Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas

ABSTRACT: Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P 0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.

RESUMO: A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P 0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.

Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas / Expressão de osteopontina e sua relação com biomarcadores prognóstico nos carcinomas mamários caninos

Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P<0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.(AU)

A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P<0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.(AU)

Osteopontin expression and its relationship with prognostic biomarkers in canine mammary carcinomas / Expressão de osteopontina e sua relação com biomarcadores prognóstico nos carcinomas mamários caninos

Osteopontin is a glycophosphoprotein implicated in different physiologic and pathologic processes and is known to be involved in progression and metastasis of various cancers in humans, but this relation is still little explored in the veterinary. The aim was to evaluate the expression of osteopontin in canine mammary carcinomas and its relation with well-established canine mammary tumor biomarkers. For that, expression of OPN, EGFR, HER2, and c-Kit were evaluated along with Ki67 rate in 43 mammary carcinomas. Osteopontin was demonstrated to be expressed by neoplastic epithelial cells in all carcinomas as well as in stromal cells from the tumor microenvironment. Relation between high osteopontin expression and EGFR positivity (P<0.001) and HER2 overexpression (P=0.012) was demonstrated. In conclusion, high OPN expression seems to be related to poor prognosis and MAPK pathway activation, given the association with EGFR and HER2, members of the MAPK signaling pathway.(AU)

A osteopontina é uma glicofosfoproteina implicada em diferentes processos fisiológicos e patológicos, sendo conhecida por estar envolvida na progressão e metástase de vários cânceres nos humanos, no entanto, essa relação é ainda pouco explorada na veterinária. O objetivo deste trabalho foi avaliar a expressão da osteopontina nos carcinomas mamários caninos e sua relação com biomarcadores bem estabelecidos para esta neoplasia. Para isto, foi avaliada a expressão de OPN, EGRH, HER2 e c-Kit juntamente com a taxa de Ki67 em 43 carcinomas mamários. A osteopontina foi expressa pelas células epiteliais neoplásicas em todos os carcinomas, assim como, nas células estromais do microambiente tumoral. Foi demonstrada uma relação entre uma alta expressão de osteopontina e positividade para EGFR (P<0.001) e superexpressão de HER2 (P=0.012). Em conclusão, alta expressão de OPN parece estar relacionada com mau prognóstico e ativação da via MAPK, devido a sua associação com EGRF e HER2, os quais são membros desta via de sinalização.(AU)

Clinicopathological and immunohistochemical features of pleomorphic lobular mammary carcinoma in a female dog

This report describes a pleomorphic lobular carcinoma (PLC) case, in an eight-year-old, female Shih Tzu dog, which attended the Veterinary Hospital of the University of Franca, Brazil. A single irregular, non-ulcerated, nonhyperaemic soft nodule, 2 cm in diameter, was observed during the clinical examination. The nodule was adhered within the deep tissues in the right caudal thoracic mammary gland. After removal of the nodule, the histopathologic examination showed that the neoplastic epithelial cells were scattered throughout the stroma in linear patterns, had abundant eosinophiliccytoplasm, eccentric nuclei and cytoplasmatic vacuoles, features compatible with the diagnosis of pleomorphic lobularcarcinoma. An immunohistochemical evaluation was performed in order to better characterise the tumour. Based on thenegative immunoreactivity for hormone receptors and human epidermal growth factor receptor-2 and positive expression for one of the basal markers, the basal-like triple-negative phenotype was characterized. The treatment indicated was chemotherapy with carboplatin; however, the disease progressed, and the patient had an overall survival of 47 days after surgery, confirming the aggressiveness of the disease.

Clinicopathological and immunohistochemical features of pleomorphic lobular mammary carcinoma in a female dog

This report describes a pleomorphic lobular carcinoma (PLC) case, in an eight-year-old, female Shih Tzu dog, which attended the Veterinary Hospital of the University of Franca, Brazil. A single irregular, non-ulcerated, nonhyperaemic soft nodule, 2 cm in diameter, was observed during the clinical examination. The nodule was adhered within the deep tissues in the right caudal thoracic mammary gland. After removal of the nodule, the histopathologic examination showed that the neoplastic epithelial cells were scattered throughout the stroma in linear patterns, had abundant eosinophiliccytoplasm, eccentric nuclei and cytoplasmatic vacuoles, features compatible with the diagnosis of pleomorphic lobularcarcinoma. An immunohistochemical evaluation was performed in order to better characterise the tumour. Based on thenegative immunoreactivity for hormone receptors and human epidermal growth factor receptor-2 and positive expression for one of the basal markers, the basal-like triple-negative phenotype was characterized. The treatment indicated was chemotherapy with carboplatin; however, the disease progressed, and the patient had an overall survival of 47 days after surgery, confirming the aggressiveness of the disease.(AU)

Prognostic factors in canine mammary carcinomas and HER2 expression relationship / Fatores de prognóstico em carcinomas mamários caninos e sua relação com expressão de HER2

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging. Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). [...](AU)

Prognostic factors in canine mammary carcinomas and HER2 expression relationship / Fatores de prognóstico em carcinomas mamários caninos e sua relação com expressão de HER2

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging. Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). [...]

Prognostic Factors in Canine Mammary Carcinomas and HER2 Expression Relationship

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging.Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). The histologica

Expression of epidermal growth factor (EGF) and its receptor in bovine endometrium throughout the luteal phase: effects of EGF on prostaglandin production in endometrial cells

Epidermal growth factor (EGF) is produced in bovine endometrium throughout the estrous cycle. However, little is known about the expression of the EGF receptor (EGFR) and the roles of EGF in bovine endometrium. To clarify whether EGF is involved in local regulation of bovine endometrial function, first we determined the EGF protein and the expression of EGFR mRNA in endometrial tissues throughout the luteal stages. EGF protein concentration was higher(P < 0.05) in the mid (days 8-12) luteal stage than in the other luteal stages. EGFR mRNA expression was higher (P < 0.05) in the mid and late (days 15-17) luteal stages than in the other luteal stages. To investigate the protein concentrations of EGF and EGFR mRNA expression in cultured bovine endometrial cells, epithelial and stromal cells were isolated between day 0 and day 4 post-ovulation from 22 uteri. Both EGF protein concentration and EGFR mRNA expression were higher (P < 0.05) in epithelial cells than in stromal cells. Then, to examine the possible role of EGF in the regulation of prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2), cultured endometrial epithelial and stromal cells were exposed to EGF (0, 1, 10 and 100 nm) for 24 h. In epithelial cells, EGF (10 and/or 100 nm) increased (P < 0.05) PGF2α and PGE2 secretion, but in stromal cells EGF (100 nm) increased (P < 0.05) PGF2α, but not PGE2 secretion. These results indicate that 1) the highest amount of EGF is produced by bovine endometrium at the mid-luteal stage, 2) endometrial EGFR mRNA expressions are higher at mid and late-luteal stages than other stages, 3) EGF is expressed mainly by uterine epithelial cells and 4) EGF has the ability to increase PGE2 and PGF2α production in both epithelial and stromal cells and therefore may play a role in local regulation of uterine function.

Expression of epidermal growth factor (EGF) and its receptor in bovine endometrium throughout the luteal phase: effects of EGF on prostaglandin production in endometrial cells

Epidermal growth factor (EGF) is produced in bovine endometrium throughout the estrous cycle. However, little is known about the expression of the EGF receptor (EGFR) and the roles of EGF in bovine endometrium. To clarify whether EGF is involved in local regulation of bovine endometrial function, first we determined the EGF protein and the expression of EGFR mRNA in endometrial tissues throughout the luteal stages. EGF protein concentration was higher(P < 0.05) in the mid (days 8-12) luteal stage than in the other luteal stages. EGFR mRNA expression was higher (P < 0.05) in the mid and late (days 15-17) luteal stages than in the other luteal stages. To investigate the protein concentrations of EGF and EGFR mRNA expression in cultured bovine endometrial cells, epithelial and stromal cells were isolated between day 0 and day 4 post-ovulation from 22 uteri. Both EGF protein concentration and EGFR mRNA expression were higher (P < 0.05) in epithelial cells than in stromal cells. Then, to examine the possible role of EGF in the regulation of prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2), cultured endometrial epithelial and stromal cells were exposed to EGF (0, 1, 10 and 100 nm) for 24 h. In epithelial cells, EGF (10 and/or 100 nm) increased (P < 0.05) PGF2α and PGE2 secretion, but in stromal cells EGF (100 nm) increased (P < 0.05) PGF2α, but not PGE2 secretion. These results indicate that 1) the highest amount of EGF is produced by bovine endometrium at the mid-luteal stage, 2) endometrial EGFR mRNA expressions are higher at mid and late-luteal stages than other stages, 3) EGF is expressed mainly by uterine epithelial cells and 4) EGF has the ability to increase PGE2 and PGF2α production in both epithelial and stromal cells and therefore may play a role in local regulation of uterine function.(AU)

Analysis of EGFR and HER-2 expressions in ductal carcinomas in situ in canine mammary glands / Análise da expressão de EGFR e HER-2 em carcinomas ductais in situ da glândula mamária canina

Biomolecular evidence has shown that ductal carcinoma in situ (DCIS) may develop into invasive carcinoma of the canine mammary gland, and mutations in proto-oncogenes HER2 and EGFR; two members of the family of epidermal growth factor receptors, may be involved in this process. The purpose of this study was the characterization of the immunohistochemical expression of the EGFR and HER2 proteins in the process of neoplastic transformation, supposedly present in ductal carcinomas in situ in canine mammary glands. Fifteen cases of DCIS were evaluated, with a higher expression of HER2 and EGFR being observed in low-grade carcinomas when compared with high-grade neoplasms, and with a high positive statistical correlation in the latter. Results suggest that aggressive tumors tend to lose the expression of EGFR and HER2 simultaneously. The loss of the expression of these markers may be related to the process of neoplastic progression in canine mammary tumors.(AU)

Evidências biomoleculares sugerem que o carcinoma ductal in situ (CDIS) pode progredir para carcinoma invasor na mama canina e que mutações nos proto-oncogenes HER-2 e EGFR, dois membros da família de receptores para fatores de crescimento epidérmicos, podem estar envolvidas neste processo. A partir disso, este trabalho teve por objetivo caracterizar a expressão imuno-histoquímica das proteínas EGFR e HER-2 no processo de transformação neoplásica supostamente presente em carcinomas ductais in situ da glândula mamária canina. Foram avaliados 15 casos de CDIS, sendo observada maior expressão de HER-2 e EGFR em carcinomas de baixo grau em comparação às neoplasias de alto grau, com correlação estatística positiva alta nestes últimos. Os resultados sugerem que tumores mais agressivos tendem a perder, simultaneamente, a expressão de EGFR e HER-2. A perda na expressão desses marcadores pode estar envolvida no processo de progressão neoplásica em tumores mamários caninos.(AU)

EFEITOS DE ALTAS DILUIÇÕES DE PHYTOLACCA DECANDRA NA VIABILIDADE E EXPRESSÃO DO FATOR HER-2 EM ADENOCARCINOMA MAMÁRIO MURINO (4T1) IN VITRO

Em estudo anterior, experimental, in vivo, observou-se que o tratamento de camundongos inoculados com células 4T1 com Phytolacca decandra em diferentes diluições homeopáticas apresentavam mudanças no crescimento tumoral e na expressão de HER-2. HER-2 (receptor de fator de crescimento epidermal, com atividade tirosina-quinase) é super-expressa em cerca de 30% dos carcinomas ductais invasivos humanos e, por isso, é alvo de tratamentos específicos. O objetivo desse estudo foi avaliar o efeito de diferentes diluições homeopáticas de Phytolacca decandra, preparadas segundo a Farmacopéia Homeopática Brasileira, na apoptose, morfologia e expressão de HER-2 em células 4T1 in vitro. Células 4T1 tratadas no momento do cultivo foram analisadas após 24 horas quanto à positividade para anexina V pelo método Countess® para a avaliação da apoptose. A expressão de HER-2 foi avaliada pelo método da imuno-citoquímica e a morfologia das células foi avaliada pelas colorações hematoxilina-eosina e Giemsa, a partir de esfregaços obtidos das células em suspensão. Células tratadas com Phytolacca decandra 200cH mostraram aumento no número de células positivas à anexina V (células em apoptose) e ocorrência de células bi ou multinucleadas. A expressão de HER-2 na superfície celular foi irregular nessas células, bem como nas células tratadas com a diluição 12cH, sugerindo infrarregulação desse receptor. Em suma, os resultados mostram alterações fenotípicas significativas em células 4T1 tratadas com Phytolacca decandra 200cH, as quais merecem estudos mais aprofundados.

In a previous study, experimental, the treatment of mice inoculated with 4T1 cells with Phytolacca decandra at different homeopathic dilutions produced changes in tumor growth and expression of HER-2. HER-2 protein (epidermal growth factor receptor, with tyrosine kinase activity) is superexpressed in about 30% of human invasive ductal carcinomas and, therefore, is targeted by specific treatments. The objective of this study was to evaluate the effect of different homeopathic dilutions of Phytolacca decandra, according to the Brazilian Homeopathic Pharmacopea, on apoptosis, morphology and expression of HER-2 in 4T1 cells in vitro. Treated 4T1 cells were analyzed after 24 hours of culture, for positivity to annexin V, using the Countess® method, for apoptosis evaluation. HER-2 expression was assessed by the immuno-cytochemistry method and cell morphology was evaluated by hematoxylin-eosin and Giemsa staining, in smears obtained from suspended cells. Cells treated with Phytolacca decandra 200cH showed increase in the number of annexin V positive cells (apoptosis cells) and occurrence of bi or multinucleated cells. HER-2 expression on the membrane was irregular in these cells, as well as in those treated with the 12cH dilution, suggesting downregulation of the receptor. In short, the results show significant phenotypic changes in 4T1 cells treated with Phytolacca decandra 200cH, which deserve further studies.

EXPRESSÃO DE HER-2 EM TUMORES MAMÁRIOS DE GATAS

As neoplasias mamárias são o terceiro tipo mais frequente de neoplasias em felinos e a busca por fatores prognósticos para este tipo de tumor nas gatas tem se intensificado nos últimos anos, dada sua alta frequência e relevância na clínica de felinos. Dentre os marcadores imunohistoquímicos utilizados para prognóstico destaca-se o receptor do fator de crescimento epidérmico humano tipo 2 (Human Epidermal Growth Factor Receptor type 2 Her-2) que em mulheres está associado a um pior prognóstico. Objetivou-se avaliar a expressão de Her-2 em hiperplasias e neoplasias mamárias, benignas e malignas, de gatas e relacionar a expressão ao tipo e grau histológico dos carcinomas mamários. Foram selecionados 38 casos de neoplasias e hiperplasias mamárias de gatas, no período de 2006 a 2016. A partir dos protocolos selecionados obteve-se informações epidemiológicas e características macroscópicas das lesões nas mamas. Os tumores foram individualmente classificados e graduados histologicamente por dois patologistas. A expressão de Her-2 foi determinada por imunohistoquímica, por meio de análise semi quantitativa das lâminas conforme escore recomendado pelo Herceptest (Dako). Utilizou-se Teste Qui-quadrado (2) para avaliação da relação entre expressão de Her-2 e tipo de lesão mamária, tipos histológicos de carcinoma e grau histológico (P<0,05). A idade média dos animais foi de 8,5 anos e as gatas jovens foram acometidas somente por hiperplasias e neoplasias benignas. Gatas sem raça definida foram mais acometidas (84,37%), seguida por siamês (9,37%) e persa (6,25%). Quanto à classificação histológica, 18,42% das lesões mamárias foram classificadas como hiperplasia, 18,42% como adenoma e 63,16% foram classificadas como carcinoma. O tipo de carcinoma mais comum foi o carcinoma tubulopapilar (66,7%), seguido por carcinoma sólido (25%) e carcinoma cribriforme (8,33%). Observou-se superexpressão de Her-2 em 4 amostras (16,7%) de carcinoma do tipo tubulopapilar, sendo dois carcinomas grau I e dois graduação II, todavia não houve relação entre a expressão de Her-2 e o tipo de lesão mamária (p=0,3127), tipo de carcinoma (0,3467) ou grau histológico (0,6485). Conclui-se que o percentual de carcinomas mamários de gatas que expressam Her-2 é menor que no câncer de mama da mulher e não houve relação entre grau e tipo histológico e expressão de Her-2. C-erbB-2/neu; felino; neoplasia mamária.

Mammary neoplasms are the third most frequent type of neoplasm in felines and the search for prognostic factors for this type of tumor in cats has intensified in recent years, given its high frequency and relevance in feline clinics. Among the immunohistochemical markers used for prognosis, the Human Epidermal Growth Factor Receptor type 2 (Her-2) receptor, which in women is associated with a worse prognosis, stands out. The objective of this study was to evaluate the expression of Her-2 in hyperplasias and benign and malignant neoplasms of cats and to relate the expression to the type and histological grade of mammary carcinomas. Thirty-eight cases of neoplasia and mammary hyperplasia of cats were selected from 2006 to 2016. From the protocols selected, epidemiological information and macroscopic characteristics of mammary lesions were obtained. Tumors were individually classified and graded histologically by two pathologists. Her-2 expression was determined by immunohistochemistry, using a semi-quantitative analysis of the slides according to the Hercepest (Dako) recommended score. The Chi-square test (2) was used to evaluate the relationship between Her-2 expression and type of mammary lesion, histological types of carcinoma and histological grade (P <0.05). The mean age of the animals was 8.5 years and young cats were affected only by benign hyperplasias and neoplasms. Undefined cats were more affected (84.37%), followed by Siamese (9.37%) and Persian (6.25%). Regarding the histological classification, 18.42% of the mammary lesions were classified as hyperplasia, 18.42% as adenoma and 63.16% were classified as carcinoma. The most common type of carcinoma was tubulopapillary carcinoma (66.7%), followed by solid carcinoma (25%) and cribriform carcinoma (8.33%). Her-2 overexpression was observed in 4 samples (16.7%) of tubulopapillary carcinoma, two carcinomas grade I and two degrees II, but there was no relationship between Her-2 expression and type of mammary lesion (P = 0.3127), carcinoma type (0.3446) or histological grade (0.6485). It is concluded that the percentage of female mammary carcinomas expressing Her-2 is lower than in women's breast cancer and there was no relation between grade and histological type and Her-2 expression.

IMUNOMARCAÇÃO DOS RECEPTORES DE EGF (EGFR E c-ErbB2) NO CARCINOMA DE CÉLULAS ESCAMOSAS EM CÃES

O carcinoma de células escamosas (CCE) é um dos tumores cutâneos malignos mais comuns em todas as espécies animais, assim como na espécie humana, podendo acometer desde animais jovens a idosos. Tem desenvolvimento associado a fatores ambientais como exposição prolongada aos raios solares e hipopigmentação epidermal. Os estudos revelam que, 80% das neoplasias malignas têm origem em estímulos ambientais, em decorrência a exposição a carcinógenos. Apesar da etiologia multifatorial, a busca pelo esclarecimento das causas e dos mecanismos de evolução do câncer deve ser incessante, pois inúmeras neoplasias podem ser prevenidas, especialmente quando induzidas por fatores exógenos. Assim, este trabalho teve por objetivo estudar o CCE cutâneo canino à luz dos diferentes padrões histomorfológicos da neoplasia, avaliando o seu imunofenótipo quanto aos receptores do fator de crescimento epidérmico EGFR e c-erbB2. Para tal, foram avaliadas os casos de CCE canino provenientes do arquivo do Setor de Patologia Animal da EVZ/UFG no período de 2006 a 2015. Para o estudo epidemiológico foram consideradas as informações de registro, incluindo raça, sexo, idade e localização anatômica. Quanto à avaliação histomorfológica e critérios de malignidade, as lesões foram classificadas de acordo com o sistema preconizado por Müller e Kirks. Para o estudo imunoistoquímico foram utilizados os anticorpos anti-EGFR (HER1) e anti-c-erbB-2 (HER2), com o intuito de melhor compreender a participação dessas proteínas nos mecanismos envolvidos na gênese, proliferação e evolução do CCE em cães. Considerando as análises histomorfológica e imunofenotípica foi testada a correlação entre suas variáveis, sendo constatada correlação entre a imunomarcação para EGFR e o grau de diferenciação do CCE (r=0,26; p=0,02). Por outro lado, não houve correlação entre a imunomarcação de c-erbB2 e a diferenciação histomorfológica (r=0,02; p=0,83). Quando testada a correlação entre as imunomarcações de EGFR e c-erbB2, também foi observada correlação positiva, porém sem significância estatística (r=0,21; p=0,06). Conclui-se que há propensão de que o aumento na imunoexpressão de EGFR seja diretamente proporcional ao grau de diferenciação do CCE, sendo que o mesmo não ocorre com a imunomarcação de c-erbB2. Também, os CCE em cães parecem apresentar aumento simultâneo da imunomarcação dos receptores de EGF.

Squamous cell carcinoma (SCC) is one of the most common malignant cutaneous tumors in all species, as well as in the human species, ranging from young animals to the elderly. It has development associated with environmental factors such as prolonged exposure to solar rays and epidermal hypopigmentation. According to the literature, 80% of malignancies originate in environmental stimuli, due to exposure to carcinogens. Despite the multifactorial etiology, the search for clarification of the causes and mechanisms of cancer evolution must be incessant, since innumerable neoplasms can be prevented, especially when induced by exogenous factors. The aim of this study was to study canine cutaneous SCC in the light of different histomorphological patterns of the neoplasia, evaluating its immunophenotype response to EGFR and c-erbB2 epidermal growth factor receptors. For that, the cases of canine SCC were analyzed from the archive of the Animal Pathology Sector of the EVZ / UFG from 2006 to 2015. For the epidemiological study, registration information was considered, including breed, sex, age and anatomical location. Regarding the histomorphological evaluation and malignancy criteria, the lesions were classified according to the system recommended by the Müller e Kirks. Anti-EGFR (HER1) and anti-c-erbB-2 (HER2) antibodies were used for the immunohistochemical study to better understand the role of these proteins in the mechanisms involved in the genesis, proliferation and evolution of SCC in dogs. Considering the histomorphological and immunophenotypic analyzes, the correlation between its variables was tested, and a correlation was verified between the EGFR immunostaining and the degree of SCC differentiation (r = 0.26, p = 0.02). On the other hand, there was no correlation between c-erbB2 immunostaining and histomorphological differentiation (r = 0.02; p = 0.83). When the correlation between EGFR and c-erbB2 immunoblots was tested, a positive correlation was also observed, but not statistically significant (r = 0.21, p = 0.06). It is concluded that there is a propensity that the increase in EGFR immunoexpression is directly proportional to the degree of SCC differentiation, and that it does not occur with c-erbB2 immunostaining. Also, SCCs in dogs seems to exhibit simultaneous increase of EGF receptor immunostaining.

Desenvolvimento in vitro de folículos pré-antrais caprinos na presença de kit ligand (kl) e proteína morfogenética óssea 15 (bmp-15) / Development of in vitro preantral follicles goats in the presence of kit ligand (kl) and protein bone morphogenetic 15 (bmp-15)

Esse estudo teve como objetivos: 1) quantificar os níveis de RNAm para os receptores de kit ligand (KL) e proteína morfogenética óssea 15 (BMP-15) em folículos ovarianos caprinos utilizando RT-PCR em tempo real; 2) investigar o papel do KL, associado ou não ao hormônio folículo estimulante (FSH) em meio dinâmico, sobre a sobrevivência, ativação e crescimento de folículos pré-antrais caprinos cultivados in situ; 3) avaliar os efeitos do KL e da BMP-15 sobre a sobrevivência, formação de antro e crescimento in vitro de folículos secundários caprinos isolados, bem como sobre a retomada da meiose de seus oócitos. [...] Verificou-se ainda níveis maiores de RNAm para o receptor tipo I da BMP-15 (BMPRIB) nas células da granulosa/teca de grandes folículos antrais em comparação àquelas de pequenos antrais. Após 16 dias de cultivo in situ, observou-se que o meio dinâmico contendo KL até o dia 8 e FSH do dia 8 ao dia 16 manteve a integridade ultraestrutural e induziu a ativação e o crescimento folicular. Quanto aos folículos isolados, a adição de 50 e/ou 100 ng/mL de KL ao cultivo, na ausência de FSH, promoveu a sobrevivência e melhorou a formação de antro, crescimento oocitário e retomada da meiose após 18 dias. Já 50 ng/mL de BMP-15 manteve a ultraestrutura e estimulou a formação de antro e o crescimento de folículos isolados. Em conclusão, os resultados deste estudo mostraram que os RNAm para c-kit, BMPRIB e BMPRII foram detectados em todas as categorias foliculares e tipos celulares investigados. A utilização de KL e FSH de forma dinâmica em cultivo in situ, bem como de KL (50 e/ou 100 ng/mL) e BMP-15 (50 ng/mL) em cultivo de folículos secundários isolados, promoveu a manutenção da sobrevivência folicular e o desenvolvimento de folículos pré-antrais caprinos.(AU)

The aims of this study were: 1) to quantify the mRNA levels for kit ligand (KL) and bone morphogenetic protein 15 (BMP-15) receptors in goat ovarian follicles by real time RT-PCR; 2) to investigate the role of KL alone or associated with follicle stimulating hormone (FSH) in a dynamic medium on the survival, activation and growth of goat preantral follicles cultured in situ; 3) to evaluate the effects of KL and BMP-15 on the in vitro survival, antrum formation and growth of goat isolated secondary follicles, and on the meiotic resumption of their oocytes. [...] Moreover, higher mRNA levels were observed for ckit in cumulus-oocyte complexes from small and large antral follicles compared with their respective granulosa/theca cells, which also occurred for BMPRII in the large antral follicles. Higher mRNA levels were also seen for BMP-15 type I receptor (BMPRIB) in the granulosa/theca cells from large antral follicles compared with those from small antral follicles. After 16 days of in situ culture, the dynamic medium containing KL until day 8 and FSH from day 8 to day 16 maintained the ultrastructural integrity and induced activation and follicular growth. For the isolated follicles, the addition of 50 and/or 100 ng/mL of KL to the culture, in the absence of FSH, promoted follicle survival and improved antrum formation,oocyte growth and meiotic resumption after 18 days. BMP-15 (50 ng/mL) maintained the ultrastructure and stimulated antrum formation and growth in isolated follicles. In conclusion, the results of this study showed that mRNAs for c-kit, BMPRIB and BMPRII were detected in all follicular categories and cellular types investigated. The use of KL and FSH dynamically in in situ culture, and the use of KL (50 and/or 100 ng/mL) and BMP-15 (50 ng/mL) in culture of isolated secondary follicles, promoted the maintenance of follicular survival and the development of caprine preantral follicles.(AU)

Desenvolvimento in vitro de folículos pré-antrais caprinos na presença de kit ligand (kl) e proteína morfogenética óssea 15 (bmp-15) / Development of in vitro preantral follicles goats in the presence of kit ligand (kl) and protein bone morphogenetic 15 (bmp-15)

Esse estudo teve como objetivos: 1) quantificar os níveis de RNAm para os receptores de kit ligand (KL) e proteína morfogenética óssea 15 (BMP-15) em folículos ovarianos caprinos utilizando RT-PCR em tempo real; 2) investigar o papel do KL, associado ou não ao hormônio folículo estimulante (FSH) em meio dinâmico, sobre a sobrevivência, ativação e crescimento de folículos pré-antrais caprinos cultivados in situ; 3) avaliar os efeitos do KL e da BMP-15 sobre a sobrevivência, formação de antro e crescimento in vitro de folículos secundários caprinos isolados, bem como sobre a retomada da meiose de seus oócitos. [...] Verificou-se ainda níveis maiores de RNAm para o receptor tipo I da BMP-15 (BMPRIB) nas células da granulosa/teca de grandes folículos antrais em comparação àquelas de pequenos antrais. Após 16 dias de cultivo in situ, observou-se que o meio dinâmico contendo KL até o dia 8 e FSH do dia 8 ao dia 16 manteve a integridade ultraestrutural e induziu a ativação e o crescimento folicular. Quanto aos folículos isolados, a adição de 50 e/ou 100 ng/mL de KL ao cultivo, na ausência de FSH, promoveu a sobrevivência e melhorou a formação de antro, crescimento oocitário e retomada da meiose após 18 dias. Já 50 ng/mL de BMP-15 manteve a ultraestrutura e estimulou a formação de antro e o crescimento de folículos isolados. Em conclusão, os resultados deste estudo mostraram que os RNAm para c-kit, BMPRIB e BMPRII foram detectados em todas as categorias foliculares e tipos celulares investigados. A utilização de KL e FSH de forma dinâmica em cultivo in situ, bem como de KL (50 e/ou 100 ng/mL) e BMP-15 (50 ng/mL) em cultivo de folículos secundários isolados, promoveu a manutenção da sobrevivência folicular e o desenvolvimento de folículos pré-antrais caprinos.

The aims of this study were: 1) to quantify the mRNA levels for kit ligand (KL) and bone morphogenetic protein 15 (BMP-15) receptors in goat ovarian follicles by real time RT-PCR; 2) to investigate the role of KL alone or associated with follicle stimulating hormone (FSH) in a dynamic medium on the survival, activation and growth of goat preantral follicles cultured in situ; 3) to evaluate the effects of KL and BMP-15 on the in vitro survival, antrum formation and growth of goat isolated secondary follicles, and on the meiotic resumption of their oocytes. [...] Moreover, higher mRNA levels were observed for ckit in cumulus-oocyte complexes from small and large antral follicles compared with their respective granulosa/theca cells, which also occurred for BMPRII in the large antral follicles. Higher mRNA levels were also seen for BMP-15 type I receptor (BMPRIB) in the granulosa/theca cells from large antral follicles compared with those from small antral follicles. After 16 days of in situ culture, the dynamic medium containing KL until day 8 and FSH from day 8 to day 16 maintained the ultrastructural integrity and induced activation and follicular growth. For the isolated follicles, the addition of 50 and/or 100 ng/mL of KL to the culture, in the absence of FSH, promoted follicle survival and improved antrum formation,oocyte growth and meiotic resumption after 18 days. BMP-15 (50 ng/mL) maintained the ultrastructure and stimulated antrum formation and growth in isolated follicles. In conclusion, the results of this study showed that mRNAs for c-kit, BMPRIB and BMPRII were detected in all follicular categories and cellular types investigated. The use of KL and FSH dynamically in in situ culture, and the use of KL (50 and/or 100 ng/mL) and BMP-15 (50 ng/mL) in culture of isolated secondary follicles, promoted the maintenance of follicular survival and the development of caprine preantral follicles.

Utilização do hormônio luteinizante (lh), fator de crescimento epidermal (egf) e ativina-a no cultivo in vitro de folículos pré-antrais caprinos isolados / Activin-A promotes the development of goat isolated secondary folliclesin vitro

[...] No experimento 1, foi verificado que a adição de LH a partir do dia 6 de cultivo aumentou as taxas de oócitos ? 110 µm, bem como a taxa de retomada da meiose (P<0,05). Por outro lado, quando o LH foi adicionado no início do cultivo foram obtidos apenas oócitos em estádio de vesícula germinativa. No experimento 2, no dia 6 de cultivo de cultivo, a adição de 50 ng/mL de EGF aumentou as taxas de formação do antro, em comparação com o controle cultivado, e após 18 dias resultou em maiores taxas de retomada da meiose quando comparado aos demais tratamentos (P<0,05). Na presença de EGF, a taxa de crescimento folicular diária foi superior em comparação ao controle cultivado (P<0,05). Após 18 dias, os níveis de RNAm para o FSH-R e aromatase P450 foram superiores ao controle não cultivado (P<0,05). No experimento 3 no dia 6 de cultivo, a taxa de formação de antro foi superior na presença de ativina-A quando comparado ao controle cultivado (P<0,05). A adição de 50 ng/mL de ativina-A aumentou a taxa de crescimento folicular do D12 para o D18, resultando em maiores taxas de retomada da meiose (P<0,05). No D6, a ativina-A (50 ng/ml) aumentou os níveis de RNAm para seu receptor (ActR-IA) quando comparado ao controle cultivado (P<0,05). Após 18 dias, a adição de 50 ng/ml de ativina-A aumentou significativamente seus próprios níveis de RNAm comparado ao controle não cultivado. Além disso, este tratamento reduziu os níveis de RNAm para aromatase P450 quando comparado ao controle cultivado (P <0,05). Em conclusão, o momento de adição de LH no meio de cultivo afetou o desenvolvimento de folículos pré-antrais caprinos cultivados. Os efeitos do EGF e os níveis de mRNA para o EGF, EGF-R, FSH-R aromatase P450 variaram de acordo com a fase de desenvolvimento folicular. A adição de ativina-A, de forma dependente, ao meio de cultivo promoveu o desenvolvimento de folículos pré-antrais caprinos após o cultivo.

[...] In experiment 1, was verified that the addition of LH from D6 onward of culture significantly increased the rates of oocytes ≥ 110 μm and the resumption of meiosis rate. On the other hand, when the LH was added since the beginning of culture, only oocytes at the germinal vesicle stage were obtained. In experiment 2, after 6 days of culture, the addition of 50 ng/ml EGF increased the rates of antrum formation compared to the cultured control, and after 18 days this treatment produced oocytes with higher rates of meiosis resumption compared to the other treatments (P<0.05). In the presence of EGF, the follicular growth rate was higher compared to the cultured control (P<0.05). After 18 days, the mRNA levels for FSH-R and P 450 aromatase were higher than the non-cultured control (P<0.05). In experiment 3, after 6 days of culture, the rate of antrum formation was higher in the presence of activin-A compared to the cultured control (P<0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate from D12 to D18, resulting in higher rates of meiosis resumption (P<0.05). On D6, the activin-A (50 ng/ml) increased the mRNA levels for its own receptor (ActR-IA) compared to the cultured control (P<0.05). After 18 days, the addition of 50 ng/ml activin-A also significantly increased its own mRNA levels compared to the non-cultured control. Furthermore, this treatment reduced the mRNA levels for P 450 aromatase compared to the cultured control (P<0.05). In conclusion, the moment of LH addition to the culture medium affects the development of cultured goat preantral follicles. The effects of EGF and mRNA levels for EGF, EGF-R, FSH-R and P 450 aromatase varied according to the follicular developmental stage. The addition of activin-A to the culture medium promoted the development of goat preantral follicles after culture.

Utilização do hormônio luteinizante (lh), fator de crescimento epidermal (egf) e ativina-a no cultivo in vitro de folículos pré-antrais caprinos isolados / Activin-A promotes the development of goat isolated secondary folliclesin vitro

[...] No experimento 1, foi verificado que a adição de LH a partir do dia 6 de cultivo aumentou as taxas de oócitos ? 110 µm, bem como a taxa de retomada da meiose (P<0,05). Por outro lado, quando o LH foi adicionado no início do cultivo foram obtidos apenas oócitos em estádio de vesícula germinativa. No experimento 2, no dia 6 de cultivo de cultivo, a adição de 50 ng/mL de EGF aumentou as taxas de formação do antro, em comparação com o controle cultivado, e após 18 dias resultou em maiores taxas de retomada da meiose quando comparado aos demais tratamentos (P<0,05). Na presença de EGF, a taxa de crescimento folicular diária foi superior em comparação ao controle cultivado (P<0,05). Após 18 dias, os níveis de RNAm para o FSH-R e aromatase P450 foram superiores ao controle não cultivado (P<0,05). No experimento 3 no dia 6 de cultivo, a taxa de formação de antro foi superior na presença de ativina-A quando comparado ao controle cultivado (P<0,05). A adição de 50 ng/mL de ativina-A aumentou a taxa de crescimento folicular do D12 para o D18, resultando em maiores taxas de retomada da meiose (P<0,05). No D6, a ativina-A (50 ng/ml) aumentou os níveis de RNAm para seu receptor (ActR-IA) quando comparado ao controle cultivado (P<0,05). Após 18 dias, a adição de 50 ng/ml de ativina-A aumentou significativamente seus próprios níveis de RNAm comparado ao controle não cultivado. Além disso, este tratamento reduziu os níveis de RNAm para aromatase P450 quando comparado ao controle cultivado (P <0,05). Em conclusão, o momento de adição de LH no meio de cultivo afetou o desenvolvimento de folículos pré-antrais caprinos cultivados. Os efeitos do EGF e os níveis de mRNA para o EGF, EGF-R, FSH-R aromatase P450 variaram de acordo com a fase de desenvolvimento folicular. A adição de ativina-A, de forma dependente, ao meio de cultivo promoveu o desenvolvimento de folículos pré-antrais caprinos após o cultivo.(AU)

[...] In experiment 1, was verified that the addition of LH from D6 onward of culture significantly increased the rates of oocytes ≥ 110 μm and the resumption of meiosis rate. On the other hand, when the LH was added since the beginning of culture, only oocytes at the germinal vesicle stage were obtained. In experiment 2, after 6 days of culture, the addition of 50 ng/ml EGF increased the rates of antrum formation compared to the cultured control, and after 18 days this treatment produced oocytes with higher rates of meiosis resumption compared to the other treatments (P<0.05). In the presence of EGF, the follicular growth rate was higher compared to the cultured control (P<0.05). After 18 days, the mRNA levels for FSH-R and P 450 aromatase were higher than the non-cultured control (P<0.05). In experiment 3, after 6 days of culture, the rate of antrum formation was higher in the presence of activin-A compared to the cultured control (P<0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate from D12 to D18, resulting in higher rates of meiosis resumption (P<0.05). On D6, the activin-A (50 ng/ml) increased the mRNA levels for its own receptor (ActR-IA) compared to the cultured control (P<0.05). After 18 days, the addition of 50 ng/ml activin-A also significantly increased its own mRNA levels compared to the non-cultured control. Furthermore, this treatment reduced the mRNA levels for P 450 aromatase compared to the cultured control (P<0.05). In conclusion, the moment of LH addition to the culture medium affects the development of cultured goat preantral follicles. The effects of EGF and mRNA levels for EGF, EGF-R, FSH-R and P 450 aromatase varied according to the follicular developmental stage. The addition of activin-A to the culture medium promoted the development of goat preantral follicles after culture.(AU)

Prognostic Factors in Canine Mammary Carcinomas and HER2 Expression Relationship

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging.Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). The histologica

Prognostic Factors in Canine Mammary Carcinomas and HER2 Expression Relationship

Background: The human epidermal growth factor type 2 (HER2) receptor is a membrane glycoprotein tyrosine kinase. In woman, HER2 expression is diagnosed in 30% of breast carcinomas and it is associated with a worse prognosis, higher rate of recurrence and mortality. In the bitch, the HER2 overexpression in canine mammary tumors is still controversial and the prognostic value remains uncertain. Thus, we aimed to verify the HER2 expression in canine mammary carcinomas and relate it to the type and histological grade, lymph node metastasis and clinical staging.Materials, Methods & Results: Ninety bitches diagnosed with mammary carcinoma were included in this study. The inclusion criteria were bitches with complete clinical examination, thoracic radiographic examination and submitted unilateral or bilateral mastectomy. Ninety-nine samples of mammary carcinoma were used and the fragments of tumor and regional lymph nodes were fixed in 10% neutral formalin for histopathological and immunohistochemistry analysis. The lesions were evaluated by two pathologists and classified according to the type and histological grade. HER2 expression was performed by semi-quantitative analysis of the slides according to the HerceptTestTM (Dako) recommended score. Simple carcinomas were the most frequent (51.51%) followed by complex carcinomas (46.47%) and in situ carcinoma (2.02%). The histologica