Epidermal Growth Factor Receptor (EGFR): immunoexpression and mutation analysis in canine mammary cancer
Data
2019-08-06
Autores
Título da revista
ISSN da revista
Título do Volume
Editora
Resumo
Os tumores mamários são a neoplasia mais frequentemente diagnosticada à população canina feminina. Apesar da sua elevada incidência, importância clínica e semelhança com o cancro de mama da mulher, os estudos direcionados para a sua caracterização são insuficientes, especialmente em relação ao Recetor do Fator de Crescimento Epidérmico (EGFR). O EGFR é um recetor transmembranar para membros da família de ligandos extracelulares do Fator de Crescimento Epidérmico e está envolvido na regulação de várias funções celulares, tais como proliferação, diferenciação, motilidade e sobrevivência celulares. Alterações no gene EGFR têm sido associadas à progressão neoplásica, levando à sobre-expressão da proteína EGFR. De acordo com a literatura recente, observa-se sobre-expressão desta proteína em 34 a 56% das neoplasias mamárias malignas caninas. A regulação positiva do gene EGFR pode ser causada por amplificações génicas e mutações na sequência de nucleótidos, como substituições “missense”, que originam proteínas aberrantes capazes de ativação independente do ligando. Neste estudo, a expressão do EGFR foi avaliada por imunohistoquímica em 111 tumores mamários malignos caninos (40 tumores primários não-metastáticos e 71 metastáticos), 68 metástases ganglionares e 12 metástases pulmonares. A sobre-expressão do EGFR foi observada em 53,2% (n=59) dos carcinomas, 47,1% (n=32) das metástases ganglionares e 25% (n=3) das metástases pulmonares. Verificou-se que a expressão do EGFR em tumores primários e metástases ganglionares estava significativamente associada (P<0,0001), com concordância em 79,4% (n=54) das amostras. Para além da expressão epitelial, 37,8% (n=42) dos tumores primários exibiram uma intensa coloração no estroma justa-neoplásico. Também foi realizada uma análise molecular; 30 amostras de tecido mamário canino congelado (20 carcinomas e 10 tecidos não-neoplásicos adjacentes) foram selecionadas para extração de DNA, PCR e sequenciação, e rastreadas quanto à presença de variantes da sequência do exão 19 do gene EGFR. Foram detetados quatro polimorfismos intrónicos em 6 amostras (4 carcinomas e 2 tecidos não-neoplásicos adjacentes). Todas as variantes já tinham sido reportadas, embora não sejam referidas em nenhuma publicação. Embora três destes quatro carcinomas também tenham demonstrado sobre-expressão do EGFR por imunohistoquímica, as restantes amostras EGFR-positivas não revelaram alterações genéticas. Foram ainda selecionadas para análise molecular sete amostras de tecido de metástases ganglionares, fixadas em formalina e embebidas em parafina (FFPE). Foram testados três métodos de extração de DNA e foi realizada uma avaliação da eficiência de cada um e da qualidade do DNA extraído. Embora exista um extenso acervo de amostras mamárias caninas FFPE, com enorme potencial para a investigação científica, é necessário otimizar as metodologias de extração de DNA. São necessários mais estudos com maior número de amostras para confirmar que as variações do gene EGFR são eventos raros no cancro de mama canino, bem como investigar a presença de variações do número de cópias (CNV) do EGFR em tumores mamários malignos caninos e verificar se existe alguma associação entre a CNV do gene EGFR e a sobre-expressão da sua proteína. Tanto quanto sabemos, este é o primeiro estudo a estabelecer uma comparação entre a imunoexpressão da proteína EGFR e as alterações genéticas do EGFR no cancro de mama canino.
Mammary tumours are the most frequent neoplasm diagnosed among the female canine population. Despite their high incidence, clinical importance and similarity with human breast cancer, studies targeting the characterization of canine mammary cancer are insufficient, especially with respect to Epidermal Growth Factor Receptor (EGFR). EGFR is a transmembrane receptor for members of the Epidermal Growth Factor family of extracellular ligands and it is involved in regulating various cellular functions, such as cell proliferation, differentiation, motility and survival. Alterations in the EGFR gene favour neoplastic progression, usually leading to overexpression of the EGFR protein. According to the recent literature, EGFR overexpression is observed in 34 to 56% of canine mammary malignant tumours. Upregulation of EGFR gene is commonly caused by gene amplifications and mutations in the nucleotide sequence, such as missense substitutions, that originate aberrant proteins capable of ligand-independent activation. In this study, EGFR expression was evaluated by immunohistochemical analysis in 111 canine malignant mammary tumours (40 non-metastatic and 71 metastatic primary tumours), 68 lymph node metastases and 12 lung metastases. EGFR overexpression was observed in 53.2% (n=59) of carcinomas, 47.1% (n=32) of node metastases and 25% (n=3) of lung metastases. EGFR expression in primary tumours and lymph node metastases was significantly associated (P<0.0001), with a concordance in 79.4% (n=54) samples. Besides epithelial expression, 37.8% (n=42) of primary tumours exhibited an intense staining in the juxtaneoplastic stroma. A molecular analysis was also carried out; 30 frozen canine mammary tissue samples (20 carcinomas and 10 adjacent non-neoplastic tissues) were selected for DNA extraction, PCR and sequencing, and screened for the presence of sequence variants of EGFR gene exon 19. Four intronic polymorphisms were detected in 6 samples (4 carcinomas and 2 adjacent nonneoplastic tissues). All variants have been reported, although no publication refers to them. Although three of these four carcinomas also showed EGFR overexpression by immunohistochemistry, the remaining positive EGFR samples fail to reveal gene alterations. Additionally, seven formalin-fixed paraffin-embedded (FFPE) tissues from lymph node metastases were also selected for molecular analysis. Three different methods were tested for DNA extraction and an evaluation of the efficiency and quality of the extracted DNA was performed. Although there is an extensive archive of FFPE canine mammary samples, with enormous potential for scientific research, it is mandatory to optimize DNA extraction methodologies. Additional studies with a larger series are necessary in order to confirm that EGFR gene variations are rare events in canine mammary cancer, as well as to investigate the presence of EGFR Copy Number Variations (CNV) in canine mammary malignant tumours and to verify if there is any association between CNV of EGFR gene and the overexpression of its protein. To the best of our knowledge, this is the first study that established a comparison between EGFR protein immunoexpression and EGFR genetic alterations in canine mammary cancer.
Mammary tumours are the most frequent neoplasm diagnosed among the female canine population. Despite their high incidence, clinical importance and similarity with human breast cancer, studies targeting the characterization of canine mammary cancer are insufficient, especially with respect to Epidermal Growth Factor Receptor (EGFR). EGFR is a transmembrane receptor for members of the Epidermal Growth Factor family of extracellular ligands and it is involved in regulating various cellular functions, such as cell proliferation, differentiation, motility and survival. Alterations in the EGFR gene favour neoplastic progression, usually leading to overexpression of the EGFR protein. According to the recent literature, EGFR overexpression is observed in 34 to 56% of canine mammary malignant tumours. Upregulation of EGFR gene is commonly caused by gene amplifications and mutations in the nucleotide sequence, such as missense substitutions, that originate aberrant proteins capable of ligand-independent activation. In this study, EGFR expression was evaluated by immunohistochemical analysis in 111 canine malignant mammary tumours (40 non-metastatic and 71 metastatic primary tumours), 68 lymph node metastases and 12 lung metastases. EGFR overexpression was observed in 53.2% (n=59) of carcinomas, 47.1% (n=32) of node metastases and 25% (n=3) of lung metastases. EGFR expression in primary tumours and lymph node metastases was significantly associated (P<0.0001), with a concordance in 79.4% (n=54) samples. Besides epithelial expression, 37.8% (n=42) of primary tumours exhibited an intense staining in the juxtaneoplastic stroma. A molecular analysis was also carried out; 30 frozen canine mammary tissue samples (20 carcinomas and 10 adjacent non-neoplastic tissues) were selected for DNA extraction, PCR and sequencing, and screened for the presence of sequence variants of EGFR gene exon 19. Four intronic polymorphisms were detected in 6 samples (4 carcinomas and 2 adjacent nonneoplastic tissues). All variants have been reported, although no publication refers to them. Although three of these four carcinomas also showed EGFR overexpression by immunohistochemistry, the remaining positive EGFR samples fail to reveal gene alterations. Additionally, seven formalin-fixed paraffin-embedded (FFPE) tissues from lymph node metastases were also selected for molecular analysis. Three different methods were tested for DNA extraction and an evaluation of the efficiency and quality of the extracted DNA was performed. Although there is an extensive archive of FFPE canine mammary samples, with enormous potential for scientific research, it is mandatory to optimize DNA extraction methodologies. Additional studies with a larger series are necessary in order to confirm that EGFR gene variations are rare events in canine mammary cancer, as well as to investigate the presence of EGFR Copy Number Variations (CNV) in canine mammary malignant tumours and to verify if there is any association between CNV of EGFR gene and the overexpression of its protein. To the best of our knowledge, this is the first study that established a comparison between EGFR protein immunoexpression and EGFR genetic alterations in canine mammary cancer.
Descrição
Dissertação de Mestrado em Biotecnologia para as Ciências da Saúde
Palavras-chave
Cancro de mama canino , expressão da proteína EGFR