Estudo histológico e imunohistoquímico da expressão de Ki-67 em carcinomas das células escamosas em gatos
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2024
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O cancro é uma doença altamente prevalente em animais de companhia, sendo uma das suas principais causas de morte. O carcinoma das células escamosas (CCE) é um dos tumores mais comuns em gatos, levando à desfiguração e incapacitação dos animais relacionada com a progressão da doença, às sequelas terapêuticas ou mesmo à morte em casos incuráveis. Para desenvolver melhores ferramentas de diagnóstico e prognóstico, é necessário estudar a adequação de vários biomarcadores, bem como o seu comportamento dentro do tumor. O objetivo deste trabalho foi avaliar a distribuição do marcador de proliferação Ki-67 em CCE de três localizações anatómicas diferentes. Foram selecionadas 25 amostras de CCE de gatos previamente diagnosticados por análise histológica, localizadas no plano nasal (N; n=8), pavilhão auricular (E; n=10) e cavidade oral (O; n=9). O grau de malignidade foi calculado de acordo com classificações previamente validadas, com 23 casos classificados como Grau 1 e dois como Grau 2. Foi realizada imunohistoquímica indireta para quantificar o Ki-67 em seções tumorais embebidas em parafina e as lâminas foram digitalizadas e analisadas com recurso ao software QuPath (v0.4.3), através da seleção manual de quatro regiões especificas de 1) tumor (T), 2) estroma adjacente (S), 3) frente invasora tumoral (IF) e 4) superfície tumoral (TS). Os dados das contagens resultantes foram processados com SPSS (v.25) utilizando testes paramétricos e não paramétricos consoante normalidade da distribuição dos dados em cada variável. As diferenças foram consideradas significativas quando p<0,05. Os resultados demonstram que dentro de uma mesma região alvo de cada amostra individual não há diferenças estastisticamente significativas no número de células positivas para Ki-67. Quanto às comparações entre áreas, não houve diferenças de proliferação entre TS e IF. No entanto, as áreas T apresentavam significativamente mais células marcadas para Ki-67 do que as regiões S correspondentes (p = 0,001), embora a inspeção visual mostrasse que, nestas últimas, a marcação positiva se devia principalmente a células inflamatórias infiltradas no estroma. Finalmente, verificaram-se diferenças na marcação da IF de CCE entre a localização N e a localização E (p = 0,032), sugerindo uma frente invasora mais agressiva. Este estudo demonstra que a distribuição do Ki-67 numa mesma região alvo de cada tumor é homogénea, o que pode facilitar a seleção das áreas a serem analisadas digitalmente. Em relação ao comportamento tumoral, os resultados revelaram que, embora haja proliferação observável nas células estromais adjacentes do CCE, tal deve-se à presença de são inferiores quando comparadas com as contagens das células tumorais. Finalmente, pode concluir-se que os CCE do plano nasal parecem ter um comportamento de infiltração mais agressivo do que os seus homólogos do pavilhão auricular. A análise de mais amostras destes locais de CCE (e outros) é necessária para consolidar estas observações e associar a coloração Ki-67 a outras ferramentas de classificação de tumores. Palavras-chave: Carcinoma de células escamosas; proliferação; gatos; Ki-67; heterogeneidade; frente invasora. células inflamatórias infiltrativas marcadas para o Ki-67, sendo que as contagens estromais
Cancer is highly prevalent in companion animals and is a major cause of death. Squamous cell carcinoma (SCC) is one of the most common feline tumours, leading to disfiguration and disability related to disease progression, therapeutic sequelae or death in incurable cases. To develop better diagnostic and prognostic tools, the suitability of several biomarkers needs to be studied, as well as their behaviour within the tumour. The aim of this work was to evaluate the distribution of the Ki-67 proliferation marker within SCC tumours from three different anatomical locations. 25 samples of previously diagnosed feline SCC located in the nasal plane (N; n=8), ear pinna (E; n=10) and oral cavity (O; n=9) were selected. Malignancy score was calculated according to previously validated classifications with 23 cases classified as Grade1 and two as Grade2. Indirect immunohistochemistry was performed to locate Ki-67 in paraffin embedded tumour sections. After visual observation, slides were digitalized and positively stained nucleus were identified using QuPath (v0.4.3) software by manually selecting four target areas of 1) tumour (T), 2) adjacent stroma (S), 3) invasive tumour front (IF) and 4) tumour surface (TS). Resulting data was processed with SPSS (v.25) using Wilcoxon and Kruskal-Walis or t-Student test according to the normality of data distribution. Differences were considered significant when p<0.05. Results show that within the same target area in the sample, there are no differences in the number of Ki-67 positive cells, showing proliferation. Regarding comparisons between areas, there were no differences between proliferation in TS and IF. However, T areas had significantly more Ki-67 positive marked cells than their correspondent S areas (p=0.001), although visual inspection showed that, in the latter, positive marking was mostly due to stroma infiltrated inflammatory cells. Finally, regarding proliferation in IF, results show that, although there were no differences between proliferation between O and N and E, N SCC had more positively marked Ki-67 cells than E SCC (p=0.032), suggesting a more aggressive invasive front. This study shows that Ki-67 distribution within the same target area of the tumour is homogenous, which may facilitate the selection of areas to be digitally analysed. Regarding tumour behaviour, results show that even though there is observable proliferation in SCC adjacent stromal cells, this is most likely due to infiltrative inflammatory cells and is always inferior to tumoral proliferation itself. Finally, we can conclude that nasal plane SCC seem to have a more aggressive infiltration behaviour than their: ear pinna counterparts. Analysis of more samples from these SCC locations (and others) is required to consolidate these findings and associate Ki-67 staining with other tumour classification tools. Palavras chave: Squamous cells carcinoma; cat; proliferation; Ki-67; heterogeneity; invasive front.
Cancer is highly prevalent in companion animals and is a major cause of death. Squamous cell carcinoma (SCC) is one of the most common feline tumours, leading to disfiguration and disability related to disease progression, therapeutic sequelae or death in incurable cases. To develop better diagnostic and prognostic tools, the suitability of several biomarkers needs to be studied, as well as their behaviour within the tumour. The aim of this work was to evaluate the distribution of the Ki-67 proliferation marker within SCC tumours from three different anatomical locations. 25 samples of previously diagnosed feline SCC located in the nasal plane (N; n=8), ear pinna (E; n=10) and oral cavity (O; n=9) were selected. Malignancy score was calculated according to previously validated classifications with 23 cases classified as Grade1 and two as Grade2. Indirect immunohistochemistry was performed to locate Ki-67 in paraffin embedded tumour sections. After visual observation, slides were digitalized and positively stained nucleus were identified using QuPath (v0.4.3) software by manually selecting four target areas of 1) tumour (T), 2) adjacent stroma (S), 3) invasive tumour front (IF) and 4) tumour surface (TS). Resulting data was processed with SPSS (v.25) using Wilcoxon and Kruskal-Walis or t-Student test according to the normality of data distribution. Differences were considered significant when p<0.05. Results show that within the same target area in the sample, there are no differences in the number of Ki-67 positive cells, showing proliferation. Regarding comparisons between areas, there were no differences between proliferation in TS and IF. However, T areas had significantly more Ki-67 positive marked cells than their correspondent S areas (p=0.001), although visual inspection showed that, in the latter, positive marking was mostly due to stroma infiltrated inflammatory cells. Finally, regarding proliferation in IF, results show that, although there were no differences between proliferation between O and N and E, N SCC had more positively marked Ki-67 cells than E SCC (p=0.032), suggesting a more aggressive invasive front. This study shows that Ki-67 distribution within the same target area of the tumour is homogenous, which may facilitate the selection of areas to be digitally analysed. Regarding tumour behaviour, results show that even though there is observable proliferation in SCC adjacent stromal cells, this is most likely due to infiltrative inflammatory cells and is always inferior to tumoral proliferation itself. Finally, we can conclude that nasal plane SCC seem to have a more aggressive infiltration behaviour than their: ear pinna counterparts. Analysis of more samples from these SCC locations (and others) is required to consolidate these findings and associate Ki-67 staining with other tumour classification tools. Palavras chave: Squamous cells carcinoma; cat; proliferation; Ki-67; heterogeneity; invasive front.
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VETERINARY MEDICINE, SQUAMOUS CELL CARCINOMA, CATS, CELL DIVISION, BIOMARKERS, MESTRADO INTEGRADO EM MEDICINA VETERINÁRIA, VETERINÁRIA, MEDICINA VETERINÁRIA, CARCINOMA DE CÉLULAS ESCAMOSAS, GATOS, DIVISÃO CELULAR, BIOMARCADORES, Mestrado Integrado em Medicina Veterinária