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André Guollo AVALIAÇÃO DO PERFIL DE EXPRESSÃO GÊNICA GLOBAL NA PROGRESSÃO DE LEUCOPLASIA VERRUCOSA PROLIFERATIVA ORAL ANDRÉ GUOLLO Dissertação apresentada à Fundação Antônio Prudente para a obtenção do título de Mestre em Ciências Área de Concentração: Oncologia Orientador: Prof. Dr. Fábio de Abreu Alves Co-Orientadora: Prof.ª Dra. Silvia Regina Rogatto São Paulo 2016 FICHA CATALOGRÁFICA Preparada pela Biblioteca da Fundação Antônio Prudente Guollo, André Avaliação do perfil de expressão gênica global na progressão de leucoplasia verrucosa proliferativa oral / André Guollo – São Paulo, 2016. 103p. Dissertação (Mestrado)-Fundação Antônio Prudente. Curso de Pós-Graduação em Ciências - Área de concentração: Oncologia. Orientador: Fábio de Abreu Alves Descritores: 1. LEUCOPLASIA/genética. 2. LEUCOPLASIA/etiologia. 3. NEOPLASIAS BUCAIS/diagnóstico. 4. INFECÇÕES POR PAPILLOMAVIRUS. “With a little help from my friends” Paul and John DEDICATÓRIA Dedico este trabalho a minha mãe Leda e meu pai Nelsi. E a todos os pacientes portadores desta condição. AGRADECIMENTOS Ao meu orientador Prof. Dr. Fábio de Abreu Alves, que abriu as portas do seu serviço para eu poder realizar minha formação, mudando minha carreira e minha vida. E pelo exemplo de profissionalismo e dedicação. A minha co-orientadora Profª Dra. Silvia Regina Rogatto, pela colaboração indispensável neste projeto, pelos conselhos, e aceitar que eu participasse da rotina do seu laboratório. E pelo exemplo de liderança. A minha família, minha mãe Leda, meu pai Nelsi, minha irmã Renata e meu irmão Felipe, por entenderem minha longa ausência, e por apoiarem minhas decisões. A minha namorada Fernanda Mariá, pelo Amor, pelo carinho, pela ajuda, e por estar sempre ao meu lado. A Profª Dra. Luísa Lina Villa, pela importante contribuição na realização da genotipagem do HPV. A todos os titulares do Departamento de Estomatologia, Dr. José Divaldo, Dr. André Caroli, Dra. Graziella Jaguar, Dr. Rodrigo Lopes e Dra. Ana Paula, por fazerem parte da minha formação. Aos colegas e amigos do Laboratório Neogene, Mateus, Hellen, Fábio, Mariana, Julia, Luiza, Sara, Isabella, Priscila, Juan, Marco, André, Flávia, Caroline, Tatiana, Maísa, Graziela, pela ajuda em todas as etapas deste projeto. Sem vocês, ele não sairia do papel. Ao Departamento de Cirurgia de Cabeça e Pescoço e ao Departamento de Anatomia Patológica. Aosfuncionários do CIPE. A Biblioteca do A.C.Camargo Cancer Center, em especial a Suely, Renato e Denis. Aos funcionários da Pós-Graduação da Fundação Antônio Prudente. RESUMO Guollo A. Avaliação do perfil de expressão gênica global na progressão de leucoplasia verrucosa proliferativa oral. São Paulo; 2016. [Dissertação de Mestrado-Fundação Antônio Prudente]. A Leucoplasia Verrucosa Proliferativa (LVP) é uma lesão multifocal que abrange amplas áreas da mucosa bucal sendo observada principalmente em mulheres acima de 50 anos. São observadas altas taxas de recidivas e cancerização.Considerando que não são conhecidos marcadores preditivos para a cancerização das LVP, torna-se evidente anecessidade da identificação de marcadores úteis para odiagnóstico precoce e alvos para terapias mais conservadoras. O objetivo deste estudo foi avaliar clinicamente pacientes com LVP e caracterizaro perfil de expressão gênica global e a presença do vírus do papiloma humano (HPV).Este é um estudo retrospectivo que analisou pacientes com LVP atendidos pelo Departamento de Estomatologia do A.C. Camargo Cancer Center, no período de 01/01/2003 a 30/01/2016. Foram incluídos 23 pacientes com idade média de 67 anos. O gênero feminino foi o mais acometido (78,2%) e o tempo de acompanhamento médio foi de 70 meses. A mucosa jugal foi o sitio mais acometido pelas lesões leucoplásicas. A taxa de cancerização foi de 65,2% e o tempo médio para o desenvolvimento do primeiro tumor foi de 20,5 meses. Houve uma associação significativa entre o tabagismo e a transformação maligna. Para a análise molecular, foram selecionados seis pacientes que apresentaram cancerização da LVP (3 amostras pareadas de cada: tecido leucoplásico - L, tecido tumoral- T e tecido normal adjacente - NA), totalizando 18 amostras. Amostras de DNA e RNA foram obtidas a partir de tecidos fixados e em blocos de parafina. A genotipagem do HPV foi realizada com o kit INNO-LiPA HPV Genotyping Extra Amp(Fujirebio, EUA). Aexpressão gênica global foi avaliada usando-se aplataforma HTA 2.0 da Affymetrix (GeneChip HTA 2.0).Foram identificados 1.650 genes diferencialmente expressos na comparação entre T e NA (568 com expressão aumentada e 1.082 reduzida), 90 genes na comparação entre L e NA (17 com expressão aumentada e 73 reduzida) e 845 genes na comparação entre Te L (293 com expressão aumentada e 552 reduzida) (P<0,05 e FoldChange>1,5). A análise de agrupamento hierárquico supervisionado (utilizando as 2.428 sondas diferencialmente expressas identificadas pelo teste ANOVA) revelou dois grupos moleculares distintos.Na análise linear (modelo de progressão NA<L<T ou NA>L>T) foram identificados 72 transcritos, 27 apresentando aumento e 45 diminuição de expressão. Estes transcritos encontravam-se em cinco vias canônicas presentes em dois programas utilizados para a análise in silico (Ingenuity Pathway Analysis e KOBAS 2.0), sendo a maior parte deles relacionados com adesão celular e citoesqueleto. Foi também detectada a inibição dos genes ESR1 e TP53, os quais foram preditos nas redes de interações moleculares. Três amostras foram positivas para HPV de alto risco, sendo uma leucoplasia e dois tumores. Os dados clínicos indicaram que a presente casuística teve o mesmo perfil clínico-patológico descrito em literatura, entretanto, os casos apresentaram uma associação significativa entre o tabagismo e a cancerização da LVP. O perfil de expressão gênica global demonstrou um padrão de progressão das alterações moleculares revelando agrupamentos distintos entre os tipos diferentes de tecidos estudados. Não houve associação entre as características clínicas-patológicas e o perfil transcriptômico das LVP. A análise linear (NA<L<T ou NA>L>T) revelou potenciais marcadores, os quais necessitam ser validados em um grupo independente de amostras. Nos casos avaliados, a infecção pelo HPV revelou ter importância na progressão maligna da LVP. SUMMARY Guollo A. [Evaluation of global gene expression profile in the progression of oral proliferative verrucous leukoplakia]. São Paulo; 2016. [Dissertação de Mestrado-Fundação Antônio Prudente]. Proliferative verrucous leukoplakia (PVL) is a multifocal lesion that covers large areas of the oral mucosa with prevalence in women over 50 years. High rates of recurrence and cancerization are observed. Since there are no predictive markers for the PVL cancerization, it is necessary to identify markers for early diagnosis as well as targets for more conservative therapies. The aim of this clinical study was to evaluate patients with PVL, to characterize its global gene expression profile and to identify the presence of human papilloma virus (HPV).This is a retrospective study that enrolled patients with PVL attended in the Stomatology Department of A.C.Camargo Cancer Center, from 01/01/2003 to 01/30/2016. Twenty-three patients with an average age of 67 years were included. Females were the most affected (78.2%) and the mean follow-up was 70 months. The buccal mucosa was the most affected site. The cancerization rate was 65.2% and the average time for development of the first tumor was 20.5 months. There was a significant association between smoking and malignant transformation. Six patients who had cancerization of PVL were selected for the molecular analysis whereas 3-paired samples (leukoplakia tissue - L, tumor tissue - T, and adjacent normal tissue – NA) were obtained from each subject, resulting in 18 samples. DNA and RNA samples were extracted from tissues fixed and embedded in paraffin. The HPV genotyping was performed by using the INNO-LiPA HPV Genotyping Extra Amp kit (Fujirebio, USA). The global gene expression was performed with platform Affymetrix HTA 2.0 (Gene Chip HTA 2.0). A total of 1,650 differentially expressed genes were identified when T was compared to NA (568 with increased expression and 1,082 reduced), whereas 90 genes were observed between L and NA (expression increased in 17 genes and reduced in 73), and 845 genes between T and L (expression increased in 293 genes and reduced in 552) (P<0.05 and Fold Change>1.5). The supervised hierarchical clustering analysis was conducted with the 2,428 differentially expressed probes identified by ANOVA and revealed two distinct molecular groups. In linear analysis (progression model NA<L<T or NA>L>T) 72 transcripts were identified, in which the expression increased in 27 transcripts and decreased in 45. These transcripts were observed into five canonical pathways of two programs used for the in silico analysis (Ingenuity Pathway Analysis and KOBAS 2.0). The majority of them was related to cell adhesion and cytoskeleton. It was also detected inhibition of ESR1 and TP53 genes that were predicted in the molecular networks. Three samples were positive for high-risk HPV, one for leukoplakia and two for tumors. Clinical data indicates that this series had the same clinico pathological profile described in literature. However, the cases presented showed a significant association between smoking and cancerization of PVL. The global gene expression profile showed a pattern of progression in the molecular changes revealing distinct clusters among different types of the studied tissues. There was no association
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