,84%) e de ureia em oito animais (7,07%) Cadelas portadoras de neoplasias benignas apresentaram menor ocorrência de alterações nesses parâmetros. No grupo controle, a média dos valores séricos de MDA foi 12,08±4,18, enquanto que no grupo pré-tratamento, a média de MDA foi de 24,80±5,74 para as cadelas com neoplasia benigna e 32,27±10,24 para as neoplasias malignas. Verificou-se aumento significativo do valor sérico de MDA em cadelas portadoras de neoplasias malignas em comparação com os animais hígidos ou com neoplasias benignas (P 0,001). Ainda, 30 dias após o tratamento observou-se uma diminuição significativa (P 0,001) no valor médio de MDA (27,37±7,86) nos animais com neoplasias malignas. Em conclusão, os resultados deste estudo evidenciam uma associação entre níveis séricos aumentados de MDA e presença de neoplasias mamárias em cadelas. Os resultados sugerem que este fator pode ser utilizado como biomarcador de estresse oxidativo em cães, com provável impacto no prognóstico dos tumores mamários, uma vez que níveis significativamente mais altos de MDA foram detectados especialmente nas cadelas portadoras de tumores malignos e apresentando anemia.

Oxidative stress in female dogs with mammary neoplasms / Estresse oxidativo em cadelas com neoplasia mamária

The result of the reaction of free radicals with biomolecules is the formation of substances with the potential of inducing oxidative damage, a condition known as oxidative stress. There are voluminous literature data reporting the association, both as a cause and as a consequence, between different diseases and oxidative stress. In this study, 144 female dogs with mammary neoplasia were analyzed. The animals were submitted to clinical evaluation for disease staging, hematological evaluation, serum biochemistry (renal and hepatic function tests), and dosage of the oxidative damage biomarker, malondialdehyde (MDA), at the time of its approach and 30 days after treatment. A control group of 100 healthy animals was also submitted to determination of serum MDA levels. The mean age of the animals affected by mammary neoplasms was 9.88±2.95 (4 to 14) years, while in healthy animals it was 2.31±1.90 years (1 to 6). Of the 144 animals, 113 (78.9%) had malignant neoplasms, and 15, 21, 46, 17 and 14 animals were in clinical stage I, II, III, IV and V respectively and the carcinoma in a mixed tumor was the most frequent histological pattern in this group (26%). Thirty-one animals were diagnosed with benign neoplasms and mammary adenoma was the most frequent histological pattern in 15 animals (51.61%). Hematological changes in the preoperative period were observed in 44 (38.9%) and 12 (38.7%) animals with malignant and benign neoplasias, respectively, and there was a positive correlation between anemia and higher levels of MDA (P=0.0008) for animals with malignant tumors. Regarding serum biochemical parameters, the most frequent alterations in animals with malignant neoplasms were elevated ALT levels in 12 animals (10.6%), creatinine in 10 animals (8.84%) and urea in eight animals (7.07%). Females with benign neoplasms presented less occurrence of changes in these parameters. In the group of healthy animals (control), the mean serum MDA values were 12.08±4.18, whereas in the pre-treatment group, mean MDA was 24.80±5.74 for bitches with benign neoplasms and 32.27±10.24 for bitches with malignant tumors. A significant increase (P<0.001) in MDA levels was observed in animals with malignant mammary neoplasms when compared to healthy animals and with benign tumors. In addition, a significant reduction (P<0.001) was observed 30 days after treatment in MDA levels (27.37±7.86) in animals with malignant tumors. In conclusion, our results indicate an association between MDA seric levels and mammary neoplasms in dogs. The results suggest that this factor can be used as a biomarker of oxidative stress with a potential impact in the prognostic of mammary tumors, since significantly higher levels of MDA were detected especially in dogs carrying malignant tumors and presenting anemia.(AU)

O resultado da reação de radicais livres com biomoléculas é a formação de substâncias que podem ser utilizadas como marcadores de dano oxidativo, condição mais conhecida como estresse oxidativo. Evidências científicas comprovam a relação, quer como causa, quer como consequência, entre muitas doenças e o estresse oxidativo. Neste estudo, 144 cadelas portadoras de neoplasia de mama, foram submetidas à avaliação clínica para estadiamento da doença, avaliação hematológica, testes de função renal e hepática e dosagem do biomarcador de dano oxidativo, malondialdeído (MDA), no momento de sua abordagem e 30 dias após realização de tratamento. Um grupo controle de 100 cadelas saudáveis foi submetido também à determinação dos níveis séricos de MDA. A idade média dos animais acometidos por neoplasias mamárias foi de 9,88±2,95 (4 a 14) anos, enquanto que nos animais saudáveis foi de 2,31±1,90 anos (1 a 6). Dos 144 animais, 113 (78, 9%) apresentavam neoplasias malignas, sendo que 15, 21, 46, 17 e 14 animais encontravam-se em estadiamento clínico I, II, III, IV e V respectivamente, e o carcinoma em tumor misto foi o padrão histológico mais frequente neste grupo (26%). Trinta e um animais tiveram diagnóstico de neoplasias benignas, sendo que 7 estavam no estádio I, 16 no estádio II e 8 no estádio III e o adenoma mamário foi o padrão histológico mais frequente em 15 animais (51,61%). Alterações hematológicas no período pré-operatório foram observadas em 44 (38,9%) e 12 (38,7%) animais portadores de neoplasias malignas e benignas, respectivamente, sendo que houve correlação positiva entre anemia e níveis mais elevados de MDA (P=0,0008), para os animais com tumores malignos. Em relação aos parâmetros bioquímicos séricos, as alterações mais frequentes nos animais com neoplasias malignas foram a elevação dos níveis de ALT em 12 animais (10,6%), de creatinina em 10 animais (8,84%) e de ureia em oito animais (7,07%) Cadelas portadoras de neoplasias benignas apresentaram menor ocorrência de alterações nesses parâmetros. No grupo controle, a média dos valores séricos de MDA foi 12,08±4,18, enquanto que no grupo pré-tratamento, a média de MDA foi de 24,80±5,74 para as cadelas com neoplasia benigna e 32,27±10,24 para as neoplasias malignas. Verificou-se aumento significativo do valor sérico de MDA em cadelas portadoras de neoplasias malignas em comparação com os animais hígidos ou com neoplasias benignas (P<0,001). Ainda, 30 dias após o tratamento observou-se uma diminuição significativa (P<0,001) no valor médio de MDA (27,37±7,86) nos animais com neoplasias malignas. Em conclusão, os resultados deste estudo evidenciam uma associação entre níveis séricos aumentados de MDA e presença de neoplasias mamárias em cadelas. Os resultados sugerem que este fator pode ser utilizado como biomarcador de estresse oxidativo em cães, com provável impacto no prognóstico dos tumores mamários, uma vez que níveis significativamente mais altos de MDA foram detectados especialmente nas cadelas portadoras de tumores malignos e apresentando anemia.(AU)

Relationship of obesity with clinical and laboratory changes in female dogs with mammary neoplasia / Relação da obesidade com alterações clínicas e laboratoriais em cadelas com neoplasia mamária

Obesity may be associated with the onset of mammary tumors in women. In companion animals, these data are still scarce, mainly associating the clinic of the patient with laboratory and histological findings. This study aimed to evaluate the clinical and laboratory aspects of female dogs with mammary neoplasia, investigating whether they are related to obesity. Four groups of spayed or non-spayed female dogs were studied, divided into (G1) female dogs without mammary tumor and normal body condition score (BCS), (G2) female dogs without mammary tumor and obese, (G3) female dogs with mammary tumor and normal BCS and (G4) female dogs with mammary tumor and obese. BCS, canine body mass index (CBMI), blood count, glucose, total cholesterol, triglycerides and cytopathological and histopathological examinations were evaluated. Mixed-breed animals were the most prevalent in this study (67.5%). There was a limitation regarding the use of CBMI related to body proportionality. Considering the 28 tumors evaluated, carcinoma in mixed tumor was the most prevalent malignant histological type, while adenomyoepithelioma was the most prevalent benign histological type. It was possible to conclude that obesity was not related to clinical and laboratory changes in the female dogs affected with mammary neoplasias. In addition, no relationship was noted between BCS and CBMI with the histological type or malignancy of mammary neoplasia, since the malignant histological types were the most prevalent tumors in both the ideal score group and the overweight to obese group.(AU)

A obesidade pode estar associada ao aparecimento de tumores mamários em mulheres. Em animais de companhia, esse dado ainda é escasso, principalmente associando a clínica da paciente aos achados laboratoriais e histológicos. Este estudo objetivou avaliar os aspectos clínicos e laboratoriais de cadelas com neoplasia mamária, investigando se estão relacionadas à obesidade. Foram estudados quatro grupos de cadelas castradas ou não castradas, divididas em (G1) cadelas sem tumor de mama e Escore de Condição Corporal normal (BCS), (G2) cadelas sem tumor de mama e obesa, (G3) cadelas com tumor de mama e ECC normal e (G4) cadelas com tumor de mama e obesa. Foram avaliados o Escore de Condição Corporal (ECC), Índice de Massa Corporal Canina (IMC), hemograma, glicose, colesterol total, triglicerídeos e exames citopatológicos e histopatológicos. Os animais de raça mista foram os mais prevalentes neste estudo (67,5%). Houve uma limitação quanto ao uso do IMC relacionado à proporcionalidade corporal. Considerando os 28 tumores avaliados, o carcinoma em tumor misto foi o tipo histológico maligno mais prevalente, enquanto o adenomioepitelioma foi o tipo histológico benigno mais prevalente. Foi possível concluir que a obesidade não esteve relacionada a alterações clínicas e laboratoriais nas cadelas acometidas por neoplasias mamárias. Além disso, não foi observada relação entre BCS e CBMI com o tipo histológico ou malignidade das neoplasias mamárias, uma vez que os tipos histológicos malignos foram os tumores mais prevalentes no grupo escore ideal e no grupo com sobrepeso e obesidade.(AU)

Risk factors associated with mammary tumors in female dogs / Fatores de risco para tumores mamários em cadelas de Uberlândia e Patos de Minas, Minas Gerais

Mammary tumors in female dogs are the most frequent and corresponds to half of the canine tumors. The objectives of this study were to determine the risk factors associated with the occurrence of mammary tumors in female dogs and to evaluate the macroscopic characteristics of these neoformations, using 386 dogs from the "Outubro Rosa Pets" events done within the cities of Uberlândia and Patos de Minas, Minas Gerais State, Brazil, in 2015 (n=194), 2016 (n=105) and 2017 (n=87). For the determination of risk factors, the binary logistic regression test (P<0.05) was performed. The occurrence of mammary tumors was 23.6% (91/386). The significant risk factors identified were increased age (P<0.001), overweight (P=0.048) and non-castration (P<0.001) with a chance of, respectively, 1.6, 2.3 and 9.3 times for the development of mammary tumors. In dogs with mammary tumors (n=91), 153 lesions were present, of which 39 female dogs had two or more lesions (42.8%). Most of the lesions were at the caudal abdominal (M4) and inguinal (M5) mammary glands (60.13%, 92/153). Relative to the size of the lesions, it was observed that in 78% of the female dogs the lesions were determined asT1 (<3cm), 16.5% were T2 (3-5cm) and 5.5% T3 (>5cm). At least 15.4% (14/91) of the dogs had one of the regional lymph nodes increased. In conclusion, the occurrence of mammary tumors in the evaluated population was 23.6% and that age, overweight and non-realization of ovariohysterectomy are risk factors associated with the development of mammary tumors.(AU)

Em cadelas os tumores mamários são os mais frequentes e correspondem a aproximadamente metade dos tumores em cães. Este estudo teve os objetivos de determinar os fatores de risco envolvidos na ocorrência de tumores mamários em cadelas e avaliar as características macroscópicas destas neoformações, utilizando 386 cadelas do evento "Outubro Rosa Pets" nos municípios de Uberlândia e Patos de Minas, Minas Gerais, Brasil, em 2015 (n=194), 2016 (n=105) e 2017 (n=87). Para a determinação dos fatores de risco utilizou-se o teste de Regressão logística binária (P<0,05). A ocorrência de tumores mamários foi de 23,6% (91/386). Os fatores de risco significativos identificados foram aumento da idade (P<0,001), sobrepeso (P=0,048) e não-castração (P<0,001) com a chance de, respectivamente, 1,6, 2,3 e 9,3 vezes de desenvolvimento de tumores mamários. Nas cadelas com tumores mamários (n=91), constatou-se a presença de 153 lesões, sendo que 39 cadelas apresentaram duas ou mais lesões (42,8%). A maioria das lesões localizaram-se nas mamas abdominais caudais (M4) e inguinais (M5) (60,13%; 92/153). Em relação ao tamanho das lesões, observou-se que 78% das cadelas eram T1 (<3cm), 16,5% T2 (3-5cm) e 5,5% T3 (>5cm). Pelo menos 15,4% (14/91) das cadelas apresentaram um dos linfonodos regionais aumentados. Conclui-se que a ocorrência dos tumores mamários na população avaliada foi de 23,6% e que a idade, sobrepeso e não ovariohisterectomia são fatores de risco para o desenvolvimento de tumores mamários.(AU)

Dosagem sérica do marcador tumoral CA 15. 3 em cadelas portadoras de neoplasias mamárias pelo método de eletroquimioluminescência / Serum dosage of the tumor marker Ca 15. 3 in bitches presenting mammary neoplasia with the chemiluminescence method

The objective of the present study was to analyze the serum levels of the tumor marker Ca15.3 in healthy bitches and those with mammary neoplasms, correlating results with tumor type, clinical staging, time until presentation, and presence of ulceration and vascularization. For the study, 30 bitches with mammary tumors and 30 healthy bitches (control group) were selected. Histopathology was performed for identification of tumor type, and blood was collected for measurement of serum concentration of the marker via the chemiluminescence method using a commercial kit. A higher frequency of malignant neoplasms was observed (76.7%), with a higher quantity of carcinoma in mixed tumor (26.7%). Regarding serum concentration of the marker Ca15.3, there was no difference in serum values when comparing the means from bitches with neoplasia and healthy bitches, nor when comparing the other characteristics. The majority of results for serum concentration of Ca15.3, whether in bitches with neoplasia or in healthy bitches, was zero. It is concluded that the measurement of the marker Ca15.3 using the chemiluminescence method and commercial kits for humans did not offer significant results that would make this method or this marker a useful tool for patient monitoring and evaluation of the prognosis of bitches with mammary neoplasms.(AU)

Decorin concentrations in canine normal and neoplastic mammary gland tissues.

In the present study, the concentration of decorin in canine normal and neoplastic mammary gland tissues was examined to understand the potential role of decorin in development and progression of canine mammary tumours. The homogenates of 48 mammary gland tumours (10 benign and 38 malignant) and 10 samples of normal canine mammary gland tissue were used in the study. The presence and quantification of decorin was examined in the homogenates using Western blot and specific canine ELISA. Western blotting confirmed the presence of decorin both in the normal mammary gland tissues and in the mammary gland tumours. The concentration of decorin was significantly higher (p < .05) in the benign tumours and non-metastatic malignant tumours than in the normal mammary gland. The concentration of decorin was significantly lower (p < .05) in the malignant tumours with metastasis to regional lymph nodes compared with benign tumours and non-metastatic malignant tumours. No significant differences were found in the level of decorin between the benign and the non-metastatic malignant tumours. Both the histological type of malignant tumours and the histological grade did not significantly affect the concentration of decorin. These findings suggest that neoplastic transformation in the canine mammary gland leads to increase in the decorin protein synthesis. The reducing decorin concentration in canine malignant mammary tumours appears to facilitate the metastatic spread of these tumours.

Uterine non-enzymatic antioxidant defence mechanisms (glutathione, vitamin C, copper and zinc) in diagnosis of canine pyometra.

The aim of the present study was to determine the concentrations of glutathione (GSH), vitamin C, copper (Cu) and zinc (Zn) in the uterine tissues in diagnosis of canine pyometra. Fourteen samples of uterine tissues from female dogs with pyometra and twelve samples of healthy uteruses (control) were used. The concentrations of GSH and vitamin C were determined in the uterine tissue homogenates using spectrophotometric methods. The concentrations of Cu and Zn were measured using atomic absorption spectrometer. The results obtained showed the significantly lower (p⟨0.05) concentration of GSH and the trend towards lower concentration of vitamin C in the pyometra samples compared to the control. The concentrations of Cu and Zn were similar in the uterine tissues from female dogs with pyometra and those from healthy female dogs. The lower GSH and vitamin C concentrations in the uterine tissues of female dogs with pyometra indicate that the non-enzymatic antioxidant mechanisms are impaired in the uterus of dogs with pyometra. These findings suggest that the imbalance of oxidative-antioxidative can play an important role in pathogenesis of canine pyometra.

The effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs.

The main aim of this study was to investigate the effect of pyometra on glycosylation of proteins in the uterine tissues from female dogs, using western blotting with selected lectins (Sambucus nigra agglutinin - SNA and Maackia amurensis agglutinin - MAL II). In addition protein pattern of examined tissues was also evaluated. The study was performed on 10 female dogs undergoing ovariohysterectomy because of pyometra and 10 clinically healthy female dogs, undergoing elective spaying (ovariohysterectomy). Uterine tissue samples of 1 cm2 were taken from the middle region of each uterine horn in both group of animals immediately after ovariohysterectomy. Tissue samples were homogenized and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting with SNA and MAL II. SDS-PAGE analysis showed differences between pyometra samples and controls in the amount of obtained protein fractions and the protein content in the individual fractions. Five protein (with a molecular weight of 193.78 kDa, 103.18 kDa, 77.67 kDa, 70.39 kDa, and 53.00 kDa) were found only in the pyometra samples. The remaining fractions differed in intensity of staining, which indicated differ abundance of a given protein. The results of western blotting with SNA and MAL II demonstrated that the pattern obtained from densitometric analysis differs between adequate healthy and pyometra samples with regard to the amount of protein fraction obtained as well as the intensity of staining of particular fraction. The pyometra tissues contained seven SNA-binding proteins (with a molecular weight 189.94 kDa, 165.51 kDa, 100.94 kDa, 59.42 KDa, 41.32 kDa, 35.16 kDa, and 32.6 kDa) that were not in the healthy tissues. Of the nine remaining fractions, six showed significantly higher (P < 0.05) intensity of staining in the healthy uterine tissues. In turn, the MAL II-binding protein with a molecular weight 75.85 kDa, 51.12 kDa, and 49.98 kDa were found only in the pyometra samples. Of the 28 remaining fractions, ten demonstrated significantly higher (P < 0.05), and five fractions had significantly lower (P < 0.05) intensity of staining in the pyometra tissues. The results obtained indicate that proteins in uterine tissues from female dogs with pyometra are differently glycosylated compared to normal uterine tissues. These findings provide the basis for further studies of the possible role of glycosylation in the pathogenesis of canine pyometra.

Immunohistochemical expression of p63 protein and calponin in canine mammary tumours.

The aim of this study was to compare the expression of p63 protein and calponin in terms of their affinity and specificity for myoepithelial cells in canine mammary tumours. The studied material included 10 benign and 32 malignant mammary tumours from female dogs treated with mastectomy. Primary mouse monoclonal antibodies directed against p63 protein clone 4A4 and calponin clone CALP were used in single- and doublestain system of immunohistochemical reaction. The investigations have shown that majority of myoepithelial cells in benign tumours and carcinomas in situ exhibited strong positive labelling for both markers. In other malignant tumours strong immunoreactivity was observed in resting myoepithelial cells (MECs) and hypertrophic myoepithelial cells (HMECs), while the immunoreactivity in spindle-stellate myoepithelial cells (SMECs) and rounded myoepithelial cells (RMECs) was moderate. The granular-diffuse nuclear expression of p63 protein was observed only in myoepithelial cells. In terms of calponin, diffuse cytoplasmic expression was noted not only in myoepithelial cell but also in some stromal fibroblasts and vascular smooth muscle cells. The epithelial cells did not exhibit specific expression of the investigated markers. The obtained results indicate that p63 is a sensitive and more specific marker of myoepithelial cells in canine mammary tumours compared with calponin. These findings suggest that the immunohistochemical analysis peformed with the use of p63 can be a valuable complement of routine histological examinations of canine mammary tumours facilitating identification of tumours with myoepithelial component.

Changes in serum neopterin and C-reactive protein concentrations in female dogs with mammary gland tumours.

The aim of this study was to measure serum neopterin and C-reactive protein (CRP) concen- trations in female dogs with mammary tumours and evaluate the association between the values of these indicators and some clinical characteristics of the tumour. Fifty three female dogs were used for this study, including 43 dogs with mammary gland tumours (10 benign and 33 malignant) and 10 healthy controls. The concentrations of neopterin and CRP were determined using the ELISA technique and commercial ELISA kits. The mean serum neopterin concentration in fe- male dogs with mammary tumours was lower than in healthy dogs, but significant difference was not found. Similarly, there were no significant differences in neopterin concentrations in female dogs based on tumour size, tumour ulceration and metastasis. The mean CRP concentration was significantly higher (p⟨0.05) in dogs with malignant tumours compared to dogs with benign tu- mours and control. Furthermore, serum CRP concentration was significantly higher (p⟨0.05) in dogs with metastatic malignant tumours compared to dogs with non-metastatic mammary tu- mours. The CRP concentration was significantly lower (p⟨0.05) in dogs with tumours less than 3 cm compared to those with larger tumours, and significantly higher in dogs with ulcerated tu- mours compared to those without ulceration. Our findings suggest that the neoplastic process in the mammary gland does not cause significant changes in serum neopterin concentrations in dogs. Higher concentrations of serum CRP in dogs with advanced stages of malignant tumours may suggest that CRP could be a potential prognostic marker in canine malignant mammary tu- mours, but this hypothesis needs further study.

Epidemiological, clinical and pathological evaluation of overall survival in canines with mammary neoplasms / Estudo epidemiológico, clínico-patológico e avaliação de sobrevida global em cadelas com neoplasias mamárias

Canine mammary neoplasms (CMNs) are the most frequent lesions and in female dogs. However, studies correlating pathological criteria with clinical evolution in female dogs with mammary neoplasms are scarce. The present study aims to present epidemiological, clinical-pathological and overall survival data to help establish the prognosis and understand the biological behavior of CMNs. A total of 1539 cases were included (85% malignant and 13% benign). Tumor size was an important prognostic factor and was associated with overall patient survival (P< 0.0001). Most dogs diagnosed with malignant neoplasms (83%) had initial clinical staging, although 17% had regional or distant metastases at the time of diagnosis and lower overall survival (P< 0.0001). Carcinoma in mixed tumor was the most frequent histological type and had a better prognosis. Solid carcinomas, micropapillary carcinomas and carcinosarcomas were considered histological types with aggressive biological behavior and were associated with a worse prognosis and lower overall survival (P< 0.0001).(AU)

Neoplasias mamárias caninas (NMCs) são as lesões mais frequentes em cadelas. Estudos que correlacionam os critérios patológicos com a evolução clínica em cadelas com neoplasias mamárias são escassos. Este estudo objetiva apresentar dados epidemiológicos, clínico-patológicos e de sobrevida global fornecendo informações que auxiliam a estabelecer o prognóstico e a compreender o comportamento biológico de NMCs. Foram incluídos 1539 casos, 85% malignos e 13% benignos. O tamanho tumoral foi um importante fator prognóstico, sendo associado com a sobrevida global das pacientes (P<0,0001). A maioria das cadelas diagnosticadas com neoplasias malignas (83%) apresentavam estadiamentos clínicos iniciais, enquanto 17% apresentavam metástases regionais ou à distância no momento do diagnóstico, denotando menor sobrevida global (P<0,0001). O carcinoma em tumor misto foi o tipo histológico mais frequente e de melhor prognóstico. Os carcinomas sólidos, carcinomas micropapilares e carcinossarcomas foram considerados tipos histológicos de comportamento biológico agressivo, sendo associados a pior prognóstico e menor sobrevida global (P<0,0001).(AU)

[Neutering of female dogs - old and new insights into Pros and Cons]. / Kastration der Hündin ­ neue und alte Erkenntnisse zu Vor- und Nachteilen.

Advice for dog owners regarding the Pros and Cons and optimal time for neutering a female dog is complex and should be based on recent and valid research data as well as individual aspects. After taking a closer look at the literature, it becomes clear that some putative robust information has to be revised or updated. An obvious advantage of neutering a female dog is that ovarian diseases and sexual steroid-dependent diseases, including metropathy, no longer occur. In addition, it is likely that an early neutering reduces the risk to a certain extent of mammary neoplasia, even if the scientific basis for this observation is weak. The effect might be less than some earlier publications suggest. Disadvantages of neutering female dogs include urinary incontinence, which was postulated decades earlier. However, reported incidences and findings regarding factors that influence urinary incontinence, including time of neutering related to puberty, are heterogeneous. Recently, several studies have been published suggesting a significantly higher risk of different forms of neoplasia and musculoskeletal disorders in neutered dogs. However, factors that may bias these findings, including nutritional condition, age and/or housing, were not addressed in most studies. Data on effects on metabolism and the immune system are currently very limited. If owners want their female dogs to be neutered, a thorough counselling is essential that includes individual aspects as well as breed- and housing-specific factors. The optimal time may be between the first and the second heat. It can be suggested, that at this time, a certain reduction of the risk of mammary neoplasia can be achieved with only a moderate potential for undesired side effects.

Preliminary study on plasma proteins in pregnant and non-pregnant female dogs.

In this study, we used a combined approach based on 2-dimensional electrophoresis (2-DE), difference in gel electrophoresis (DIGE), and mass spectrometry (MS) to identify the plasma protein composition in pregnant female dogs and compared it with non-pregnant female dogs. We used the plasma samples obtained from four female dogs during I, II, and III thirds of pregnancy, three days after parturition, as well as from four non-pregnant female dogs in diestrus phase. Analysis of 2-DE gel image exhibited of 249 protein spots. The intensity of staining of 35 spots differed significantly (P < 0.05) between the non-pregnant and pregnant female dogs. We used matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI TOF MS) to identify 47 spots corresponding to 52 different proteins. Five identified protein spots, including zinc finger BED domain-containing protein 5, hemoglobin subunit beta-2, integrator complex subunit 7, apolipoprotein A-I, and glutamyl aminopeptidase were differentially presented in the plasma of pregnant and non-pregnant female dogs. To the best of our knowledge, this is the first report on the plasma protein profile of pregnant and non-pregnant female dogs. In this study, we identified proteins that have not been previously identified in dogs. Our findings showed that numerous protein spots were differentially presented in the plasma of female dogs during normal pregnancy. Although we identified only a limited number of differentially presented proteins, our study demonstrated that the plasma protein profile changed during pregnancy in female dogs, which suggests its importance in maintaining pregnancy. Further studies are necessary to define complete plasma protein profile of pregnant female dogs and to identify all proteins that are differentially presented in the pregnant animals compared with the non-pregnant ones. In addition, studies are warranted to explain the role of those proteins in maintaining the pregnancy and their usefulness in detection of early pregnancy. Furthermore, our results indicated that DIGE technique is useful in the comparison of samples originated from different states and time points in dogs.

The Effect of Low Monotonic Doses of Zearalenone on Selected Reproductive Tissues in Pre-Pubertal Female Dogs--A Review.

The growing interest in toxic substances combined with advancements in biological sciences has shed a new light on the problem of mycotoxins contaminating feeds and foods. An interdisciplinary approach was developed by identifying dose-response relationships in key research concepts, including the low dose theory of estrogen-like compounds, hormesis, NOAEL dose, compensatory response and/or food tolerance, and effects of exposure to undesirable substances. The above considerations increased the researchers' interest in risk evaluation, namely: (i) clinical symptoms associated with long-term, daily exposure to low doses of a toxic compound; and (ii) dysfunctions at cellular or tissue level that do not produce clinical symptoms. Research advancements facilitate the extrapolation of results and promote the use of novel tools for evaluating the risk of exposure, for example exposure to zearalenone in pre-pubertal female dogs. The arguments presented in this paper suggest that low doses of zearalenone in commercial feeds stimulate metabolic processes and increase weight gains. Those processes are accompanied by lower proliferation rates in the ovaries, neoangiogenesis and vasodilation in the ovaries and the uterus, changes in the steroid hormone profile, and changes in the activity of hydroxysteroid dehydrogenases. All of the above changes result from exogenous hyperestrogenizm.

Prognostic significance of tissue and serum HER2 and MUC1 in canine mammary cancer.

The aim of our study was to compare serum levels and protein tissue of human epidermal growth factor receptor-2 proto-oncogene (HER2) and mucin 1 (MUC1) using an antigen-capture enzyme-linked immunosorbent assay and immunohistochemistry (IHC) in canine mammary carcinomas and investigate how the 2 markers correlate with dogs with metastasis and without metastasis to a regional lymph node. Forty-eight female dogs were selected, including 14 with non-metastatic cancer, 14 with lymph node metastasis, and 20 healthy animals. Serum samples were collected from all the animals and tissues from 28 dogs with malignant mammary tumor with or without metastasis for evaluated HER2 and MUC1 expression. Tissue sample were evaluated for MUC1 and HER2 immunoexpression by IHC. The results showed measurable serum levels of MUC1 and HER2 in all groups. While serum MUC1 levels were significantly higher in animals with metastasis than the other 2 groups, no increase was observed in HER2 serum levels. The MUC1 IHC results showed that only membrane immunostaining was significantly different between the groups. Statistically, there was an association between immunostaining and the serum HER2 levels. Our results indicate that serum concentrations of HER2 and the IHC staining pattern for HER2 in primary tumor do not correlate with the presence of regional metastasis. However, increased concentrations of MUC1 in the serum of dogs with mammary cancer are associated with the presence of metastasis to regional lymph nodes. A membrane pattern of IHC staining for MUC1 in the primary tumor suggests that metastases to regional lymph node are present.