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Doctoral Thesis
DOI
Document
Author
Full name
Marcus Fernando Kodama Pertille Ramos
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2019
Supervisor
Committee
Júnior, Ulysses Ribeiro (President)
Zilberstein, Bruno
Júnior, Wilson Luiz da Costa
Montagnini, André Luis
Title in Portuguese
Caracterização dos subtipos moleculares do câncer gástrico por expressão gênica e proteica
Keywords in Portuguese
Biomarcadores
Hibridização in situ
Imuno-histoquímica
Neoplasias gástricas
Perfil de expressão gênica
Pesquisa médica translacional
Abstract in Portuguese
INTRODUÇÃO: Recentemente, a classificação molecular do câncer gástrico (CG) emergiu como opção promissora para definir subgrupos prognósticos, distinguir o comportamento biológico, além de permitir a identificação de potenciais alvos terapêuticos para drogas específicas. Por meio de técnicas moleculares, o CG foi dividido em 4 subtipos: instabilidade de microssatélites (MSI), vírus Epstein-Barr (EBV)-positivo, genomicamente estável (GS) e instabilidade cromossômica (CIN). Os custos associados à complexidade técnica das metodologias moleculares são ainda um obstáculo para sua implantação na prática de rotina. OBJETIVOS: Determinar os grupos da classificação molecular por meio da expressão proteica de marcadores associados a cada subtipo. Comparar as diferentes classificações moleculares existentes e propor um novo modelo. MÉTODOS: Foram avaliados, retrospectivamente, 287 pacientes com CG submetidos à gastrectomia-D2 curativa, por meio da construção de tissue microarray. A expressão das proteínas de reparo do DNA, E-caderina e p53 foram avaliadas por imuno-histoquímica (IH), determinando os grupos MSI, GS e CIN, respectivamente. O EBV foi detectado por hibridização in situ (ISH). RESULTADOS: Após avaliação histopatológica, 179 (62,4%) pacientes foram classificados como CIN, 58 (20,2%) MSI, 30 (10,5%) EBV e 20 (7%) como GS. Os subtipos associaram-se com características distintas, tais como: gênero masculino (EBV, p=0.101); idade avançada (MSI, p=0,017), menor relação neutrófilo-linfócito (CIN, p=0,029), gastrectomia total (EBV, p < 0,001), localização distal (MSI, p=0,004), Laurén difuso (GS, p < 0,001), e estádio avançado (GS, p=0,014). O subtipo MSI apresentou melhor sobrevida livre de doença (SLD) (82,8%), seguido pelo EBV e CIN (ambos com 70%) e GS (50%) (p=0.005). A sobrevida global (SG) foi maior nos tumores MSI (75,9%), seguido pelo EBV (73,3%), CIN (65.4%) e GS (45%, mediana de 25 meses) (p=0.007). Em análise multivariada, gastrectomia total, pT, pN e os subtipos tumorais foram fatores significativos associados à SLD (MSI p=0,012; EBV p=0,037; CIN p=0,018; GS referência). Do mesmo modo, o tipo de cirurgia, pT, e os subtipos tumorais foram fatores independentes associados a SG (MSI p=0,010; EBV p=0,006; CIN p=0,025; GS referência). Com base no risco de recidiva dos pacientes do estudo, nova classificação, que inclui 5 subtipos, foi proposta. Evidenciou-se que a classificação por risco e cluster tiveram melhor acurácia para identificar recidivas e óbitos respectivamente. CONCLUSÃO: A análise IH/ISH foi capaz de determinar subtipos de CG com características clinicopatológicas e prognósticos distintos, reproduzindo os subtipos obtidos pela classificação molecular
Title in English
Characterization of the molecular subtypes of gastric cancer by gene and protein expression
Keywords in English
Biomarkers
Gene expression profile
Immunohistochemistry
In situ hybridization
Stomach neoplasms
Translational medical research
Abstract in English
BACKGROUND: Recently, the molecular classification of gastric cancer (CG) emerged as a promising option to define prognostic subgroups, to distinguish biological behavior, and to identify potential therapeutic targets for specific drugs. Through molecular techniques, GC was divided into 4 subtypes: microsatellite instability (MSI), Epstein-Barr virus (EBV) positive, genomically stable (GS) and chromosomal instability (CIN). The costs associated with the technical complexity of the molecular methodologies are still an obstacle to its implementation in routine practice. OBJECTIVES: To determine molecular classification groups by means of protein expression of markers associated with each subtype. To compare the different existing molecular classifications and propose a new model. METHODS: We retrospectively evaluated 287 CG patients submitted to curative D2-gastrectomy through the construction of tissue microarray. Expression of the DNA repair proteins, E-cadherin and p53 were evaluated by immunohistochemistry (IH), determining the MSI, GS and CIN subtypes, respectively. EBV was detected by in situ hybridization (ISH). RESULTS: After the histopathological evaluation, 179 (62.4%) patients were classified as CIN, 58 (20.2%) MSI, 30 (10.5%) EBV and 20 GS (7%) as GS. The subtypes presented associations with distinct characteristics, such as: male gender (EBV, p=0.101); advanced age (MSI, p=0.017), Laurén diffuse type (GS, p < 0.001), lower neutrophil-lymphocyte ratio (CIN, p=0.029), total gastrectomy (EBV, p < 0.001), distal location (MSI, p=0.004), and advanced stage (GS, p=0.014). The MSI subtype presented better disease-free survival (DFS) (82.8%), followed by the EBV and CIN subtypes (both with 70%) and GS (50%) (p=0.005). Overall survival (OS) was higher in MSI tumors (75.9%), followed by EBV (73.3%), CIN (65.4%) and GS (45%, median of 25 months) (p=0.007). In multivariate analysis, total gastrectomy, tumor invasion, lymph node metastasis, and tumor subtypes were significant factors associated with SLD (MSI p=0.012, EBV p=0.037, CIN p=0.018, GS reference). Likewise, type of surgery, pT, and tumor subtypes were independent factors associated with OS (p=0.010, p=0.010, EBV p=0.006, CIN p=0.025, GS reference). Based on the risk of recurrence, a new classification including 5 subtypes was proposed. It was evidenced that the risk classification and cluster had better accuracy to identify recurrences and deaths respectively. CONCLUSION: The IH / ISH analysis was able to determine CG groups with clinicopathological characteristics and distinct prognoses, reproducing the subtypes obtained by the molecular classification
 
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Publishing Date
2019-07-03
 
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