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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.

Fibrolamellar hepatocellular carcinoma (FL-HCC) is a malignant liver tumor which is thought to be a variant of conventional hepatocellular carcinoma (HCC). It accounts for a small proportion of HCC cases and occurs in a distinctly different group of patients which are young and usually not in the setting of chronic liver disease. The diagnosis of FL-HCC requires the integration of clinical information, imaging studies, and histology. In terms of the treatment options, the only potentially curative treatment option for patients who have resectable disease is surgery either liver resection (LR) or liver transplantation (LT). When performed in a context of aggressive therapy, long-term outcomes after surgery, particularly liver resection for FL-HCC, were favorable. The clinical outcome of patients with unresectable disease is suboptimal with median survival of less than 12 months. The aim of this review is to update the available evidence on diagnosis, treatment options, outcome predictors, and recent developments of patients with this rare disease and to provide a summarized overview of the available literature.

INTRODUÇÃO: O carcinoma hepatocelular (CHC) é o mais frequente tipo de câncer primário do fígado e a sua incidência vem aumentando nas últimas décadas, , tornando-o hoje a terceira causa de morte por câncer no mundo. Em cerca de 70 a 80% dos pacientes, o CHC é precedido pelo desenvolvimento de cirrose hepática. Existe um consenso de que a ressecção cirúrgica do tumor é a única terapêutica efetivamente comprovada. Esta ressecção pode ser realizada tanto através de uma hepatectomia como pelo transplante hepático. Atualmente, apenas 30 a 40% dos pacientes se beneficiam dos tratamentos ditos curativos e, mesmo entre esses pacientes, a sobrevida em cinco anos continua baixa, em torno de 60 a 70%, com taxa de recorrência do tumor em torno de 50% em três anos. Alguns estudos mostraram um pior prognóstico para os pacientes com CHC cuja etiologia é a infecção por vírus B ou C. Isso nos leva à questão sobre a existência de uma diferença entre as diversas etiologias do CHC e o seu prognóstico. OBJETIVOS: Comparar o prognóstico (sobrevida global e livre de doença em cinco anos) de pacientes submetidos à hepatectomia para o tratamento do CHC com relação às diversas etiologias da hepatopatia e estudar fatores prognósticos nesse grupo de pacientes. MÉTODO: Foi realizado um levantamento de prontuários dos pacientes submetidos à hepatectomia entre 2000 e 2014 para tratamento de CHC, seguido de análise estatística desse banco de dados, visando a avaliação de parâmetros clínicos, laboratoriais e cirúrgicos. Os pacientes foram divididos em grupos de acordo com a etiologia da hepatopatia, sendo feita uma análise de sobrevida para comparação. RESULTADOS: Não houve diferença estatisticamente significante de prognóstico entre os grupos de pacientes divididos conforme a etiologia do CHC. A sobrevida global e livre de doença em cinco anos dos pacientes dessa amostra foi de 49,9% e 40,7%, respectivamente. As variáveis prognósticas estatisticamente significantes para sobrevida global foram nível sérico de alfafetoproteína (p=0,043), nível sérico de CA19.9 (p=0,028), invasão da cápsula tumoral (p=0,030), margem livre (p=0,004) e presença de complicações pós-operatórias (p < 0,001). CONCLUSÕES: Pelos dados dessa amostra, pudemos constatar que não houve diferença em relação ao prognóstico entre os grupos de pacientes das diversas etiologias de CHC. As variáveis nível sérico de alfafetoproteína e de CA 19.9, invasão da cápsula tumoral, margem livre e complicações pósoperatórias podem ser consideradas preditoras de pior prognóstico / INTRODUCTION: Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and its incidence is increasing around the world in the last decades, making HCC the third cause of death by cancer in the world. In about 70 to 80% of patients, HCC is preceded by cirrhosis of the liver. It is believed that hepatic resection is the single proven curative treatment. This resection can be done in the form of a hepatectomy or liver transplantation. Nowadays, only 30 to 40% of HCC patients can benefit from these curative treatments and, among them, survival in five years is still around 60 to 70%, with tumor recurrence rate around 50% in three years. Some studies have shown a worse prognosis for HCC patients whose etiology is viral. That brings us to the question about the existence of a difference between the various etiologies of HCC and its prognosis. OBJECTIVES: To compare the prognosis (overall and disease-free survival at five years) of patients undergoing hepatectomy for the treatment of HCC with respect to various etiologies of liver disease and to study prognostic factors in this group of patients. METHOD: We performed a review of medical records of patients undergoing hepatectomy between 2000 and 2014 for the treatment of HCC, followed by statistical analysis of this database for evaluation of clinical, laboratory and surgical parameters. Patients were divided into groups according to the etiology of liver disease followed by overall and disease-free survival analysis for comparison. RESULTS: There was no statistically significant difference in the outcomes of the groups of patients divided according to the etiology of HCC. Overall and disease-free survival at five years of patients in this sample was 49.9% and 40.7%, respectively. Statistically significant prognostic variables for overall survival were serum alpha-fetoprotein (p = 0.043), serum CA19.9 (p = 0.028), invasion of the tumor capsule (p = 0.030), resection margins (p = 0.004) and presence of postoperative complications (p < 0.001). CONCLUSIONS: From the data of this sample, we could verify that there was no prognostic differences between the groups of HCC patients of the various etiologies. The variables serum alphafetoprotein and CA 19.9, invasion of the tumor capsule, resection margins and presence of postoperative complications can be considered predictive of worse prognosis

Gho Wai-Man. / Thesis submitted in: August 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 114-123). / Abstracts in English and Chinese. / ABSTRACT --- p.I / 摘要 --- p.IV / ACKNOWLEDGEMENT --- p.VI / TABLE OF CONTENT --- p.VII / LIST OF TABLES --- p.XII / LIST OF FIGURES --- p.XIII / ABBREVIATIONS --- p.XVI / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Epidemiology --- p.2 / Chapter 1.3 --- Etiological factors --- p.6 / Chapter 1.3.1 --- Viral Hepatitis Infection --- p.6 / Chapter 1.3.1.1 --- Hepatitis B Virus (HBV) --- p.7 / Chapter 1.3.1.2 --- Hepatitis C Virus (HCV) --- p.9 / Chapter 1.3.2 --- Aflatoxin Exposure --- p.10 / Chapter 1.3.3 --- Alcohol Abuse --- p.11 / Chapter 1.3.4 --- Liver Cirrhosis --- p.12 / Chapter 1.4 --- Genetic alterations in hcc --- p.16 / Chapter 1.4.1 --- Chromosomal Gain --- p.16 / Chapter 1.4.2 --- Chromosomal Loss --- p.17 / Chapter 1.5 --- Discovery of common activating transcription factor 5 (atf5) down-regulations in hcc --- p.19 / Chapter 1.5.1 --- Chromosome 19 Aberration in HCC --- p.19 / Chapter 1.5.2 --- Discovery of High Frequency of　ATF5 Down-regulations --- p.19 / Chapter 1.5.3 --- Activating Transcription Factor Family --- p.20 / Chapter 1.6 --- Aim of thesis --- p.28 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.29 / Chapter 2.1 --- Materials --- p.30 / Chapter 2.1.1 --- Chemicals --- p.30 / Chapter 2.1.2 --- Buffers --- p.31 / Chapter 2.1.3 --- Cell culture --- p.31 / Chapter 2.1.4 --- Nucleic acids --- p.32 / Chapter 2.1.5 --- Enzymes --- p.32 / Chapter 2.1.6 --- Equipment --- p.32 / Chapter 2.1.7 --- Kits --- p.33 / Chapter 2.1.8 --- Software and Web Resource --- p.33 / Chapter 2.2 --- Dna extraction --- p.34 / Chapter 2.2.1 --- Cell Lines --- p.34 / Chapter 2.2.2 --- Primary HCC --- p.34 / Chapter 2.2.3 --- Lymphocytic DNA --- p.35 / Chapter 2.3 --- Rna extraction --- p.36 / Chapter 2.4 --- Dna sequencing --- p.38 / Chapter 2.4.1 --- Polymerase Chain Reaction (PCR) --- p.38 / Chapter 2.4.2 --- Cycle Sequencing --- p.39 / Chapter 2.5 --- Dual-labeled fluirescence in situ hybridization (fish) --- p.41 / Chapter 2.5.1 --- FISH Probe Preparation --- p.41 / Chapter 2.5.1.1 --- Preparation of Human Bacterial Artificial Chromosome (BAC) --- p.41 / Chapter 2.5.1.2 --- Nick Translation --- p.41 / Chapter 2.5.2 --- FISH --- p.42 / Chapter 2.6 --- 5-aza-2'-deoxycytidine & trichostatin a treatment on cell lines --- p.43 / Chapter 2.7 --- Bisulfite modificaiton of dna --- p.43 / Chapter 2.8 --- Methylation-specific pcr (msp) --- p.44 / Chapter 2.9 --- Bisulfite dna sequencing --- p.44 / Chapter 2.10 --- Quantitative reverse transcription pcr (qrt-pcr) --- p.46 / Chapter 2.11 --- In-vitro and in-vivo functinal examination --- p.49 / Chapter 2.11.1 --- ATF5 Transfection --- p.49 / Chapter 2.11.2 --- Cell Growth Assay --- p.50 / Chapter 2.11.3 --- Xenograft Development --- p.51 / Chapter 2.12 --- codelink expression microarray --- p.51 / Chapter 2.13 --- Statistical analysis --- p.53 / Chapter CHAPTER 3 --- INACTIVATION OF MECHANISMS UNDERLYING ATF5 DOWN-REGULATION --- p.54 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Materials and methods --- p.58 / Chapter 3.2.1 --- Cell Lines --- p.58 / Chapter 3.2.2 --- Mutational Analysis --- p.58 / Chapter 3.2.3 --- Copy Number Loss --- p.59 / Chapter 3.2.4 --- Epigenetic Control --- p.59 / Chapter 3.3 --- Results --- p.67 / Chapter 3.3.1 --- Sequencing Analysis of A TF5 Gene --- p.67 / Chapter 3.3.2 --- FISH Analysis of ATF5 Copy Number --- p.73 / Chapter 3.3.3 --- Epigenetic Control of A TF5 Expression --- p.73 / Chapter 3.4 --- Discussion --- p.82 / Chapter CHAPTER 4 --- FUNCTIONAL EXAMINATION AND INVESTIGATION OF DOWNSTREAM TARGETS MODULATED BY ATF5 --- p.85 / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Materials and methods --- p.88 / Chapter 4.2.1 --- Cell Lines --- p.88 / Chapter 4.2.2 --- Plasmids and Transfection --- p.88 / Chapter 4.2.3 --- Cell Growth Assay --- p.88 / Chapter 4.2.4 --- Xenograft Development --- p.88 / Chapter 4.2.5 --- CodeLink Expression Microarray --- p.89 / Chapter 4.2.6 --- Quantitative RT-PCR --- p.90 / Chapter 4.2.7 --- Statistical analysis --- p.90 / Chapter 4.3 --- Results --- p.91 / Chapter 4.3.1 --- Cell Proliferation --- p.91 / Chapter 4.3.1.1 --- In-Vitro Examination --- p.91 / Chapter 4.3.1.2 --- In-Vivo Examination --- p.91 / Chapter 4.3.2 --- Microarray A nalysis --- p.91 / Chapter 4.3.3 --- Correlation of A TF5 with Id-1 Expression --- p.103 / Chapter 4.4 --- Discussion --- p.106 / Chapter CHAPTER 5 --- PROPOSED FUTURE INVESTIGATIONS --- p.110 / Chapter 5.1 --- inactivation mechanisms of atf5 gene --- p.111 / Chapter 5.2 --- Molecular pathways modulated by atf5 --- p.112 / Chapter CHAPTER 6 --- REFERENCES --- p.114

Ho Kar Fai, William. / Thesis submitted in: July 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 121-129). / Abstracts in English and Chinese. / Abstract --- p.I / Acknowledgement --- p.V / Table of Contents --- p.VI / Abbreviations --- p.VIII / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- The recent situation of hepatitis B infection and HBV-induced HCC in Hong Kong / Chapter 1.2 --- Natural history of HBV infection in human / Chapter 1.3 --- The genomic organization of HBV / Chapter 1.4 --- Potential oncogenic mechanism of HBV-induced hepatocarcinogenesis / Chapter 1.5 --- Aim of the present study / Chapter Chapter 2 --- Materials and methods --- p.16 / Chapter 2.1 --- Transformation in E.coli for subtracted normal-counterpart library / Chapter 2.2 --- PCR amplification of subtracted clones / Chapter 2.3 --- Sequencing of subtracted clones with dye-terminator cycle sequencing technology / Chapter 2.4 --- Sequence analysis and database construction / Chapter 2.5 --- Molecular cloning and characterization of novel gene / Chapter 2.6 --- In silico structural and functional analysis of Z313 / Chapter 2.7 --- Cloning and sequencing analysis of zinc finger protein 313 (Z313) / Chapter 2.7.1 --- PCR amplification of target gene -Z313 / Chapter 2.7.2 --- Mini-preparation of plasmid DNA / Chapter 2.7.3 --- Cycle sequencing of cloned cDNA -Z313 with dye-primer technology / Chapter 2.8 --- Multiple Tissue Northern (MTN) blot hybridisation / Chapter 2.9 --- RT-PCR analysis of Z313 / Chapter 2.10 --- Subcellular localization study of Z313 by Green Fluorescent Protein (GFP) / Chapter 2.10.1 --- Directional cloning of Z313 into pEGFP-Cl / Chapter 2.10.2 --- Mini-preparation of plasmid DNA / Chapter 2.10.3 --- Transient transfection of plasmids in different cell lines / Chapter 2.10.4 --- Microscope observation of GFP transfected cells / Chapter Chapter 3 --- Results --- p.49 / Chapter 3.1 --- PCR selection of subtracted clones for sequencing analysis / Chapter 3.2 --- Partial sequencing of selected subtracted clones / Chapter 3.3 --- DNA homology searching using program - BLASTN / Chapter 3.4 --- Catalogue of the 467 ESTs from the subtracted normal-counterpart library / Chapter 3.5 --- Classification and frequency of the subtracted normal-counterpart cDNA clones / Chapter 3.6 --- Identification of putative differentially expressed genes in HCC surrounding normal liver / Chapter 3.7 --- Categorization of ESTs exclusively appeared in the subtracted normal- counterpart library / Chapter 3.8 --- In silico structural and functional analysis of zinc finger protein313 (Z313) / Chapter 3.9 --- Molecular cloning of zinc finger protein 313 (Z313) / Chapter 3.10 --- Northern analysis of zinc finger protein 313 (Z313) / Chapter 3.11 --- RT-PCR analysis of zinc finger protein 313 (Z313) / Chapter 3.12 --- Subcellular localization study of zinc finger protein 313 (Z313) / Chapter Chapter 4 --- Discussion --- p.104 / Chapter 4.1 --- EST analysis on the subtracted normal-counterpart cDNA clones / Chapter 4.1.1 --- Characterization of ESTs generated from the subtracted normal-counterpart library / Chapter 4.1.2 --- Putative differentially expressed genes in HCC surrounding normal liver related to hepatocellular carcinoma / Chapter 4.2 --- Molecular cloning and characterization of zinc finger protein313 (Z313) / Chapter 4.3 --- Future aspects / References --- p.121

INTRODUÇÃO: O carcinoma hepatocelular (CHC) é o mais frequente tipo de câncer primário do fígado e a sua incidência vem aumentando nas últimas décadas, , tornando-o hoje a terceira causa de morte por câncer no mundo. Em cerca de 70 a 80% dos pacientes, o CHC é precedido pelo desenvolvimento de cirrose hepática. Existe um consenso de que a ressecção cirúrgica do tumor é a única terapêutica efetivamente comprovada. Esta ressecção pode ser realizada tanto através de uma hepatectomia como pelo transplante hepático. Atualmente, apenas 30 a 40% dos pacientes se beneficiam dos tratamentos ditos curativos e, mesmo entre esses pacientes, a sobrevida em cinco anos continua baixa, em torno de 60 a 70%, com taxa de recorrência do tumor em torno de 50% em três anos. Alguns estudos mostraram um pior prognóstico para os pacientes com CHC cuja etiologia é a infecção por vírus B ou C. Isso nos leva à questão sobre a existência de uma diferença entre as diversas etiologias do CHC e o seu prognóstico. OBJETIVOS: Comparar o prognóstico (sobrevida global e livre de doença em cinco anos) de pacientes submetidos à hepatectomia para o tratamento do CHC com relação às diversas etiologias da hepatopatia e estudar fatores prognósticos nesse grupo de pacientes. MÉTODO: Foi realizado um levantamento de prontuários dos pacientes submetidos à hepatectomia entre 2000 e 2014 para tratamento de CHC, seguido de análise estatística desse banco de dados, visando a avaliação de parâmetros clínicos, laboratoriais e cirúrgicos. Os pacientes foram divididos em grupos de acordo com a etiologia da hepatopatia, sendo feita uma análise de sobrevida para comparação. RESULTADOS: Não houve diferença estatisticamente significante de prognóstico entre os grupos de pacientes divididos conforme a etiologia do CHC. A sobrevida global e livre de doença em cinco anos dos pacientes dessa amostra foi de 49,9% e 40,7%, respectivamente. As variáveis prognósticas estatisticamente significantes para sobrevida global foram nível sérico de alfafetoproteína (p=0,043), nível sérico de CA19.9 (p=0,028), invasão da cápsula tumoral (p=0,030), margem livre (p=0,004) e presença de complicações pós-operatórias (p < 0,001). CONCLUSÕES: Pelos dados dessa amostra, pudemos constatar que não houve diferença em relação ao prognóstico entre os grupos de pacientes das diversas etiologias de CHC. As variáveis nível sérico de alfafetoproteína e de CA 19.9, invasão da cápsula tumoral, margem livre e complicações pósoperatórias podem ser consideradas preditoras de pior prognóstico / INTRODUCTION: Hepatocellular carcinoma (HCC) is the most frequent type of primary liver cancer and its incidence is increasing around the world in the last decades, making HCC the third cause of death by cancer in the world. In about 70 to 80% of patients, HCC is preceded by cirrhosis of the liver. It is believed that hepatic resection is the single proven curative treatment. This resection can be done in the form of a hepatectomy or liver transplantation. Nowadays, only 30 to 40% of HCC patients can benefit from these curative treatments and, among them, survival in five years is still around 60 to 70%, with tumor recurrence rate around 50% in three years. Some studies have shown a worse prognosis for HCC patients whose etiology is viral. That brings us to the question about the existence of a difference between the various etiologies of HCC and its prognosis. OBJECTIVES: To compare the prognosis (overall and disease-free survival at five years) of patients undergoing hepatectomy for the treatment of HCC with respect to various etiologies of liver disease and to study prognostic factors in this group of patients. METHOD: We performed a review of medical records of patients undergoing hepatectomy between 2000 and 2014 for the treatment of HCC, followed by statistical analysis of this database for evaluation of clinical, laboratory and surgical parameters. Patients were divided into groups according to the etiology of liver disease followed by overall and disease-free survival analysis for comparison. RESULTS: There was no statistically significant difference in the outcomes of the groups of patients divided according to the etiology of HCC. Overall and disease-free survival at five years of patients in this sample was 49.9% and 40.7%, respectively. Statistically significant prognostic variables for overall survival were serum alpha-fetoprotein (p = 0.043), serum CA19.9 (p = 0.028), invasion of the tumor capsule (p = 0.030), resection margins (p = 0.004) and presence of postoperative complications (p < 0.001). CONCLUSIONS: From the data of this sample, we could verify that there was no prognostic differences between the groups of HCC patients of the various etiologies. The variables serum alphafetoprotein and CA 19.9, invasion of the tumor capsule, resection margins and presence of postoperative complications can be considered predictive of worse prognosis

Jiang, Jieying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 170-186). / Abstract also in Chinese. / ACKNOWLEDGEMENT --- p.II / ABBREVIATIONS --- p.III / ABSTRACT OF THESIS ENTITLED: --- p.VI / 摘要 --- p.IX / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Individual variations of blood sex steroid levels and their determinants --- p.1 / Chapter 1.1.1 --- Introduction to Sex steroids --- p.1 / Chapter 1.1.2 --- Androgens --- p.1 / Chapter 1.1.2.1 --- Types of androgens --- p.1 / Chapter 1.1.2.2 --- Androgens plasma concentration and relative biological potencies --- p.2 / Chapter 1.1.2.3 --- Androgen biosynthesis and metabolism --- p.3 / Chapter 1.1.2.4 --- Testosterone transportation in plasma --- p.6 / Chapter 1.1.2.5 --- Measurement of free testosterone --- p.7 / Chapter 1.1.2.6 --- The hypothalamus-pituitary-testicular axis and testosterone secretion --- p.8 / Chapter 1.1.2.7 --- Androgen action --- p.10 / Chapter 1.1.2.8 --- Androgen biological function and diseases in men --- p.11 / Chapter 1.1.3 --- Estrogen biological function and diseases in men --- p.12 / Chapter 1.1.4 --- Factors influencing circulating sex steroid levels --- p.13 / Chapter 1.1.4.1 --- Genetic determinants affecting sex steroid levels --- p.15 / Chapter 1.2 --- Genetic variants in sex steroid metabolic pathway and hepatocellular carcinoma (HCC) --- p.18 / Chapter 1.2.1 --- Epidemiology of HCC --- p.18 / Chapter 1.2.2 --- Etiological factors of HCC --- p.22 / Chapter 1.2.3 --- The male predominance in HCC --- p.24 / Chapter 1.2.4 --- Genetic predisposition to HCC --- p.26 / Chapter CHAPTER 2 --- PART A STUDY: GENETIC VARIATIONS IN SEX STEROID METABOLIC PATHWAY AND ASSOCIATION WITH SEX STEROID LEVELS --- p.28 / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Candidate genes association with sex steroid levels --- p.28 / Chapter 2.1.2 --- Genes involved in androgen metabolism --- p.29 / Chapter 2.1.2.1 --- SRD5A --- p.29 / Chapter 2.1.2.2 --- HSD3B1 --- p.30 / Chapter 2.1.2.3 --- HSD17B2 --- p.31 / Chapter 2.1.2.4 --- AKR1C3 and AKRlC4 --- p.31 / Chapter 2.1.2.5 --- AKR1D1 --- p.32 / Chapter 2.1.3 --- Genes involved in estrogen metabolism --- p.32 / Chapter 2.1.3.1 --- CYP19A1 --- p.32 / Chapter 2.1.3.2 --- Other genes involved in estrogen metabolism --- p.33 / Chapter 2.1.4 --- Association of sex steroid related genes and blood concentrations of sex steroid levels --- p.33 / Chapter 2.1.4.1 --- Genes involved in androgen metabolic pathway and association with sex steroid levels --- p.33 / Chapter 2.1.4.2 --- Genes involved in estrogen metabolic pathway and association with sex steroid levels --- p.36 / Chapter 2.1.5 --- Aims of the study (Part A) --- p.37 / Chapter 2.2 --- Materials and methods --- p.38 / Chapter 2.2.1 --- Study subjects and biological samples --- p.38 / Chapter 2.2.2 --- TagSNP selection --- p.39 / Chapter 2.2.3 --- Genotyping of tagging SNPs --- p.41 / Chapter 2.2.4 --- Genotyping methods comparison --- p.52 / Chapter 2.2.5 --- Statistics --- p.53 / Chapter 2.3 --- Results --- p.54 / Chapter 2.3.1 --- Characteristics of study population --- p.54 / Chapter 2.3.2 --- Replication study for the association of CYP19A1 --- p.55 / Chapter 2.3.2.1 --- Association of the SNP rs2470152 and rs2899470 with serum estrogen and testosterone levels --- p.55 / Chapter 2.3.2.2 --- Halotype analysis and haplotype association in the tertile groups --- p.61 / Chapter 2.3.2.3 --- Haplotype construction of 3 SNPs --- p.63 / Chapter 2.3.3 --- SRD5A1 --- p.65 / Chapter 2.3.3.1 --- Association of SRD5A1 and sex steroid levels --- p.65 / Chapter 2.3.3.2 --- Haplotype analysis and haplotype association in the tertile groups --- p.71 / Chapter 2.3.4 --- SRD5A2 --- p.72 / Chapter 2.3.4.1 --- Association of SRD5A2 and sex steroid levels --- p.72 / Chapter 2.3.4.2 --- Haplotype association analysis of SRD5A2 in tertile groups --- p.76 / Chapter 2.3.5 --- HSD3B1 --- p.77 / Chapter 2.3.5.1 --- Association of HSD3B1 and sex steroid levels --- p.77 / Chapter 2.3.6 --- HSD17B2 --- p.80 / Chapter 2.3.6.1 --- Association of HSD17B2 and sex steroid levels --- p.80 / Chapter 2.3.6.2 --- Halotype association analysis of HSD17B2 in the tertile groups --- p.87 / Chapter 2.3.7 --- AKR1C4 --- p.89 / Chapter 2.3.7.1 --- Association of AKR1C4 and sex steroid levels --- p.89 / Chapter 2.3.7.2 --- Halotype association analysis of AKR1C4 in the tertile groups --- p.93 / Chapter 2.3.8 --- AKR1D1 --- p.94 / Chapter 2.3.8.1 --- Association of AKR1D1 and sex steroid levels --- p.94 / Chapter 2.3.8.2 --- Haplotype association analysis of AKR1D1 in the tertile groups --- p.99 / Chapter 2.3.9 --- AKR1C3 --- p.100 / Chapter 2.3.9.1 --- Association of AKR1C3 and sex steroid levels --- p.100 / Chapter 2.3.9.2 --- Haplotype association analysis of AKR1C3 in the tertile groups --- p.104 / Chapter 2.3.10 --- Overall association of polymorphisms in sex steroid metabolism genes and metabolites levels in blood --- p.105 / Chapter 2.4 --- Discussion --- p.106 / Chapter 2.4.1 --- SRD5A and sex steroid levels --- p.106 / Chapter 2.4.2 --- HSD17B2 and sex steroid levels --- p.110 / Chapter 2.4.3 --- "AKR1D1, AKR1C4, AKR1C3 and catabolic intermediates of sex steroids" --- p.112 / Chapter 2.4.4 --- HSD3B1 and sex steroid levels --- p.114 / Chapter 2.4.4 --- CYP19A1 and sex steroid levels --- p.114 / Chapter CHAPTER 3 --- PART B STUDY: GENETIC VARIATIONS IN SEX STEROID METABOLIC PATHWAY AND ASSOCIATION WITH HCC --- p.119 / Chapter 3.1 --- Introduction --- p.119 / Chapter 3.1.1 --- Previous genetic association studies of HCC on sex steroid metabolic pathways --- p.119 / Chapter 3.1.2 --- Previous genetic association studies of HCC in other pathways --- p.120 / Chapter 3.1.3 --- "Association of sex steroid related genes and other cancers, like prostate cancer" --- p.121 / Chapter 3.1.4 --- Aims of the study (Part B) --- p.123 / Chapter 3.2 --- Materials and method --- p.125 / Chapter 3.2.1 --- "Study subjects, Genomic DNA extraction" --- p.125 / Chapter 3.2.2 --- Tissue specimen and cell lines --- p.125 / Chapter 3.2.3 --- TagSNP selection --- p.126 / Chapter 3.2.4 --- Genotyping of tagging SNPs --- p.126 / Chapter 3.2.5 --- Statistics --- p.127 / Chapter 3.2.6 --- Extraction of RNA and Reverse-Transcription-PCR --- p.128 / Chapter 3.3 --- Results --- p.130 / Chapter 3.3.1 --- SRD5A1 --- p.130 / Chapter 3.3.1.1 --- SRD5A1 polymorphisms and risk of HCC --- p.130 / Chapter 3.3.2 --- SRD5A2 --- p.134 / Chapter 3.3.2.1 --- SRD5A2 polymorphisms and risk of HCC --- p.134 / Chapter 3.3.2.2 --- Haplotype analysis --- p.136 / Chapter 3.3.3 --- HSD3B1 --- p.137 / Chapter 3.3.3.1 --- HSD3B1 polymorphisms and risk of HCC --- p.137 / Chapter 3.3.3.2 --- Haplotype analysis --- p.139 / Chapter 3.3.4 --- HSD17B2 --- p.140 / Chapter 3.3.4.1 --- HSD17B2 polymorphisms and risk of HCC --- p.140 / Chapter 3.3.4.2 --- Haplotype analysis --- p.143 / Chapter 3.3.5 --- CYP19A1 --- p.144 / Chapter 3.3.5.1 --- CYP19A1 polymorphisms and risk of HCC --- p.144 / Chapter 3.3.5.2 --- Haplotype analysis --- p.146 / Chapter 3.3.6 --- AKR1C4 --- p.147 / Chapter 3.3.6.1 --- AKR1C4 polymorphisms and risk of HCC --- p.147 / Chapter 3.3.6.2 --- Haplotype analysis --- p.148 / Chapter 3.3.7 --- AKR1D1 --- p.149 / Chapter 3.3.7.1 --- AKR1D1 polymorphisms and risk of HCC --- p.149 / Chapter 3.3.7.2 --- Haplotype analysis --- p.150 / Chapter 3.3.8 --- AKR1C3 --- p.151 / Chapter 3.3.8.1 --- AKR1C3 polymorphisms and risk of HCC --- p.151 / Chapter 3.3.8.2 --- Haplotype analysis --- p.152 / Chapter 3.3.9 --- mRNA expression study of the 5 α -reductase isoforms --- p.153 / Chapter 3.3.9.1 --- Expression of SRD5A1 and SRD5A2 mRNAin HCC patients --- p.153 / Chapter 3.3.9.2 --- Expression of SRD5A1 and SRD5A2 mRNAin prostate and HCC cell lines --- p.154 / Chapter 3.3.10 --- Overall association of polymorphisms in sex steroid metabolism genes and risk of HCC --- p.154 / Chapter 3.3.11 --- GMDR analysis --- p.156 / Chapter 3.4 --- Discussion --- p.159 / Chapter 3.4.1 --- 5 α-reductase and risk of HCC --- p.159 / Chapter 3.4.1.1 --- SRD5A2 --- p.160 / Chapter 3.4.1.2 --- SRD5A1 --- p.161 / Chapter 3.4.2 --- Other genes and association with HCC --- p.162 / Chapter 3.4.2.1 --- HSD17B2 and risk of HCC --- p.162 / Chapter 3.4.2.2 --- "HSD3B1, AKR1C3, AKR1C4, AKR1D1 and risk of HCC" --- p.163 / Chapter 3.4.2.3 --- CYP19A1 and risk of HCC --- p.164 / Chapter 3.4.3 --- Gene-Gene interactions associated with HCC --- p.165 / Chapter CHAPTER 4 --- CONCLUSIONS AND PROSPECT FOR FUTURE WORK --- p.166 / Chapter 4.1 --- Conclusion --- p.166 / Chapter 4.2 --- Future works and prospect --- p.169 / REFERENCES --- p.170

Hepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC. / Hepatocellular carcinoma (HCC) is the second most common cause of cancerrelated mortality worldwide. AKT pathway has been found activated in 50% of HCC cases, making it promising target. Therefore we assess efficacy of the allosteric AKT inhibitor or the combination of Sorafenib with AKT inhibitor compared to untreated control and to standard treatment, Sorafenib, in vitro and in vivo. AKT inhibitor blocked phosphorylation of AKT in vitro and strongly inhibited cell growth with significantly higher potency than Sorafenib. Similarly, apoptosis and cell migration were strongly reduced by AKT inhibitor in vitro. To mimic human advanced HCC, we used diethylnitrosamine-induced cirrhotic rat model with fully developed HCC. MRI analyses showed that AKT inhibitor significantly reduced overall tumor size. Furthermore, number of tumors was decreased by AKT inhibitor, which was associated with increased apoptosis and decreased proliferation. Tumor contrast enhancement was significantly decreased in the AKT inhibitor group. Moreover, on tumor tissue sections, we observed a vascular normalization and a significant decrease in fibrosis in surrounding liver of animals treated with AKT inhibitor. Finally, pAKT/AKT levels in AKT inhibitor treated tumors were reduced, followed by down regulation of actors of AKT downstream signalling pathway: pmTOR, pPRAS40, pPLCγ1 and pS6K1. In conclusion, we demonstrated that AKT inhibitor blocks AKT phosphorylation in vitro and in vivo. In HCC-rat model, AKT inhibitor was well tolerated, showed anti-fibrotic effect and had stronger antitumor effect than Sorafenib. Our results confirm the importance of targeting AKT in HCC.

Première partie: Les adénomes hépatocellulaires (AHC) sont des tumeurs bénignes qui se développent le plus souvent chez la femme jeune suite à la prise de contraceptifs oraux. Ils sont classés, sur le plan moléculaire, en: 1) AHC inflammatoires (AHCI), caractérisés par des mutations de différents gènes (IL6ST, GNAS ou STAT3) entrainant une activation de la voie de signalisation de l'interleukine 6; 2) les AHC avec mutations biallèliques inactivatrices d'HNF1A (H-AHC); 3) les AHC mutés pour CTNNB1 (bAHC) qui présentent un risque accru de transformation maligne et 4) les AHC non classés (UAHC). La glycogénose de type I (GSD I) est une maladie métabolique héréditaire rare qui prédispose au développement des AHC. Cependant les principales études moléculaires des AHC ont été réalisées sur des tumeurs sporadiques, et le profil moléculaire des AHC associée à la GSDI reste à déterminer.L'objectif de notre a été de caractériser, par des techniques de séquencage et de PCR quantitative, les altérations moléculaires d'une série d'AHC développés chez les patients atteints de GSD. Nous avons mis en évidence que le profil des AHC développés dans un contexte de GSDI était différent de celui des AHC sporadiques, avec aucun H-AHC et une tendance vers une augmentation de la fréquence des mutations de CTNNB1. Nous avons également observé que les foies non tumoraux des patients atteints de GSDI présentaient différentes anomalies métaboliques (répression de la néoglucogenèse et activation de la glycolyse et de la synthèse des acides gras) également retrouvées dans les H-AHC sporadiques. Ces anomalies sont d'autre connues pour favoriser la cancérogénèse. Notre étude a ainsi démontré que la GSDI prédisposait à des AHC de sous type moléculaire particulier, elle a également identifié diverses anomalies métaboliques observées dans les foies non tumoraux des patients atteints de GSDI qui pourraient contribuer au développement des tumeurs. Deuxième partie: Le carcinome hépatocellulaire (CHC), développé à partir des hépatocytes, est la tumeur maligne hépatique primitive la plus fréquente. Il représente le cinquième cancer le plus fréquent dans le monde et la troisième cause de mortalité liée au cancer. Il se développe dans environ 80% des cas dans un contexte d'hépatopathie chronique, sur parenchyme cirrhotique. Le processus de transformation maligne de la cirrhose en CHC est séquentiel, et passe par différentes étapes prénéoplasiques, comme les macronodules de bas ou haut grade. Il a récemment été mis en évidence que l'anomalie moléculaire la plus fréquente dans le CHC était les mutations du promoteur de TERT, gène codant pour la télomérase. Le but de notre étude a été d'étudier si ces mutations étaient également présente dans une série de 96 nodules développés sur cirrhose. Dans 31 cas, nous avons également déterminer le statut mutationnel de 10 gènes fréquemment mutés dans le CHC. L'ensemble des nodules a fait l'objet d'une relecture par 6 anatomopathologistes spécialisés en pathologie hépatique, et l'expression de marqueurs immunohistochimiques de malignité (Glypican 3, heat Shock protein 70, Glutamine Synthetase) a été évaluée. Nous avons identifié des mutations dans 6% des macronodules de bas grade, 20% des macronodules de haut grade, et dans environ 50% des nodules transformés en CHC. L’existence de mutations était corrélée à plusieurs critères morphologiques et immunohistochimiques de malignité. Aucune mutation dans un des 10 autres gènes fréquemment mutés dans le CHC n’a été retrouvée. Nos résultats démontrent que la fréquence des mutations du promoteur de TERT augmente au cours de la carcinogénèse hépatique, et qu’il s’agit d’une anomalie très précoce qui pourrait constituer un biomarqueur d'évaluation du risque de transformation des lésions prénéoplasiques développées sur cirrhose. / First Part: Hepatocellular adenomas (HCA) are benign tumors which most often develop in young women taking oral contraceptives. They are classified as : 1) inflammatory HCA (IHCA), characterized by mutations of genes (IL6ST, GNAS, STAT3) involved in the interleukin 6 pathway; 2) HCA with biallelic inactivating mutations of HNF1A (H-HCA) ; 3) CTNNB1 mutated HCA (bAHC), which harbour a high risk of malignant transformation and 4) unclassified HCA (UHCC). Glycogen storage disease type 1 (GSD1) is a rare hereditary metabolic disease that predispose to HCE development. The main molecular studies of HCA were pervformed on sporadic cases, and the molecular profile of HCA associated to GSDI remains to be investigated. The aim of our study was to characterize, by gene sequencing and gene expression profiling, a series of HCA developed in patients with GSD1. The molecular profile of GSD1 HCA was different to that of sporadic HCA, with a lack of H-HCA and a high frequency of b-HCA. We also observed that non tumoral livers of GSD1 patients featured several metabolic alterations (gluconeogenesis repression, glycolysis and fatty acid synthesis activation) that were also observed in sporadic H-HCA and may favor carcinogenesis. Alltogether, our study demonstrated that GSD1 predispose to particular HCA subtypes and the metabolic alterations observed in non tumorl liver of GSD1 patients may contribute to tumor formation. Second Part: Hepatocellular carcinoma (HCCIt is the fifth most frequent cancer and the third cause of cancer related death worldwide. If TERT promoter mutations are, so far, the most frequent recurrent molecular alterations of HCC, genetic determinants of the early steps of carcinogenesis on cirrhosis are still poorly understood. We aimed to evaluate the occurrence of telomerase reverse transcriptase (TERT) promoter mutations in a series of 96 macrondouels developed in a cirrhotic background. For 30 cases, 10 genes frequently mutated in HCC were also screeened. Six liver pathologists reviewed all the samples, and mmunohistochemistry (IHC) analyses were performed for glypican 3, glutamine synthase, and heat shock protein 70. TERT promoter mutations were highly related to the step-wise hepatocarcinogenesis because mutations were identified in 6% of low grade dysplastic nodules, 19% of high grade dysplastic nodules, 61% of high grade nodules with foci of transformation into HCC, and 42% of small HCC. Mutations in the 10 genes recurrently mutated in HCC were only identified in 28% of the small HCC. In conclusion, Frequency of TERT promoter mutations rapidly increases during the different steps of the transformation of premalignant lesions into HCC on cirrhosis. Consequently, somatic TERT promoter mutation is a new biomarker predictive of transformation of premalignant lesions into HCC.