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INTRODUÇÃO: O transplante (TX) de fígado corresponde ao tratamento de escolha em pacientes com cirrose e carcinoma hepatocelular (CHC) precoce irressecável. A recidiva do CHC pós-transplante, entretanto, ainda apresenta impacto na sobrevida dos pacientes transplantados com este tumor. As taxas de recidiva, nos estudos mais recentes, variam de 8 a 20%. O tamanho e número de nódulos, a presença de invasão vascular e de nódulos satélites no explante, são fatores de risco relacionados à recidiva tumoral pós-transplante. No Brasil, observamos um crescimento importante do número de transplantes de fígado, inclusive por CHC. Entretanto, existem poucos estudos nacionais analisando os resultados do transplante hepático por CHC. Os objetivos do nosso estudo foram analisar as características demográficas, clínicas e a evolução dos pacientes submetidos a transplante hepático com CHC no Brasil, avaliando os fatores prognósticos relacionados com a recidiva do CHC pós-transplante e sobrevida e estudar o desempenho dos critérios de seleção para transplante utilizados no nosso país, os \"Critérios de Milão Brasil\" (CMB). MÉTODOS: Estudo de coorte retrospectivo, multicêntrico, para analisar os resultados do transplante de fígado em pacientes com CHC, após a implantação do sistema MELD. Foram incluídos 1.119 pacientes transplantados com CHC, de 07/2006 até 07/2015, em 13 centros de transplante, no Brasil. Características clínicas, demográficas, exames laboratoriais e de imagem e dados anatomopatológicos, foram retrospectivamente analisados e correlacionados com a sobrevida e recidiva do CHC pós-transplante. RESULTADOS: A maioria dos pacientes era do sexo masculino (81%), com uma idade média no TX de 58 anos. A etiologia mais associada ao tumor foi a Hepatite C (VHC), presente em 60% dos casos. O tempo médio de espera em lista foi de 9,8 meses. Setenta e oito pacientes (8%) foram incluídos por \"Down-staging\". Nos exames de imagem do diagnóstico, a maioria dos casos (67%) apresentava um nódulo, com tamanho médio de 30 mm; 85% estavam dentro dos Critérios de Milão (CM), 8% fora dos CM, mas dentro dos \"Critérios de Milão Brasil\" (CMB) e 7% fora de ambos os critérios. O tratamento do CHC em lista foi realizado em 67% dos pacientes. Na análise do explante, 44% apresentavam tumor uninodular, com tamanho médio de 26 mm e a maioria (71%) tinha CHC moderadamente diferenciado. A invasão vascular foi observada em 26% dos casos e nódulos satélites em 22%. No explante, 70% dos pacientes estavam dentro dos CM, 20,5% fora dos CM, mas dentro dos CMB e 9,5%fora de ambos os critérios. A sobrevida global foi de 79% em 1 ano, 72,5% em 3 anos e 63%, em 5 anos, com um tempo médio de seguimento de 28 meses. Excluindo os pacientes que foram a óbito no pós-operatório ( < 30 dias pós-transplante), a sobrevida global foi de 89% em 1 ano e 75%, em 5 anos. A recidiva do CHC pós-TX ocorreu em 8% (86/1.119) dos casos, em um tempo médio de 12 meses. A sobrevida livre de recidiva (SLR) foi de 94,4% em 1 ano e 88,3%, em 5 anos. A recidiva do CHC foi extra-hepática em 55% dos casos, hepática em 27% e hepática e extra-hepática em 18%. Os pacientes transplantados que evoluíram com recidiva tumoral apresentaram alta mortalidade, com uma sobrevida em 1 ano de 34% e em 5 anos de 13%. Em relação aos fatores prognósticos, os pacientes transplantados dentro dos Critérios de Milão apresentaram melhor sobrevida e SLR quando comparados aos pacientes transplantados fora dos CM, mas dentro dos CMB, tanto quando analisamos os dados do diagnóstico, quanto através da análise do explante. Os pacientes transplantados após realização de \"Down-staging\" apresentaram taxas de recidiva e sobrevida semelhantes aos pacientes transplantados sem \"Down-staging\". Os níveis séricos elevados de alfa-fetoproteína (AFP) foram um fator prognóstico importante de sobrevida e recidiva tumoral. Os melhores pontos de corte de AFP encontrados para avaliação do risco de recidiva e sobrevida foram: AFP > 400 ng/ml, no momento do diagnóstico e AFP > 200 ng/ml pré-transplante. Realizamos, também, uma comparação dos \"Critérios de Milão Brasil\" com os Critérios de Milão, através do índice IDI (Integrated Discrimination Index) e os CMB apresentaram performance inferior aos CM, na capacidade de classificar corretamente os pacientes em relação ao risco de recidiva tumoral. Os níveis séricos elevados de AFP, o estádio fora dos Critérios de Milão no momento do diagnóstico e no explante e a presença e invasão vascular no explante, foram fatores de risco independentes de recidiva do CHC pós-transplante e pior sobrevida. A idade > 60 anos e a etiologia da hepatopatia (VHC), também foram fatores prognósticos negativos de sobrevida. CONCLUSÕES: A presença de recidiva tumoral teve grande impacto na sobrevida do paciente transplantado com CHC. O estadiamento tumoral no diagnóstico e no explante, avaliado através dos Critérios de Milão, os níveis séricos elevados de AFP e a presença de invasão vascular no explante foram fatores prognósticos importantes de recidiva do CHC pós-transplante e sobrevida. Os pacientes transplantados após \"Down-staging\" apresentaram evolução pós-transplante semelhante a dos pacientes transplantados sem \"Down-staging\". Os pacientes transplantados fora dos CM, mas dentro dos CMB, apresentaram pior sobrevida, quando comparados aos pacientes dentro dos CM. Os CMB apresentaram desempenho inferior aos CM na capacidade de classificar corretamente os pacientes em relação ao risco de recidiva tumoral / INTRODUCTION: Liver transplantation (LT) is the treatment of choice for patients with cirrhosis and unresectable early hepatocellular carcinoma (HCC). HCC post-transplant recurrence, however, still has an impact on survival. In recent studies, the incidence of HCC recurrence after transplantation ranged from 8% to 20%. Tumor number, size, vascular invasion and satellite nodules have emerged as risk factors for HCC recurrence. In Brazil, in the last decade, we observed a significant increase in the number of liver transplants performed, including in patients with HCC. However, there are few national studies analyzing the results of liver transplantation for HCC. The aim of this multicentric study was to analyze the demographic characteristics, clinical features and outcomes of patients submitted to liver transplantation with HCC in Brazil, evaluate prognostic factors related to HCC post-transplant recurrence and survival, and study the performance of the national selection criteria for liver transplantation, the \"Brazilian Milan Criteria\" (BMC). METHODS: We conducted a national, multicentric, retrospective study to analyze the results of liver transplantation in patients with HCC, in \"MELD era\". Medical records of 1,119 transplanted patients with HCC between 07/2006 and 07/2015, from 13 transplant centers in Brazil, were collected. Patient and tumor characteristics, radiologic and pathologic data were retrospectively analyzed and correlated with post-transplant HCC recurrence and survival. RESULTS: Of the 1,119 HCC transplanted patients, median age was 57 years and 81% were male. Etiology of liver disease was HCV in 60%. Median time on transplant list was 9.8 months. Seventy-eight patients (8%) were included after \"Down-staging\". At diagnosis, most patients had uninodular HCC (67%) and median tumor burden was 30 mm. At diagnosis, in imaging studies, 85% of patients were within the Milan criteria (MC), 8% out of the MC but within the \"Brazilian Milan Criteria\" (BMC) and 6% out of both criteria. During the waiting list period, HCC treatment was performed in 67%. In explant analysis, tumor was uninodular in 46% and moderately differentiated in the majority of cases (71%). Median HCC size was 26 mm. Vascular invasion and satellite nodules were observed in 26% and 22% of patients, respectively. In explant, 70% of patients were within Milan Criteria, 20.5% outside MC but within BMC and 9.5% out of both criteria. Mean follow-up was 28 months, an overall survival was 79% in 1 year, 72.5% in 3 years and 63% in 5 years. Excluding patients who died within 30 days after surgery, overall survival was 89% in 1 year and 75% in 5 years. HCC post-transplant recurrence occurred in 86/1,119 (8%) cases, at a mean time of 12 months. Recurrence-free survival (RFS) was 94.4% in 1 year and 88.3% in 5 years. Sites of recurrence were extrahepatic in 55%, hepatic in 27% and both hepatic and extrahepatic in 18%. Transplanted patients with tumor recurrence presented high mortality, with 1-year survival rate of 34% and 5-year survival rate of 13%. Analyzing the prognostic factors, patients transplanted under Milan Criteria, in radiologic or explant analysis, presented better survival and RFS when compared to patients transplanted outside MC, but within BMC. Patients submitted to liver transplantation after \"Down-staging\" present long-term survival and RFS similar to patients transplanted without \"Down-staging\". Alpha-fetoprotein (AFP) levels were an important pre-transplant prognostic factor for tumor survival and recurrence. The best AFP cut off points found for relapse risk and survival assessment were: AFP at diagnosis > 400 ng / ml and AFP pre-transplant > 200 ng / ml. We also performed a comparison of the \"Brazilian Milan Criteria\" with the Milan Criteria through the Integrated Discrimination Index (IDI). The BMC presented a lower performance than the MC, in the ability to correctly classify patients in relation to the risk of relapse. Elevated AFP levels before liver transplantation, tumor outside Milan Criteria at diagnosis and in explant, and vascular invasion, were independent risk factors for post-transplant HCC recurrence and worse survival. Age > 60 years and etiology of liver disease (HCV), were also negative prognostic factors for survival. CONCLUSIONS: The presence of tumor recurrence had a major impact on survival of transplanted patients with HCC. Tumor staging, evaluated by Milan Criteria on imaging studies or explant analysis, high serum AFP levels and presence of vascular invasion in explant were important prognostic factors for post-transplant HCC recurrence and survival. Patients transplanted after Down-staging presented long-term outcomes similar to patients transplanted under conventional criteria. Patients transplanted outside Milan Criteria, but within \"Brazilian Milan Criteria\" presented worse survival, when compared to patients within MC. The BMC showed lower performance than MC in the ability to correctly classify patients in relation to the risk of tumor recurrence

INTRODUÇÃO: A cirrose é a oitava causa de mortalidade no mundo, e sua progressão e estadiamento são de extrema importância nos pacientes com doença terminal do fígado. A presença de cirrose é reconhecida como risco aumentado de carcinoma hepatocelular (CHC) e o seu aparecimento está diretamente relacionado ao grau de fibrose do fígado. Na última década, notou-se o desenvolvimento e aperfeiçoamento dos métodos de predição do grau de fibrose e cirrose, através de métodos não-invasivos, com o objetivo de substituir a biópsia hepática. A população em lista de espera para transplante de fígado apresenta graus diferentes de fibrose hepática, que pode não estar diretamente relacionada ao MELD. Além disso, esses pacientes apresentam CHC no momento da triagem para transplante de fígado. Não existe avaliação desta população por elastografia. OBJETIVO: O objetivo deste trabalho foi avaliar os pacientes em triagem para transplante de fígado, com e sem carcinoma hepatocelular, pela elastografia hepática com Fibroscan® e ARFI. MÉTODO: Foram estudados 103 pacientes adultos do ambulatório de triagem da Disciplina de Transplante de Órgãos do Aparelho Digestivo HC/FMUSP, no período de outubro de 2012 à dezembro de 2013. A amostragem foi por conveniência e foram avaliados dados clínicos, epidemiográficos, laboratoriais, imagem, elastográficos e o desfecho. Análise de elastografia transitória (ET) foi feita pelo Fibroscan® TM (Echosens, França) e força impulso por radiação acústica (ARFI) (Siemens Acuson S2000, Alemanha) nos grupos com e sem CHC comprovados de acordo com orientação de diagnóstico pelas diretrizes européias (EASL) e americanas (AASLD). Para a análise estatística foi realizado o teste de Mann-Whitney, teste não paramétrico aplicado para duas amostras independentes; o teste de Fisher e o método ANOVA através do teste de Kruskal-Wallis ou teste de Tukey para comparações múltiplas. Foi realizado também a curva ROC para avaliação dos testes diagnósticos e ponto de corte. O valor considerado de p significativo foi <0,05. RESULTADOS: Entre os pacientes avaliados, a maioria foi de homens (68%), com idade média de 53 ± 11,5 anos. A etiologia mais comum foi o vírus da hepatite C (VHC) em 34,9%. A classificação pelo escore Child-Turcotte-Pugh (CTP) mostrou: pacientes classe A em 38,4%, classe B em 47,2% e classe C em 14,2%. O valor do MELD médio dos pacientes foi de 14,75 (± 6,45) e a mediana de 14 (variando, 6 - 32). Na população estudada de 103 pacientes, a ET (Fibroscan®) foi realizada com sucesso em 75 de 103 pacientes e ARFI em 78 de 78 pacientes. A etiologia VHC e elevados valores de alfa-feto proteína foram fatores de risco para a presença de CHC. Os valores de MELD mais elevados foram significativos nos pacientes que evoluíram a óbito. A curva ROC mostrou respectivamente sensibilidade e especificidade para a AFP de 50% e 86% (valor de corte 9,1); ET (valor de corte 9 kPa) 92% e 17%; e ARFI 21% e 92% (valor de corte 2,56 m/s). O valor médio da ET nos pacientes com CHC foi de 30,4 ± 21,0 kPa, do ARFI do parênquima hepático foi de 1,97 ± 0,64 e ARFI do nódulo hepático foi de 1,89 ± 0,74. CONCLUSÃO: Os pacientes em triagem para transplante de fígado com carcinoma hepatocelular apresentam valores elevados de elastografia tanto pelo Fibroscan® quanto pelo ARFI®. A elastografia apresenta-se como uma importante ferramenta não invasiva para o acompanhamento de cirróticos graves podendo ajudar no manejo do carcinoma hepatocelular / INTRODUCTION: Cirrhosis is the eighth leading cause of mortality worldwide and its progression and staging are extremely important in patients with end liver disease. The presence of cirrhosis is recognized as an increased risk of hepatocellular carcinoma (HCC) and its incidence is directly related to the degree of liver fibrosis. In the last decade, was noted the development and improvement of methods for predicting the degree of fibrosis and cirrhosis using non-invasive methods, aiming to replace the liver biopsy. The population on the liver transplant waiting list presents different degrees of liver fibrosis, which may not be directly related to MELD. In addition, these patients have HCC at the time of screening for liver transplantation. There is no evaluation of this population by elastography. OBJECTIVE: The aim of this study was to evaluate patients on screened to the list for liver transplantation, with and without hepatocellular carcinoma, by liver elastography with Fibroscan and ARFI. METHOD: Were studied 103 adult patients from the screening for liver transplantation waiting list on the Liver and Gastrointestinal Transplant Division HC/FMUSP from October 2012 to December 2013. Sampling for convenience and evaluation clinical data, epidemiological, laboratory, imaging, elastography findings and outcome. Analysis of transient elastography (TE) by Fibroscan TM (Echosens, France) and Acoustic Radiation Force Impulse (ARFI) by (Siemens Acuson S2000, Germany) in patients with and without HCC proven in accordance with guidelines of diagnosis EASL/AASLD. Fisher\'s ANOVA or Kruskal-Wallis tests Whitney-Mann Test were performed. Tukey and define cut-of for examinations with ROC curves. The p value considered was < 0.05. RESULTS: Among the patients, the majority were men (68%), mean age 53 ± 11.5 years. This is the most common cause of hepatitis C virus (HCV) 34.9%. The classification by Child-Turcotte-Pugh score (CTP) showed: class A patients in 38.4%, 47.2% in class B and class C in 14.2%. The average value of MELD patients was 14.75 (± 6.45) and a median of 14 (range, 6-32). In the study population of 103 patients, the ET (Fibroscan) was successfully performed in 75 of 103 patients and ARFI in 78 of 78 patients. The HCV etiology and high levels of alpha-fetoprotein were risk factors for the presence of HCC. MELD values were significant higher in patients who died. The ROC curve shown respectively sensitivity and specificity for AFP of 50% and 86% (cutoff 9.1); ET (9 cutoff kPa) 92% to 17%; and ARFI 21% and 92% (cut-off 2.56 m / s). The average value of ET in HCC patients was 30.4 ± 21.0 kPa, the ARFI parenchymal liver was 1.97 ± 0.64 and ARFI liver nodules was 1.89 ± 0.74. CONCLUSION: Patients in screening for liver transplantation with hepatocellular carcinoma have elevated values of both elastography by Fibroscan as the ARFI®. Elastography is presented as an important non-invasive tool for monitoring severe cirrhosis may help in management of hepatocellular carcinoma

L'insuline, hormone clé du contrôle de l'homéostasie métabolique, exerce également des effets trophiques sur la croissance et la prolifération cellulaire. Des études épidémiologiques ont récemment montré que les individus obèses ou diabétiques de type 2 ont un risque plus élevé de développer des cancers et elles ont également suggéré que l’insuline jouerait un rôle dans ce développement tumoral. Ainsi, une signalisation adéquate en aval du récepteur de l’insuline est indispensable pour éviter des processus physiopathologiques. La signalisation de l’insuline est contrôlée par des mécanismes de rétrocontrôle, dont l’adaptateur moléculaire Grb14 qui agit comme un inhibiteur endogène de l’activité catalytique du RI. L’objectif de ma thèse a été d’étudier les conséquences métaboliques et mitogéniques de l’inhibition de Grb14 in#vivo spécifiquement dans le foie de souris. Dans une première étude, nous montrons que sept jours après l’invalidation de Grb14, les souris présentent une activation des voies de signalisation de l’insuline, qui s’accompagne d’une amélioration de la tolérance au glucose et de la production hépatique de glucose. Cependant, de façon paradoxale, la voie de la lipogenèse est très fortement diminuée. En décryptant le mécanisme moléculaire impliqué, nous montrons que l’inhibition de Grb14 permet la libération de la protéine p62/sqstm1 qui active le facteur de transcription Nrf2, ce qui entraine une inhibition du récepteur nucléaire pro-lipogénique LXR. De façon intéressante, l’invalidation de Grb14 chez des souris ob/ob permet de restaurer la glycémie et la stéatose hépatiques à des valeurs comparables aux témoins. Cette étude a ainsi permis de mettre en évidence une nouvelle voie de régulation de la lipogenèse hépatique. Dans une deuxième étude, nous nous sommes intéressés à l'action mitogénique de l'insuline. Nous montrons que 48 heures après l'inhibition de Grb14, les hépatocytes, qui sont des cellules quiescentes, entrent massivement dans le cycle cellulaire. Ce processus est dépendant de l’expression du RI et est médié par la signalisation PI3K/Akt/mTORC1 et la voie Rb/E2F1. Ces données révèlent ainsi que l'insuline est un puissant facteur mitogène dans le foie et que son action est étroitement contrôlée par l’adaptateur Grb14. D’un point de vue physiopathologique, nous avons pu mettre en évidence une diminution de significative de 58% de l’expression de Grb14 dans une collection de 70 CHC humains, apportant ainsi une explication moléculaire à une action pro-tumorigène de l’insuline dans le foie. L’ensemble de ces deux études permet de placer Grb14 au centre de la régulation des actions métaboliques et mitogéniques de l’insuline dans le foie. / Insulin is a key hormone controling metabolic homeostasis which also exerts having trophic effects on cell growth and proliferation. Epidemiological studies have recently shown that obese and type 2 diabetes patients are at higher risk of developing cancers, suggesting that insulin could be involved in tumor development. Proper signaling downstream the insulin receptor is thus essential to prevent pathophysiological processes. Insulin signaling is controlled by feedback mechanisms including the molecular adapter Grb14 which acts as an endogenous inhibitor of the IR catalytic activity. The aim of my PhD was to investigate the metabolic and mitogenic consequences of liver specific Grb14 inhibition in mouse. In the first study, we showed that after seven days of Grb14 invalidation, liver insulin signaling is enhanced, resulting in improved glucose tolerance and diminished hepatic glucose production. However, paradoxically, lipogenesis was greatly decreased. Deciphering the molecular mechanism, we show that Grb14 inhibition leads to the release of its partner p62/SQSTM1, inducing the activation of the Nrf2 transcription factor, which ultimatly inhibited the pro-lipogenic LXR nuclear receptor. Interestingly, Grb14 invalidation in ob/ob mice can restore blood glucose and hepatic steatosis comparable to control values. The study thus highlighted a new pathway controlling lipogenesis that could be targetted to improve metabolic diseases. In the second study, we were interested in insulin mitogenic action. We showed that 48 hours after Grb14 inhibition, hepatocytes that are quiescent cells, massively go through one cell cycle. This process depend on IR expression and is mediated by the PI3K/Akt/mTORC1 pathway and the Rb/E2F1 complex. Our data thus suggest that insulin is a potent mitogenic factor in the liver whose action is closely controlled by the Grb14 adapter in physiological conditions. Importantly, Grb14 expression is significantly decreased in a collection of human HCC, hence bringing out a molecular basis for a pro-tumorigenic action of hyperinsulinemia. Together these two studies reveal that Grb14 is a crucial gatekeeper of insulin metabolic and mitogenic actions in the liver.

Les adénomes hépatocellulaires (AHC) sont des tumeurs bénignes rares dérivant de la prolifération d'hépatocytes et se développent dans un contexte hépatique par ailleurs normal, principalement chez des femmes suite à la prise de contraceptifs oraux. D'autres études ont identifié des facteurs de risque supplémentaires comme le syndrome métabolique et des mutations germinales d’HNF1α. Les travaux réalisés dans le laboratoire ont permis le classement des AHC en cinq sous-groupes : 1) les AHC-H avec des mutations inactivatrices du facteur de transcription HNF1α, 2) AHC-I avec une activation de la voie inflammatoire, 3) B-AHC portant des mutations activatrices du gène CTNNB1, codant pour la β-caténine 4) Sh-AHC avec une activation de la voie sonic hedgehog et 5) U-AHC sans altération moléculaire spécifique trouvée à ce jour. Les AHC montrent un faible risque de transformation maligne en carcinome hépatocellulaire (CHC). Le CHC est la deuxième cause de décès liés au cancer dans le monde entier, ce qui en fait un problème majeur de santé publique. Le CHC se développe dans 90% des cas sur un foie cirrhotique et est lié à une infection virale (VHB et HCV), à une consommation d'alcool et au syndrome métabolique. L'une des modifications génétiques les plus fréquentes dans les CHC est la réactivation de la télomérase (TERT), une enzyme clé nécessaire à la maintenance des télomères. Tout d'abord, dans nos travaux, nous avons cherché à établir une corrélation génotype-phénotype des AHC activés pour la β-caténine pour mieux comprendre l'hétérogénéité des tumeurs et le risque de transformation maligne. Notre étude a permis d'identifier trois sous-groupes d’AHC mutés CTNNB1 selon le type de mutation : mutations fortement, modérément et faiblement activatrices. De façon intéressante, chaque type de mutation présentait immuno marquage spécifique de la GS et un risque de transformation maligne diffèrent. En plus, nous avons identifié la mutation du promoteur de TERT comme un événement tardif au cours de la tumorigenèse nécessaire à la transformation des AHC-B en CHC. Deuxièmement, en raison de l’importance de la réactivation de TERT dans les tumeurs du foie, nous avons testé un nouveau traitement potentiel basé sur des oligonucléotides antisens conduisant à l’inhibition de l’expression transcriptionelle de TERT. En accord avec l'érosion progressive des séquences télomériques qui se produisent à chaque division cellulaire, nous avons pu observer un effet sur la prolifération cellulaire après traitement à long terme. Nous avons montré que les effets observés étaient liés au raccourcissement des télomères. De plus, nous avons identifié AAV2 comme un nouveau virus lié au développement de rares cas de CHC. Nos résultats sont soutenus par l'expansion clonale des cellules tumorales avec insertion virale, la surexpression des gènes insérés et l'insertion observée uniquement dans des gènes déjà connus pour être impliqués dans la tumorigenèse comme TERT, TNFSF10, MLL4, CCNA2 et CCNE1. En conclusion, dans nos études, nous avons affiné la classification des AHC, avec des possibles applications dans la prise en charge clinique des patients pour identifier le meilleur traitement selon le type de mutation et le risque de transformation maligne. De plus, nous avons obtenu des résultats prometteurs concernant l'utilisation de TERT comme cible thérapeutique. Cependant, des études supplémentaires sont nécessaires pour mieux évaluer les effets du traitement par ASO contre le TERT sur la physiologie cellulaire et les avantages possibles avec des combinaisons thérapeutiques. Enfin, nos résultats remettent en question la sécurité des thérapies génique basées sur l'AAV actuellement en cours, mais des études supplémentaires sont actuellement en cours au laboratoire pour comprendre les conséquences et les mécanismes impliqués dans de l'insertion virale. / Hepatocellular adenomas (HCA) are rare benign tumors deriving from proliferation of hepatocytes occurring in normal liver background mainly of female patients with history of oral contraceptive assumption. Further studies identified additional risk factors as metabolic syndromes and germline mutations. Works performed in the laboratory allowed the classification of HCA in five subgroups: 1) H-HCA with inactivating mutation of transcription factor HNF1α, 2) I-HCA with activation of the inflammatory pathway, 3) B-HCA carrying activating mutations of CTNNB1 gene, coding for β-catenin 4) Sh-HCA with activation of the sonic hedgehog pathway and 5) U-HCA with no specific alteration found to date. HCA show an average low risk of malignant transformation in hepatocellular carcinoma (HCC). HCC is the second cause of cancer-related deaths worldwide making it a major problem of public health. HCC development occurs in 90% of the cases on cirrhotic background and is linked to virus infection (HBV and HCV), alcohol intake and metabolic syndromes. One of the most frequent genetic alterations in HCC is the reactivation of TERT, a key enzyme necessary for telomeres maintenance. First, in our works we aimed to perform a correlation between genotype and phenotype of β-catenin activated HCA to better understand tumor heterogeneity and risk of malignant transformation. Our study allowed the identification of three subgroups of CTNNB1 mutated HCA according to the mutation type: highly, moderately and weakly activating mutations. Interestingly each type of mutation showed specific IHC staining for GS and risk of malignant transformation. Additionally we identified TERT promoter mutation as a late event in tumorigenesis necessary for HCA transformation in HCC with a significant correlation with CTNNB1 alterations. Second, due to the relevance of TERT reactivation in tumors, we tested a new potential treatment based on antisense oligonucleotides leading to TERT transcript ablation and lack of protein production. According to the slow erosion of the telomere sequences that occurs at each cell division, we were able to observe some effect on cell proliferation with long-term treatment. We showed that the observed effects were linked to telomere shortening. Further, we identified AAV2 as a new virus linked to tumor formation. Our results are supported by the clonal expansion of cells with viral insertion, overexpression of the inserted genes and insertion observed only in genes already known to be involved in tumorigenesis like TERT, TNFSF10, MLL4, CCNA2 and CCNE1. Taken together, in our studies we refined HCA classification, with possible direct applications in the clinical care of the patients in identifying the best treatment according to mutation type and risk of malignant transformation. Additionally we obtained promising results regarding using TERT as a therapeutic target. Still, additional studies are needed to better assess the effects of ASO treatment against TERT on cell physiology and possible advantages in using combination therapies. Finally, our findings challenge the safety of AAV based targeted therapies currently ongoing but additional studies to understand the effective consequences and mechanisms of viral insertion are object of current work in the laboratory.

Le profil d'expression aberrant des micro(mi)ARN est une caractéristique typique de nombreux cancers, dont le carcinome hépatocellulaire (CHC), une tumeur hépatique maligne primaire qui se classe seconde dans le monde en termes de mortalité par cancer. Notre équipe a récemment montré la baisse d’expression de miR-4510 dans des échantillons de patients atteints de CHC et son activité « suppresseur de tumeur ». L'analyse de données protéomiques recueillies à partir de cellules Huh7 transfectées par miR-4510 a révélé une diminution importante de plusieurs oncogènes, dont la sérine / thréonine protéine kinase RAF1. J’ai également découvert que le taux de protéine RAF1 était significativement surexprimé chez les patients atteints de CHC. Le rôle de RAF1 et de miR-4510 dans le CHC étant mal compris, j’ai étudié la fonction du couple RAF1/miR-4510 dans la tumorigenèse du foie. Mes analyses ont montré que miR-4510 régule négativement les taux de protéine RAF1 et d'ARNm. Une analyse par le système de double fluorescence-FunREG a révélé que miR-4510 interagit directement avec la région 3’ non-traduite de l’ARN de RAF1 via un site unique. La déplétion de RAF1 dans deux lignées tumorales de CHC par miR-4510 ou ARN interférant désactive leur caractère tumorigène in vitro et in vivo. Collectivement, mes données suggèrent que miR-4510 participe à la carcinogenèse du foie via son action directe sur RAF1 et la régulation de la voie MAPK/ERK. En conclusion, mon étude soutient l’hypothèse selon laquelle un traitement à base de miR-4510 pourrait être efficace pour traiter les patients atteints de CHC de type avancé ou réfractaire à la chimiothérapie. / Aberrant micro(mi)RNA expression signature is a hallmark of many cancers including hepatocellular carcinoma (HCC), a primary malignant liver disease which ranks second in cancer mortality worldwide. Our team previously reported the downregulation of miR-4510 in HCC samples and identified this miRNA as a strong tumor suppressor in liver. Proteomic data analysis collected from Huh7 cells transfected by miR-4510 showed a significant decrease of multiple oncogenes including RAF1 serine/threonine protein kinase. I also found that RAF1 protein level is significantly increased in HCC patients. The role of RAF1 and miR-4510 in HCC being poorly understood, I studied the function of RAF1/miR-4510 pair in tumorigenesis of the liver. My results showed that miR-4510 overexpression significantly decreases both RAF1 protein and mRNA levels and inhibits MAPK/ERK signaling. The dual fluorescence-FunREG assay revealed that miR-4510 directly interacts with RAF1 3’-untranslated region through a unique site. Silencing of RAF1 in two hepatic cell lines by miR-4510 or a specific small interfering RNA suppressed important tumorigenic features (proliferation, migration….) both in vitro and in vivo. Collectively, my data suggest that miR-4510 participates in liver carcinogenesis through RAF1 targeting and MAPK/ERK signaling inactivation. In addition, my study suggests that miR-4510-based therapy may represent a promising strategy to treat patients with advanced or refractory HCC.

There are multiple immune mechanisms identified for persistence of hepatitis B virus (HBV) infection. This thesis considers the vital role that inhibitory receptors play in contributing to impairment of the adaptive immune system in chronic hepatitis B (CHB), and the potential role they play in the innate immune system, focusing on the inhibitory receptor leucocyte-associated immunoglobulin-like receptor (LAIR)-1. The unique aspect of this work is that for the first time LAIR-1 expression has been investigated on natural killer (NK) cells in CHB. Our striking findings of increased LAIR-1 expression on peripheral NK cells in CHB and an inverse correlation between expression and effector function suggest this inhibitory receptor could have a potential role in exhaustion of NK cells in CHB. We therefore additionally explored the expression of LAIR-1 on circulating NK cells from patients with hepatocellular carcinoma (HCC) and non-alcoholic fatty liver disease (NAFLD). The particular relevance of LAIR-1 to liver disease is that one of its major ligands is collagen. We demonstrated a downregulation of LAIR-1 expression on intrahepatic NK cells, which we postulate might occur following repetitive engagement with abundant collagen within the liver. In line with this, intrahepatic NK cells with a liver-resident (CXCR6+) phenotype had even lower LAIR-1 expression than liver infiltrating (non-resident, CXCR6-) NK cells. Furthermore, preliminary experiments display attenuation of the cytotoxic degranulation capacity (CD107a) by circulating NK cells from CHB patients upon exposure to plate-bound collagen. We demonstrate differential expression of LAIR-1 on NK cells in viral hepatitis, HCC and NAFLD and between peripheral and intrahepatic NK cells. Preliminary experiments demonstrate a role in inhibiting NK cell function suggesting this as a novel therapeutic target to harness the capacity of NK cells to control chronic infection and cancer.

L’infection chronique par le virus de l’hépatite C (VHC) conduit au développement de la fibrose et de la cirrhose qui risque d’évoluer vers le carcinome hépatocellulaire (CHC). La protéine de capside du VHC interagit avec de nombreuses protéines de l’hôte et en particulier avec Smad3, protéine majeure de la voie de signalisation du transforming growth factor beta (TGF-Β). Mon travail de thèse consistait à étudier les conséquences biologiques de l’interaction entre la protéine de capside avec la voie de signalisation du TGF-Β. Le VHC présente une grande variabilité génétique et des travaux du laboratoire ont montré l’existence de séquences différentes de protéines de capside du virus entre les régions tumorales (cT) et cirrhotiques (cNT) d’un même sujet. Nous avons montré que ces différentes protéines de capside exprimées dans des hépatocytes orientent les réponses biologiques du TGF-Β vers la promotion tumorale en diminuant l’apoptose et en augmentant la transition épithelio-Mésenchymateuse (TEM) en particulier le variant cT. Cet effet est attribué à la capacité de la protéine de capside de diminuer l’activité transcriptionnelle de Smad3. De plus, les variants de la protéine de capside activent le TGF-Β latent via l’augmentation de l’expression de la trombospondine. L’un des marqueurs classiquement exprimé au cours d’une TEM est l’alpha-Actine musculaire lisse (αSMA). Nous avons montré qu’une autre isoforme, la γSMA, était polymérisée dans les cellules hépatiques développant une TEM. L’expression de γSMA a été retrouvée sur des coupes de CHC et a pu être significativement corrélée à la fois avec des marqueurs de la TEM, des marqueurs progéniteurs et avec l’agressivité de la tumeur.Ce travail apporte une meilleure compréhension du rôle de la protéine de capside dans la fibrose hépatique liée à l’infection virale. En effet, la protéine de capside du VHC agit à la fois de façon autocrine dans les hépatocytes en modulant les réponses du TGF-Β vers la promotion tumorale et de façon paracrine, en affectant l’activation des cellules étoilées en myofibroblastes par le TGF-Β activé. / Chronic HCV infection) may progress to liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC). HCV core binds several cellular proteins and in particular Smad3, a major protein of transforming growth factor beta (TGF-Β) signalling.. The aim of this study was to determine the implication of HCV core protein in TGF-Β responses. High genetic variability is a characteristic of HCV and it was previously shown that HCV core protein isolated from tumour (cT) or adjacent non-Tumour (cNT) livers displayed different sequences. Both were able to shift TGF-B responses from tumour suppressor to tumour promotor by decreasing hepatocyte apoptosis and increasing epithelial-Mesenchymal transition (EMT). Core cT was more potent than core cNT to promote this effect that was mainly attributed to the capacity of HCV core to alleviate Smad3 activity. Moreover, HCV core protein activated the latent form of TGF-Β through increased thrombospondin expression. It is commonly accepted that αSMA (alpha smooth muscle actin) is a hallmark of EMT. In the current study another SMA isoform, γSMA was found to be polymerized during hepatocyte EMT. γSMA was expressed in HCC tissues and correlated with EMT, stem cell and aggressiveness markers. In conclusion, this work contributed to a better understanding of the HCV core role in hepatitis fibrosis and HCC related to HCV. Indeed, HCV core might act both as an autocrine and paracrine way by modulating TGF-Β responses within hepatocytes and by activating hepatic stellate cells in stromal environment through its capacity to activate TGF-Β.

OBJETIVOS: Analisar a expressão de EGFR, proteínas da via de sinalização ou relacionadas aos seus efeitos em carcinoma hepatocelular (CHC) primários, metastáticos e em tecido hepático não tumoral em autópsias. Correlacionar os achados a dados clínico-patológicos e marcadores de classes moleculares. Avaliar a heterogeneidade de expressão em CHC metastáticos e fatores de disseminação extra-hepática. MÉTODOS: Oitenta autópsias de pacientes com CHC ao exame foram incluídas em estudo retrospectivo transversal. Foram analisados sexo, idade, raça, etilismo, infecção por vírus da hepatite B (VHB) e C (VHC), infecção pelo HIV, tratamento prévio, causas básica e imediata de óbito, peso do fígado, cirrose, número e tamanho dos nódulos, padrão macroscópico, grau histológico, variantes histológicas, padrão arquitetural, invasão de grandes veias e metástases extra-hepáticas. Imuno-histoquímica foi realizada em micromatrizes teciduais para pesquisa de EGFR, pEGFR(Tyr 1173), HER2, ERK1/2, MAPKAPK-2, pMAPK, Ag Ki67, caspase 3, citoqueratina 19 (CK19), mTOR, ciclina D1, Met, vimentina, p53 e beta-catenina. A expressão de EGFR foi avaliada em intensidade (0-3+) e distribuição (0-100%) em um sistema de escores de 0 a 300. Hiperexpressão foi definida para escores >= 200. Amostras de fígado normal foram incluídas como controles. Amostras de CHC primário foram pareadas às suas metástases e consideradas concordantes quando na mesma categoria de expressão. No tecido não tumoral foram semi-quantificadas a reação ductular expressando CK19 e a densidade da população de células estromais perissinusoidais pela vimentina. Estatística foi realizada através dos testes do qui-quadrado ou exato de Fisher ao nível de significância de 0,05. Para as correlações de escores e variáveis categóricas foi utilizado o coeficiente de Spearman. RESULTADOS: Foram incluídos 62 casos do sexo masculino e 18 do sexo feminino (58,1 ± 10,9 anos). Infecção pelo VHC foi a principal causa em 49% (39/80), seguida por etilismo em 30% (24/80) e infecção por VHB em 19% (15/80). Cirrose foi identificada em 90% (72/80) dos casos. Os tumores mostraram-se avançados em 95% (76/80). Invasão de grandes veias foi detectada em 19% (15/80) e metástases extra-hepáticas em 38% (30/80). MAPKAPK2, pEGFR (Tyr1173) e HER2 tiveram expressão fraca ou ausente. A expressão de EGFR foi mais frequente no fígado não neoplásico (26/26) (P < 0,05) - e nos controles normais (8/8) do que nas amostras tumorais primárias (60/75) e nas metástases (12/17). Nenhuma amostra dos controles apresentou hiperexpressão de EGFR, a qual foi mais frequente na cirrose (65% - 17/26) do que nos tumores avançados (36% - 26/72) (P < 0,05). EGFR hiperexpresso foi mais frequente nos tumores de grau 1/2 (P < 0,01) e nos casos com menos de quatro nódulos hepáticos (P = 0,014). A expressão de EGFR correlacionou-se à expressão de caspase 3 (P < 0,01). A expressão das quinases ERK1 e ERK2 foi correlacionada à proliferação celular pelo Ag Ki67 (P < 0,01), porém não ao escore de expressão de EGFR. CK19, p53 e beta-catenina nuclear foram correlacionaram-se às lesões de maior grau e a maiores taxas de proliferação celular (P<0,01). Met, EGFR e caspase 3 foram correlacionados a lesões mais diferenciadas. Vimentina teve forte correlação com CK19 (P < 0,01). A concordância de expressão entre tumores hepáticos e respectivas metástases variou de 50 a 85%. Para o EGFR foi de 61%. A expressão endotelial 2-3+ de pMAPK foi mais frequente nas metástases (P = 0,09). A disseminação extra-hepática foi mais frequente nos casos com baixa densidade de células perissinusoidais positivas para vimentina (P = 0,054) e nos casos sem reação ductular no tecido não neoplásico (P = 0,095). CONCLUSÕES: O EGFR tem papel relevante nas etapas iniciais e intermediárias do CHC, sendo sua expressão reduzida nas formas avançadas. Diferentes classes de CHC podem estar associadas a ativação da via do EGFR. A presente análise imuno-histoquímica ampla parece validar pelo menos dois grupos de CHC que nesta série de autópsias parecem ter sido separados pelo grau histológico. Confirma-se a hiperexpressão das quinases como evento importante na progressão tumoral, porém não necessariamente associada à hiperexpressão de EGFR. A heterogeneidade de expressão entre o CHC primário e suas metástases variou de 15 a 45% / OBJECTIVES: To analyze the expression of EGFR and proteins related to its signaling pathway or to its effects in hepatocellular carcinoma (HCC), metastases and surrounding liver tissue in a series of autopsies. To correlate expression patterns to clinicopathological data and other markers of molecular classification. To assess the heterogeneity of expression in metastatic HCC and factors related to extrahepatic spread. METHODS: Eighty autopsies of patients with HCC were included in a cross-sectional retrospective study. We analyzed gender, age, race, alcohol intake, infection with hepatitis B (HBV) and C virus (HCV), HIV infection, prior treatment, basic and immediate causes of death, the weight of the liver, cirrhosis, number and size of nodules, gross pattern, histological grade, histological variants, architectural pattern, invasion of large veins and extrahepatic metastases. Immunohistochemistry was performed on tissue microarrays to survey EGFR, pEGFR(Tyr 1173), HER2, ERK1/2, MAPKAPK-2, pMAPK, Ag Ki67, caspase 3, cytokeratin 19 (CK19), mTOR, cyclin D1, Met, vimentin, p53 and beta-catenin. EGFR expression was evaluated in intensity (0-3+) and distribution of membrane staining (0-100%) in a 0 - 300 score. Overexpression was defined for scores >= 200. Normal liver samples were included as controls. Intra-hepatic HCC samples were matched to their respective metastases and expression was considered concordant when they were assigned to the same category. Ductular reaction expressing CK19 and the density of perisinusoidal vimentin positive stromal cells were semi-quantified in non-tumor tissue. Statistics was performed using the chi- square or Fisher exact test at a significance level of 0.05. For the correlations of scores and categorical data we used the Spearman coefficient. RESULTS: Sixty-two males and eighteen females were included (age 58.1 ± 10.9). HCV was the major cause in 49% (39/80), followed by alcoholism in 30% (24/80) and HBV infection in 19% (15/80). Cirrhosis was identified in 90% (72/80) and advanced tumors in 95% (76/80). Large vein invasion was detected in 19% (15/80) and extra-hepatic metastases in 38% (30/80). MAPKAPK2, pEGFR (Tyr1173) and HER2 expression were weak or absent. The EGFR expression was more frequent in non-tumoral liver (26/26) (P <.05) and in normal controls (8/8) than in primary HCC tumor samples (60/75) and in metastatic HCC (12/17). No samples taken from the controls showed overexpression of EGFR, which was more common in cirrhotic tissue (65% - 17/26) than in advanced tumors (36% - 26/72) (P <0.05). EGFR overexpression was more frequent in grade 1/2 tumors (P <0.01) and in cases with less than four hepatic nodules (P = 0.014). EGFR expression was correlated to the expression of caspase 3 (P <0.01). The expression of the kinases ERK1 and ERK2 was correlated to Ag Ki67 cell proliferation index (P <0.01), but not to the EGFR expression score. CK19, p53 and nuclear beta- catenin were correlated to high grade lesions and to higher rates of cell proliferation (P <0.01). Met, EGFR and caspase 3 were correlated with more differentiated lesions. Vimentin was strongly correlated with CK19 (P <0.01). The concordance of expression between liver tumors and their metastases ranged from 50 to 85% (61% for EGFR). The 2-3+ expression of pMAPK in tumor endothelial cells was more common in metastases (P = 0.09). Extrahepatic dissemination was more frequent in cases with low density of vimentin positive perisinusoidal cells (P = 0.054) and in cases with no detectable ductular reaction in non-neoplastic tissue (P = 0.095). CONCLUSIONS: EGFR plays an important role in the early and intermediate stages of HCC progression, with lower expression in advanced tumors. Different classes of HCC may be associated with activation of EGFR. The present comprehensive immunohistochemical approach seems to validate at least two molecular classes of HCC, and histological grade seems to be able to discriminate these groups. We herein confirmed overexpression of kinases as a key event in tumor progression, but not necessarily associated with overexpression of EGFR. The heterogeneity of expression between primary HCC and its metastases ranged from 15 to 45%

INTRODUÇÃO: O transplante (TX) de fígado corresponde ao tratamento de escolha em pacientes com cirrose e carcinoma hepatocelular (CHC) precoce irressecável. A recidiva do CHC pós-transplante, entretanto, ainda apresenta impacto na sobrevida dos pacientes transplantados com este tumor. As taxas de recidiva, nos estudos mais recentes, variam de 8 a 20%. O tamanho e número de nódulos, a presença de invasão vascular e de nódulos satélites no explante, são fatores de risco relacionados à recidiva tumoral pós-transplante. No Brasil, observamos um crescimento importante do número de transplantes de fígado, inclusive por CHC. Entretanto, existem poucos estudos nacionais analisando os resultados do transplante hepático por CHC. Os objetivos do nosso estudo foram analisar as características demográficas, clínicas e a evolução dos pacientes submetidos a transplante hepático com CHC no Brasil, avaliando os fatores prognósticos relacionados com a recidiva do CHC pós-transplante e sobrevida e estudar o desempenho dos critérios de seleção para transplante utilizados no nosso país, os \"Critérios de Milão Brasil\" (CMB). MÉTODOS: Estudo de coorte retrospectivo, multicêntrico, para analisar os resultados do transplante de fígado em pacientes com CHC, após a implantação do sistema MELD. Foram incluídos 1.119 pacientes transplantados com CHC, de 07/2006 até 07/2015, em 13 centros de transplante, no Brasil. Características clínicas, demográficas, exames laboratoriais e de imagem e dados anatomopatológicos, foram retrospectivamente analisados e correlacionados com a sobrevida e recidiva do CHC pós-transplante. RESULTADOS: A maioria dos pacientes era do sexo masculino (81%), com uma idade média no TX de 58 anos. A etiologia mais associada ao tumor foi a Hepatite C (VHC), presente em 60% dos casos. O tempo médio de espera em lista foi de 9,8 meses. Setenta e oito pacientes (8%) foram incluídos por \"Down-staging\". Nos exames de imagem do diagnóstico, a maioria dos casos (67%) apresentava um nódulo, com tamanho médio de 30 mm; 85% estavam dentro dos Critérios de Milão (CM), 8% fora dos CM, mas dentro dos \"Critérios de Milão Brasil\" (CMB) e 7% fora de ambos os critérios. O tratamento do CHC em lista foi realizado em 67% dos pacientes. Na análise do explante, 44% apresentavam tumor uninodular, com tamanho médio de 26 mm e a maioria (71%) tinha CHC moderadamente diferenciado. A invasão vascular foi observada em 26% dos casos e nódulos satélites em 22%. No explante, 70% dos pacientes estavam dentro dos CM, 20,5% fora dos CM, mas dentro dos CMB e 9,5%fora de ambos os critérios. A sobrevida global foi de 79% em 1 ano, 72,5% em 3 anos e 63%, em 5 anos, com um tempo médio de seguimento de 28 meses. Excluindo os pacientes que foram a óbito no pós-operatório ( < 30 dias pós-transplante), a sobrevida global foi de 89% em 1 ano e 75%, em 5 anos. A recidiva do CHC pós-TX ocorreu em 8% (86/1.119) dos casos, em um tempo médio de 12 meses. A sobrevida livre de recidiva (SLR) foi de 94,4% em 1 ano e 88,3%, em 5 anos. A recidiva do CHC foi extra-hepática em 55% dos casos, hepática em 27% e hepática e extra-hepática em 18%. Os pacientes transplantados que evoluíram com recidiva tumoral apresentaram alta mortalidade, com uma sobrevida em 1 ano de 34% e em 5 anos de 13%. Em relação aos fatores prognósticos, os pacientes transplantados dentro dos Critérios de Milão apresentaram melhor sobrevida e SLR quando comparados aos pacientes transplantados fora dos CM, mas dentro dos CMB, tanto quando analisamos os dados do diagnóstico, quanto através da análise do explante. Os pacientes transplantados após realização de \"Down-staging\" apresentaram taxas de recidiva e sobrevida semelhantes aos pacientes transplantados sem \"Down-staging\". Os níveis séricos elevados de alfa-fetoproteína (AFP) foram um fator prognóstico importante de sobrevida e recidiva tumoral. Os melhores pontos de corte de AFP encontrados para avaliação do risco de recidiva e sobrevida foram: AFP > 400 ng/ml, no momento do diagnóstico e AFP > 200 ng/ml pré-transplante. Realizamos, também, uma comparação dos \"Critérios de Milão Brasil\" com os Critérios de Milão, através do índice IDI (Integrated Discrimination Index) e os CMB apresentaram performance inferior aos CM, na capacidade de classificar corretamente os pacientes em relação ao risco de recidiva tumoral. Os níveis séricos elevados de AFP, o estádio fora dos Critérios de Milão no momento do diagnóstico e no explante e a presença e invasão vascular no explante, foram fatores de risco independentes de recidiva do CHC pós-transplante e pior sobrevida. A idade > 60 anos e a etiologia da hepatopatia (VHC), também foram fatores prognósticos negativos de sobrevida. CONCLUSÕES: A presença de recidiva tumoral teve grande impacto na sobrevida do paciente transplantado com CHC. O estadiamento tumoral no diagnóstico e no explante, avaliado através dos Critérios de Milão, os níveis séricos elevados de AFP e a presença de invasão vascular no explante foram fatores prognósticos importantes de recidiva do CHC pós-transplante e sobrevida. Os pacientes transplantados após \"Down-staging\" apresentaram evolução pós-transplante semelhante a dos pacientes transplantados sem \"Down-staging\". Os pacientes transplantados fora dos CM, mas dentro dos CMB, apresentaram pior sobrevida, quando comparados aos pacientes dentro dos CM. Os CMB apresentaram desempenho inferior aos CM na capacidade de classificar corretamente os pacientes em relação ao risco de recidiva tumoral / INTRODUCTION: Liver transplantation (LT) is the treatment of choice for patients with cirrhosis and unresectable early hepatocellular carcinoma (HCC). HCC post-transplant recurrence, however, still has an impact on survival. In recent studies, the incidence of HCC recurrence after transplantation ranged from 8% to 20%. Tumor number, size, vascular invasion and satellite nodules have emerged as risk factors for HCC recurrence. In Brazil, in the last decade, we observed a significant increase in the number of liver transplants performed, including in patients with HCC. However, there are few national studies analyzing the results of liver transplantation for HCC. The aim of this multicentric study was to analyze the demographic characteristics, clinical features and outcomes of patients submitted to liver transplantation with HCC in Brazil, evaluate prognostic factors related to HCC post-transplant recurrence and survival, and study the performance of the national selection criteria for liver transplantation, the \"Brazilian Milan Criteria\" (BMC). METHODS: We conducted a national, multicentric, retrospective study to analyze the results of liver transplantation in patients with HCC, in \"MELD era\". Medical records of 1,119 transplanted patients with HCC between 07/2006 and 07/2015, from 13 transplant centers in Brazil, were collected. Patient and tumor characteristics, radiologic and pathologic data were retrospectively analyzed and correlated with post-transplant HCC recurrence and survival. RESULTS: Of the 1,119 HCC transplanted patients, median age was 57 years and 81% were male. Etiology of liver disease was HCV in 60%. Median time on transplant list was 9.8 months. Seventy-eight patients (8%) were included after \"Down-staging\". At diagnosis, most patients had uninodular HCC (67%) and median tumor burden was 30 mm. At diagnosis, in imaging studies, 85% of patients were within the Milan criteria (MC), 8% out of the MC but within the \"Brazilian Milan Criteria\" (BMC) and 6% out of both criteria. During the waiting list period, HCC treatment was performed in 67%. In explant analysis, tumor was uninodular in 46% and moderately differentiated in the majority of cases (71%). Median HCC size was 26 mm. Vascular invasion and satellite nodules were observed in 26% and 22% of patients, respectively. In explant, 70% of patients were within Milan Criteria, 20.5% outside MC but within BMC and 9.5% out of both criteria. Mean follow-up was 28 months, an overall survival was 79% in 1 year, 72.5% in 3 years and 63% in 5 years. Excluding patients who died within 30 days after surgery, overall survival was 89% in 1 year and 75% in 5 years. HCC post-transplant recurrence occurred in 86/1,119 (8%) cases, at a mean time of 12 months. Recurrence-free survival (RFS) was 94.4% in 1 year and 88.3% in 5 years. Sites of recurrence were extrahepatic in 55%, hepatic in 27% and both hepatic and extrahepatic in 18%. Transplanted patients with tumor recurrence presented high mortality, with 1-year survival rate of 34% and 5-year survival rate of 13%. Analyzing the prognostic factors, patients transplanted under Milan Criteria, in radiologic or explant analysis, presented better survival and RFS when compared to patients transplanted outside MC, but within BMC. Patients submitted to liver transplantation after \"Down-staging\" present long-term survival and RFS similar to patients transplanted without \"Down-staging\". Alpha-fetoprotein (AFP) levels were an important pre-transplant prognostic factor for tumor survival and recurrence. The best AFP cut off points found for relapse risk and survival assessment were: AFP at diagnosis > 400 ng / ml and AFP pre-transplant > 200 ng / ml. We also performed a comparison of the \"Brazilian Milan Criteria\" with the Milan Criteria through the Integrated Discrimination Index (IDI). The BMC presented a lower performance than the MC, in the ability to correctly classify patients in relation to the risk of relapse. Elevated AFP levels before liver transplantation, tumor outside Milan Criteria at diagnosis and in explant, and vascular invasion, were independent risk factors for post-transplant HCC recurrence and worse survival. Age > 60 years and etiology of liver disease (HCV), were also negative prognostic factors for survival. CONCLUSIONS: The presence of tumor recurrence had a major impact on survival of transplanted patients with HCC. Tumor staging, evaluated by Milan Criteria on imaging studies or explant analysis, high serum AFP levels and presence of vascular invasion in explant were important prognostic factors for post-transplant HCC recurrence and survival. Patients transplanted after Down-staging presented long-term outcomes similar to patients transplanted under conventional criteria. Patients transplanted outside Milan Criteria, but within \"Brazilian Milan Criteria\" presented worse survival, when compared to patients within MC. The BMC showed lower performance than MC in the ability to correctly classify patients in relation to the risk of tumor recurrence