Li, Sai Kam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 162-179). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract in Chinese (摘要） --- p.ii / Acknowledgements --- p.iii / Table of Content --- p.iv / Abbreviations --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xvii / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- Hepatitis B Virus / Chapter 1.1.1 --- General information --- p.1 / Chapter 1.1.2 --- Classification --- p.2 / Chapter 1.1.3 --- Virus life cycle and genome --- p.3 / Chapter 1.1.4 --- Hepatitis B virus X protein (HBx) --- p.7 / Chapter 1.2 --- Enigmatic functions of HB --- p.x / Chapter 1.2.1 --- HBx as a transactivator --- p.10 / Chapter 1.2.2 --- HBx as a cell cycle regulator --- p.12 / Chapter 1.2.3 --- HBx as an apoptosis modulator --- p.13 / Chapter 1.3 --- Etiology of HBV-mediated hepatocarcinogenesis --- p.14 / Chapter 1.4 --- Clinical mutants of HBV --- p.16 / Chapter 1.5 --- Hypothesis and aims of the research --- p.16 / Chapter 1.6 --- Basis of Tet-On system --- p.18 / Chapter CHPATER 2 --- EXPERIMENT MATERIALS / Chapter 2.1 --- Cell culture / Chapter 2.1.1 --- Cell-lines --- p.21 / Chapter 2.1.2 --- Culture medium --- p.22 / Chapter 2.1.3 --- Culture medium supplements --- p.23 / Chapter 2.2 --- Reagents for subcloning / Chapter 2.2.1 --- Reagents for polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.2 --- Reagents for restriction enzyme digestion --- p.24 / Chapter 2.2.3 --- Reagents for ligation --- p.25 / Chapter 2.2.4 --- Reagents for electrophoresis --- p.25 / Chapter 2.2.5 --- Reagents for E. coli DH5a preparation --- p.25 / Chapter 2.2.6 --- Materials for bacterial culture work --- p.27 / Chapter 2.3 --- Reagents for subcellular localization study / Chapter 2.3.1 --- Reagents for cell staining --- p.28 / Chapter 2.3.2 --- Reagents for mounting slides --- p.29 / Chapter 2.3.3 --- Materials for site-directed mutagenesis --- p.29 / Chapter 2.4 --- Reagents for cell cycle analysis and cellular proliferation / Chapter 2.4.1 --- Reagents for cell cycle analysis --- p.29 / Chapter 2.4.2 --- Reagents for cellular proliferation study --- p.30 / Chapter 2.5 --- Reagents for protein expression study / Chapter 2.5.1 --- Cell lysis buffer --- p.30 / Chapter 2.5.2 --- Reagents for SDS-PAGE --- p.30 / Chapter 2.5.3 --- Reagents for Western blot --- p.33 / Chapter 2.5.4 --- Antibodies --- p.34 / Chapter 2.6 --- Reagents for gene expression study / Chapter 2.6.1 --- Reagents for RNA extraction --- p.36 / Chapter 2.6.2 --- Reagents for first strand cDNA synthesis --- p.37 / Chapter 2.6.3 --- Reagents for real-time PCR --- p.37 / Chapter 2.7 --- Reagents for establishment of Tet-On inducible stable cell-lines / Chapter 2.7.1 --- Reagents for MTT assay --- p.38 / Chapter 2.7.2 --- Reagents for selection of stable clones --- p.38 / Chapter 2.8 --- Vectors used in the project / Chapter 2.8.1 --- Vectors for subcellular localization study --- p.39 / Chapter 2.8.2 --- Vectors for establishment of Tet-on inducible cell-lines --- p.39 / Chapter 2.9 --- Primers used in the project / Chapter 2.9.1 --- Primers used for subcloning --- p.42 / Chapter 2.9.2 --- Primers used for site-directed mutagenesis --- p.43 / Chapter 2.9.3 --- Primers used in real-time chain polymerase reaction --- p.43 / Chapter CHAPTER 3 --- RESEARCH METHODS / Chapter 3.1 --- Subcloning of HBx and mutant genes into a green fluorescence protein (GFP) expression vector / Chapter 3.1.1 --- Amplification of HBxWt，HBxΔC44 and HBxAN60 genes --- p.45 / Chapter 3.1.2 --- Purification of PCR products --- p.46 / Chapter 3.1.3 --- Restriction enzyme digestion --- p.47 / Chapter 3.1.4 --- Ligation of gene products with pEGFP-C 1 vector --- p.47 / Chapter 3.1.5 --- Preparation of chemically competent bacterial cells E. coli strain DH5α --- p.47 / Chapter 3.1.6 --- Transformation of the ligation product into competent cells --- p.48 / Chapter 3.1.7 --- PCR confirmation of successful ligation --- p.48 / Chapter 3.1.8 --- Small scale preparation of bacterial plasmid DNA --- p.49 / Chapter 3.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.50 / Chapter 3.1.10 --- Large scale preparation of target recombinant plasmid DNA --- p.50 / Chapter 3.2 --- Subcellular localization pattern study / Chapter 3.2.1 --- Cell transfection --- p.51 / Chapter 3.2.2 --- Mitochondria and nucleus staining --- p.52 / Chapter 3.2.3 --- Epi-fluorescence microscopy --- p.53 / Chapter 3.2.4 --- Analysis of fluorescence images --- p.53 / Chapter 3.2.5 --- In vitro site-directed mutagenesis --- p.53 / Chapter 3.3 --- Cell cycle phase analysis with flow cytometry / Chapter 3.3.1 --- Cell transfection --- p.55 / Chapter 3.3.2 --- Cell staining --- p.55 / Chapter 3.3.3 --- Flow cytometry --- p.55 / Chapter 3.4 --- Cellular proliferation quantification by BrdU proliferation assay / Chapter 3.4.1 --- Cell transfection --- p.57 / Chapter 3.4.2 --- BrdU ELISA measurement --- p.57 / Chapter 3.5 --- Protein expression / Chapter 3.5.1 --- Cell lysate collection --- p.58 / Chapter 3.5.2 --- Quantification of protein samples --- p.59 / Chapter 3.5.3 --- SDS-PAGE --- p.59 / Chapter 3.5.4 --- Western blot --- p.60 / Chapter 3.5.5 --- Western blot luminal detection --- p.60 / Chapter 3.6 --- Gene expression / Chapter 3.6.1 --- Primer design --- p.61 / Chapter 3.6.2 --- Cell transfection --- p.61 / Chapter 3.6.3 --- RNA extraction --- p.61 / Chapter 3.6.4 --- Reverse transcription for first strand complementary DNA (cDNA) --- p.63 / Chapter 3.6.5 --- Quantitative real-time PCR --- p.63 / Chapter 3.7 --- Establishment of Tet-On inducible stable cell-lines / Chapter 3.7.1 --- Subcloning of HBx gene into pTRE2 vector --- p.64 / Chapter 3.7.2 --- Construction of WRL68/Tet-On stable cell-lines --- p.64 / Chapter 3.7.3 --- Construction of WRL68/Tet-On HBx and mutants expression cell-lines --- p.68 / Chapter 3.7.4 --- Characterization of Tet-On gene expression monoclones --- p.69 / Chapter 3.8 --- Statistical analyses --- p.70 / Chapter CHPATER 4 --- STUDY ON MITOCHONDRIA TARGETING / Chapter 4.1 --- Establishment of pEGFP-Cl-HBx and mutants constructs --- p.71 / Chapter 4.2 --- Transactivation C-terminus domain is essential for granular localization --- p.73 / Chapter 4.3 --- Wild-type HBx localizes in mitochondria --- p.76 / Chapter 4.4 --- C-terminal transactivation domain is sufficient for mitochondria targeting --- p.79 / Chapter 4.5 --- Mapping of the HBx region crucial for mitochondria targeting --- p.81 / Chapter 4.6 --- The 111-117 amino acids in HBx do not work as a signal peptide --- p.83 / Chapter 4.7 --- Site-directed mutagenesis identifies the key amino acid at 115 in HBx for mitochondrial targeting --- p.85 / Chapter CHAPTER 5 --- CELL PROLIFERATION AND REGULATION / Chapter 5.1 --- Alteration of S-phase distribution in cell cycle --- p.88 / Chapter 5.2 --- Analysis of DNA synthesis using BrdU proliferation ELISA --- p.92 / Chapter 5.3 --- Differential molecular regulation of cell cycle --- p.94 / Chapter 5.4 --- Regulation of the mRNA expression levels of cyclin-dependent kinases inhibitors p2raf/cipl and p27kipl --- p.98 / Chapter CHAPTER 6 --- TRANSACTIVATION AND RAS/RAF/MAPK PHOSPHORYLATION / Chapter 6.1 --- Determination of p53-dependency of p21、vaf/cipl expression --- p.101 / Chapter 6.2 --- Ras/Raf/MAPK pathway activation by HBx variants / Chapter 6.2.1 --- ERK1/2 phophorylation by HBx variants --- p.104 / Chapter 6.2.2 --- ERK inhibition blocks the regulation effect on p53Wt and p21waf/cipl --- p.107 / Chapter 6.3 --- Transactivation activity on oncogenes/ proto-oncogenes / Chapter 6.3.1 --- Effect on c-myc (NM´ؤ002467) mRNA expression --- p.109 / Chapter 6.3.2 --- Effect on RhoC (NM_017744) and Rabl4 (NM´ؤ016322) mRNA expression --- p.112 / Chapter CHAPTER 7 --- CONSTRUCTION OF TET-ON INDUCIBLE CELL-LINES / Chapter 7.1 --- Establishment of WRL/Tet-On monoclonal cell-lines Page / Chapter 7.1.1 --- Determination of geneticin selection dosage --- p.116 / Chapter 7.1.2 --- Selection of the best WRL/TOn clone using luciferase assay --- p.118 / Chapter 7.2 --- Establishment of inducible WRL/TOn/Gene monoclonal cell-lines / Chapter 7.2.1 --- Determination of hygromycin selection dosage --- p.120 / Chapter 7.2.2 --- Selection of positive WRL/TOn/Gene clones with viral genes --- p.122 / Chapter 7.3 --- Characterization of TOXDC1 cell-line / Chapter 7.3.1 --- Cell morphology --- p.125 / Chapter 7.3.2 --- Growth pattern of TOXDC1 --- p.126 / Chapter 7.3.3 --- HBxAC44 induced p21waf/cipl mRNA expression --- p.127 / Chapter 7.3.4 --- Doxycycline concentration dependent HBxAC44 expression in TOXDC1 --- p.129 / Chapter CHAPTER 8 --- DISCUSSION / Chapter 8.1 --- Selection of cell model / Chapter 8.1.1 --- Selection of cell models --- p.130 / Chapter 8.1.2 --- Selection of truncation mutant --- p.131 / Chapter 8.2 --- Differential sub-cellular localization of HBx and its variants / Chapter 8.2.1 --- Mechanisms of mitochondria targeting --- p.132 / Chapter 8.2.2 --- Mitochondria as site of HBx-induced apoptosis --- p.134 / Chapter 8.2.3 --- Stimulation of calcium release from mitochondria by wild-type HBx --- p.135 / Chapter 8.3 --- Cell cycle distribution profiling and its regulations / Chapter 8.3.1 --- Cell cycle pattern and cell proliferation --- p.136 / Chapter 8.3.2 --- Differential cell cycle molecular pathway activation --- p.138 / Chapter 8.4 --- Ras/Raf/MAPK mediated transactivation by HBxWt and its mutants / Chapter 8.4.1 --- p53-mediated p21waf/cipl expression --- p.142 / Chapter 8.4.2 --- ERK-mediated p21waf/cipl and wild-type p53 mRNA expression --- p.143 / Chapter 8.4.3 --- Regulation of oncogenes/ proto-oncogenes expression --- p.147 / Chapter 8.5 --- General discussions on differential effects of HBxWt and HBxAC44 --- p.149 / Chapter 8.6 --- Establishment of Tet-On/HBxAC44 cell-line TOXDC1 --- p.153 / Chapter 8.7 --- Conclusions --- p.154 / Chapter 8.8 --- Future Prospects / Chapter 8.8.1 --- From mitochondria targeting to calcium signaling --- p.157 / Chapter 8.8.2 --- Construction of a complete cell cycle regulation pathway --- p.158 / Chapter 8.8.3 --- Elucidation of the transcriptional transactivation regulation --- p.159 / Chapter 8.8.4 --- To make the best use of the Tet-on stable cell-line TOXDC1 --- p.159 / Chapter 8.8.5 --- Study with other carboxy-terminal truncation mutants --- p.160 / Chapter 8.8.6 --- In vivo study --- p.160 / REFERENCES --- p.162

Leung, Chung Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 100-113). / Abstracts in English and Chinese. / Abstract --- p.I / 摘要 --- p.III / Acknowledgements --- p.V / List of figures --- p.VI / List of tables --- p.VIII / Abbreviations --- p.IX / Table of Contents --- p.XII / Chapter Chapter 1: --- Introduction / Chapter 1.1 --- Epidemiology of hepatocellular carcinoma (HCC) --- p.1 / Chapter 1.2 --- Etiology of heptocellular carcinoma (HCC) --- p.1 / Chapter 1.3 --- HBV genome structure --- p.2 / Chapter 1.4 --- HBV pathogenesis --- p.2 / Chapter 1.5 --- Hepatitis B virus X protein (HBx) --- p.3 / Chapter 1.6 --- Oxidative stress and antioxidant --- p.5 / Chapter 1.6.1 --- Glutathione (GSH) --- p.5 / Chapter 1.6.2 --- Superoxide dismutase (SOD) --- p.7 / Chapter 1.6.3 --- Oxidative stress in HBV-related liver disease and HCC --- p.8 / Chapter 1.7 --- Apoptosis and necrosis --- p.9 / Chapter 1.7.1 --- Apoptotic pathways --- p.9 / Chapter 1.8 --- Role of HBx in apoptosis --- p.10 / Chapter 1.9 --- Transcriptional activity by HBx --- p.12 / Chapter 1.10 --- Chemotherapy drug resistance --- p.13 / Chapter 1.11 --- Objectives of study --- p.14 / Chapter Chapter 2: --- Methods and materials / Chapter 2.1 --- Construction of plasmid --- p.23 / Chapter 2.1.1 --- PCR amplification of wild-type and mutant HBx --- p.23 / Chapter 2.1.2 --- Agarose gel extraction --- p.25 / Chapter 2.1.3 --- Restriction enzyme digestion --- p.26 / Chapter 2.1.4 --- Ligation of vectors and gene of interest --- p.26 / Chapter 2.1.5 --- Preparation of competent cells for transformation --- p.27 / Chapter 2.1.6 --- Transformation of plasmid in competent cells --- p.27 / Chapter 2.1.7 --- Plasmid extraction by mini-prep --- p.28 / Chapter 2.1.8 --- DNA sequencing of the inserted genes --- p.29 / Chapter 2.2 --- Transfection --- p.30 / Chapter 2.2.1 --- Cell line --- p.30 / Chapter 2.2.2 --- Lipofectamine transfection --- p.31 / Chapter 2.2.3 --- Construction of stably-transfected cell lines --- p.31 / Chapter 2.3 --- Detection of expression of transfected gene in mRNA level by RT-PCR --- p.32 / Chapter 2.3.1 --- RNA extraction --- p.32 / Chapter 2.3.2 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.33 / Chapter 2.3.3 --- Agarose gel electrophoresis --- p.36 / Chapter 2.4 --- Detection of expression of transfected gene in protein level by Western blot --- p.36 / Chapter 2.4.1 --- Sample preparation --- p.36 / Chapter 2.4.2 --- Measurement of protein concentration --- p.36 / Chapter 2.4.3 --- Sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) --- p.37 / Chapter 2.4.4 --- Transfer of proteins to nitrocellulose membrane --- p.38 / Chapter 2.4.5 --- Immunoblotting of protein --- p.38 / Chapter 2.5 --- Measurement of reduced glutathione (GSH) concentration in cell lines --- p.39 / Chapter 2.5.1 --- Sample preparation --- p.39 / Chapter 2.5.2 --- Measurement of GSH concentration --- p.39 / Chapter 2.6 --- Superoxide dismutase (SOD) activity in cell lines --- p.40 / Chapter 2.6.1 --- Sample preparation --- p.40 / Chapter 2.6.2 --- Measurement of total SOD activity --- p.41 / Chapter 2.6.3 --- Measurement of Cu/ZnSOD and MnSOD by Western blot --- p.42 / Chapter 2.7 --- Cell proliferation assay --- p.43 / Chapter 2.7.1 --- Drugs and concentration --- p.43 / Chapter 2.7.2 --- "MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay" --- p.43 / Chapter 2.7.3 --- Cell proliferation and cytotoxicity of the drugs --- p.44 / Chapter 2.8 --- Detection of apoptosis by flow-cytometry --- p.44 / Chapter 2.8.1 --- Cell culture --- p.44 / Chapter 2.8.2 --- Cell fixation --- p.45 / Chapter 2.8.3 --- Cell staining --- p.45 / Chapter 2.8.4 --- Flow cytometry analysis --- p.46 / Chapter 2.9 --- Detection of protein involved in apoptotic pathway --- p.46 / Chapter 2.9.1 --- Antibodies --- p.46 / Chapter 2.9.2 --- Sample Preparation --- p.47 / Chapter 2.9.3 --- Measurement of protein concentration --- p.48 / Chapter 2.9.4 --- Western blotting --- p.49 / Chapter Chapter 3: --- Establishment of HBx transfected stable cell lines / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Results --- p.56 / Chapter 3.2.1 --- Plasmid construction --- p.56 / Chapter 3.2.2 --- Stable transfection of cell lines --- p.57 / Chapter 3.2.3 --- Morphology of wild type and mutant HBx-transfected cell lines --- p.58 / Chapter 3.3 --- Discussion --- p.58 / Chapter Chapter 4: --- Antioxidant level in HBx transfected cell lines / Chapter 4.1 --- Introduction --- p.68 / Chapter 4.2 --- Results --- p.70 / Chapter 4.2.1 --- Glutathione (GSH) concentration in different cell lines --- p.70 / Chapter 4.2.2 --- Superoxide dismutase (SOD) activity in different cell lines --- p.71 / Chapter 4.2.2.1 --- Total SOD activity --- p.71 / Chapter 4.2.2.2 --- Cu/ZnSOD --- p.71 / Chapter 4.2.2.3 --- MnSOD --- p.72 / Chapter 4.3 --- Discussion --- p.72 / Chapter Chapter 5: --- Involvement of HBx in apoptotic pathway / Chapter 5.1 --- Introduction --- p.81 / Chapter 5.2 --- Results --- p.82 / Chapter 5.2.1 --- Cell proliferation of HBx transfected cells --- p.82 / Chapter 5.2.2 --- Apoptosis of HBx transfected cells --- p.83 / Chapter 5.2.3 --- Cytotoxicity of fluorouracil (5FU) and doxorubicin (DOX) in HBx transfected cells --- p.84 / Chapter 5.2.4 --- Detection of anti-apoptotic proteins cIAP2 and Bcl-2 in HBx-transient and stably transfected cells --- p.84 / Chapter 5.3 --- Discussion --- p.85 / Chapter Chapter 6: --- Concluding remarks and general discussion / Chapter 6.1 --- General discussion --- p.93 / Chapter 6.2 --- Future work --- p.97 / Chapter 6.3 --- Summary --- p.99 / References --- p.100 / Appendix 1 --- p.114

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 155-161.

A análise das proteínas transportadoras de compostos biliares, antes restrita à fisiologia e à fisiopatologia de colestases, recentemente passou a incluir neoplasias hepato-biliares. O presente estudo teve como objetivo caracterizar a expressão das proteínas ABC de transporte biliar BSEP, MDR3, MRP2 e MRP3 em amostras retrospectivamente colecionadas de 80 casos de autópsias de carcinoma hepatocelular (CHC) e 56 casos de ressecção cirúrgica de colangiocarcinoma (CC). Áreas representativas das neoplasias foram organizadas em tissue microarrays e submetidas à pesquisa imuno-histoquímica (IHQ) com o anticorpo policlonal anti-BSEP (HPA019035) e os anticorpos monoclonais anti-BSEP (F6), anti-MDR3 (P3 II-26), anti-MRP2 (M2 III-6) e anti-MRP3 (DTX-1) com amplificação de sinal mediante uso de sistema de polímeros curtos conjugados à peroxidase. A comparação entre a positividade das reações imuno-histoquímicas para cada anticorpo e as variáveis anatomopatológicas foi realizada através dos testes de qui-quadrado de Pearson ou Exato de Fisher. A positividade das reações IHQ cujos anticorpos propiciaram melhor distinção do sinal positivo vs coloração inespecífica de fundo e detecção de casos positivos e/ou melhor capacidade de discriminar as duas neoplasias hepáticas foi comparada com a positividade observada para as reações IHQ com os anticorpos anti-CEA policlonal, anti-Hep-par-1 e anti-Arginase-1. A expressão canalicular de BSEP nos CHC foi observada em 77,3% (58/75) com o anticorpo monoclonal e 75,9% (60/79) com o anticorpo policlonal. Não foi detectada associação significativa da expressão de BSEP em relação ao tamanho, número dos nódulos e grau de diferenciação de CHC, tendo apenas sido significativamente reduzida (P < 0,05) tal reação nos casos de padrão arquitetural mais complexo. A reatividade dos CHC para o anticorpo monoclonal anti-BSEP foi aparentemente menor que a obtida com a expressão canalicular de CEA, Hep-par-1 e Arginase-1 no CHC, mas esses valores não atingiram significância estatística. Todos os casos de colangiocarcinoma foram negativos para reações IHQ para pesquisa de BSEP, resultado significativamente diferente (P=0,0001) do obtido com uso do Ac policlonal anti-CEA (padrão circunferencial) e Hep-par-1, não tendo sido demonstrada diferença significativa (P=0,222) da expressão de BSEP e de Arginase-1. A expressão canalicular de MDR3 foi observada em 56,4% (44/78) dos casos de CHC, não tendo sido detectada associação significativa quanto ao tamanho e número de nódulos. Foi observada expressão significativamente menor de MDR3 nos casos de CHC de padrão mais complexo (P=0,009), e nos casos de maior grau histológico (P=0,005). A expressão de MDR3 em CHC foi significativamente menor que a de CEA, Hep-par-1 e Arginase-1 (P < 0,05). Todos os casos de colangiocarcinoma foram negativos para a avaliação da expressão de MDR3, diferindo significativamente em relação a expressão de CEA (P=0,001), mas não em comparação a Hep-par-1 e Arginase-1 (P > 0,05). As reações IHQ para detecção de MRP2 exibiram positividade canalicular em 92,3% dos casos de CHC e em 96,3% nos casos de CC. A detecção da alta expressão de MRP2 no CHC foi constante (P > 0,05) em comparação ao tamanho, número dos nódulos, padrão arquitetural e grau histológico de diferenciação de CHC assim como, também não apresentou associação (P > 0,05) com a localização, padrão de crescimento e grau de diferenciação do CC. A reação IHQ para MRP3 resultou positiva em 15/80 casos de CH (18,8%). A reatividade IHQ para MRP foi detectada em 24/54 (44,5%) de CC. Diferente dos transportadores descritos acima, a expressão de MRP3 foi preferencialmente basolateral. A positividade para MRP3 não variou (P > 0,05) em relação ao número, tamanho dos nódulos, padrão arquitetural (inclusive os sólidos), e grau de diferenciação (inclusive os menos diferenciados). A proteína MRP3 esteve expressa regularmente (P > 0,05) em todos os casos de CC, apresentando-se reduzida apenas no subtipo histológico ductular (P=0,023). Em conclusão, o excelente contraste de reação, a frequência razoavelmente alta de positividade de CHC e a plena negatividade de CC para BSEP levam-nos a recomendar a introdução do anticorpo monoclonal anti-BSEP no painel adotado para o diagnóstico diferencial dessas duas neoplasias. A alta expressão de MRP2 no CHC e no CC é conservada independentemente dos parâmetros anatomopatológicos avaliados. A expressão do transportador MRP3 mostrou variação dentre os subtipos histológicos de CC, aspecto que torna promissoras pesquisas futuras para avaliação mais detalhada da expressão deste marcador nos colangiocarcinomas / The assessment of biliary transporters, previously restricted to the physiology and pathophysiology of cholestasis, has recently included hepato-biliary neoplasms. The present study aimed to characterize the expression of BSEP, MDR3, MRP2 and MRP3 biliary transport proteins in retrospectively collected samples from 80 cases of autopsy of hepatocellular carcinoma (HCC) and 56 cases of surgical resection of cholangiocarcinoma (CC). Representative areas of the neoplasms were organized into tissue microarrays and submitted to immunohistochemical (IHC) reaction with polyclonal antibody anti-BSEP (HPA019035) and monoclonal antibodies anti-BSEP (F6), MDR3 (P3 II-26), MRP2 (M2 III-6) and MRP3 (DTX-1). Signal amplification was achieved with a short polymer system conjugated to peroxidase. The comparison between the positivity of the immunohistochemical reactions for each antibody and the pathological variables was performed using the Pearson chi-square test or the Fisher\'s exact test. The performance of antibodies which provided a better distinction of the positive signal vs nonspecific background staining and yield better discrimination between the two hepatic neoplasms was compared with that achieved with the already accepted HCC markers polyclonal anti-CEA, Hep-par-1 and Arginase-1. The canalicular expression of BSEP in HCC was observed in 77.3% (58/75) with the monoclonal antibody and 75.9% (60/79) with the polyclonal antibody. BSEP expression levels were not significantly different according to tumor size, number of nodules and degree of differentiation. The frequency of positive reaction of HCC cases with the monoclonal anti-BSEP was apparently lower than that achieved with the canalicular expression of CEA, Hep-par-1 and Arginase-1, but these values did not reach statistical significance. All cases of cholangiocarcinoma were negative for IHC reactions to BSEP, which was significantly different (P=0.0001) from the results obtained with polyclonal anti-CEA (circumferential pattern) and Hep-par-1, but not from the resultas achieved with Arginase-1 (P=0.222). The canalicular expression of MDR3 was observed in 56.4% (44/78) of HCC cases. Among histological variables, only the finding of more complex architecture (P=0.009) and higher histological grade (P=0.005) of HCC yielded, significantly lower expression of MDR3. The expression of MDR3 in HCC was significantly lower than that of CEA, Hep-par-1 and Arginase-1 (P < 0.05). All cases of cholangiocarcinoma were negative for the evaluation of MDR3 expression, differing significantly with that achieved with polyclonal anti-CEA (P=0.001) but not with that achieved with Hep-par-1 or with Arginase-1 (P > 0.05). The IHC reactions with the MRP2 antibody exhibited canalicular positivity in 92.3% of HCC cases and 96.3% in CC cases. High expression of MRP2 in HCC was constant (P > 0.05) despite changes in size, number of nodules, architectural pattern and histological degree of HCC differentiation, as well as no association (P > 0.05) with the location, pattern of growth and degree of differentiation of CC. The IHC reaction for MRP3 was positive in 15/80 cases of HCC (18.8%) and in 24/54 (44.5%) of CC. Unlike the carriers described above, the hepatocellular expression of MRP3 was preferentially basolateral. Positivity for MRP3 did not vary (P > 0.05) in relation to number, nodule size, architectural standard (including solids), and degree of differentiation. The MRP3 protein was expressed regularly (P > 0.05) in different presentations of CC, but significant lower frequency of positivity was found in the ductular histological subtype (P=0.023). In conclusion, the excellent signal-to-noise ratio, reasonably high frequency of HCC positivity and full negativity of CC to BSEP lead us to recommend the introduction of the anti-BSEP monoclonal antibody in the panel adopted for the differential diagnosis of these two neoplasms. The high expression of MRP2 in HCC and in CC is conserved independently of the pathological parameters evaluated herein. The frequency of expression of the MRP3 transporter varied among the histological subtypes of CC, which makes promising future research for a more detailed assessment of the expression of this marker in the cholangiocarcinomas

O carcinoma hepatocelular (CHC) é a neoplasia maligna primária mais comum do fígado, sendo a quinta mais frequente e a terceira causa de morte por câncer no mundo. Atualmente, nenhum protocolo com resultados satisfatórios no tratamento de CHC foi preconizado. Neste estudo foi determinado o potencial regenerativo e imunomodulador das células-tronco mesenquimais (CTM) associadas ou não aos quimioterápicos doxorrubicina e gencitabina, no modelo ortotópico de CHC em camundongos C57BL/6J. Foi realizado in vitro a determinação da atividade citotóxica dos quimioterápicos doxorrubicina e gencitabina em células de CHC murino (Hepa1c1c7), quantificação da peroxidação de lipídeos da membrana celular (TBARS) e análise das fases do ciclo celular e a expressão de marcadores por citometria de fluxo. A IC50% em células de carcinoma hepatocelular murino (Hepa1c1c7) foi de 1,85 uM para doxorrubicina e 20,8 ?M para gencitabina. A quantificação de TBARS na linhagem celular de CHC murino (Hepa1c1c7) tratados com os quimioterápicos doxorrubicina e gencitabina demonstrou efeito deletério apenas quando tratados nas concentrações (55 a 250 uM) com a doxorrubicina e (1,75 a 7 uM) com gencitabina. Após tratamento com os quimioterápicos, ocorreram modificações nas populações celulares, com o aumento da fase sub-G1 e G0/G1 e diminuição fases S e G2/M. As CTM, apresentaram-se aderentes aos frascos de cultura com morfologia semelhante a fibroblastos e expressão positiva para marcadores CD44, CD73, CD90 e CD105 e negativa para células-tronco hematopoiéticas da medula óssea: CD11b, CD45 e CD117. O modelo experimental tumoral ortotópico de CHC foi estabelecido no 21° dia após a inoculação das células tumorais. Os efeitos da terapêutica com a CTM mostraram aspectos significativos na redução da massa tumoral (30,5%), os demais grupos experimentais tratados com quimioterapia sistêmica associada à terapia celular também evidenciaram resultados significativos na redução da massa tumoral e atenuação dos efeitos de toxicidade sistêmica. As células tumorais extraídas dos tumores hepáticos encontram-se preferencialmente na fase sub-G1 nos grupos de animais tratados com CTM em associação aos quimioterápicos gencitabina (29%) e doxorrubicina (21%), enquanto o tratamento isolado com a doxorrubicina foi de 22% e de 15% para a gencitabina, além de induzir significativamente diminuição das fases S e G2/M. No grupo de animais tratado com CTM em associação ao quimioterápico gencitabina há um aumento da expressão das caspases 3 e 8, e dos marcadores CD44 e IL-1R. O grupo de animais tratados com CTM apresentou aumento na expressão dos marcadores CD90 e CD14. Os resultados obtidos no tratamento do modelo ortotópico de CHC com CTM associadas aos quimioterápicos doxorrubicina e gencitabina apresentaram eficácia na redução da massa tumoral e atenuação dos efeitos colaterais causados pelo tratamento quimioterápico sistêmico, além do seu potencial efeito imunomodulador / Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver, the most frequent fifth and the third leading cause of cancer death in the world. Currently, no protocol with satisfactory results in the treatment of CHC was recommended. In this study we determined the regenerative potential immunomodulatory and mesenchymal stem cells (MSCs) associated or not to chemotherapy doxorubicin and gemcitabine, in the orthotopic model of CHC in C57BL / 6J mice. Was determined in vitro the cytotoxic activity of doxorubicin and gemcitabine chemotherapy in a murine cells (Hepa1c1c7), quantification of cell membrane lipid peroxidation (TBARS) and analysis of the phases of the cell cycle and expression of markers by flow cytometry. IC50% in murine carcinoma cells (Hepa1c1c7) was 1.85 uM and 20.8 uM for doxorubicin to gemcitabine. The quantification of TBARS in cell line (Hepa1c1c7) treated with the chemotherapeutic doxorubicin and gemcitabine showed a deleterious effect only when treated at concentrations (55-250 uM) doxorubicin and (1.75 to 7 uM) with gemcitabine. After treatment with chemotherapeutic agents, there were changes in the cell population, with increased phase sub-G1 and G0 / G1 phase and reduced S and G2 / M. MSC presented to adhere to culture flasks with morphology similar to fibroblasts and positive expression of markes: CD44, CD73, CD90 and CD105 and negative hematopoietic stem cells from bone marrow: CD11b, CD45 and CD117. The tumor orthotopic experimental model of HCC was established on day 21 after tumor cell inoculation. The effects of therapy CTM showed significant aspects in reducing the tumor mass (30.5%), the other experimental groups treated with systemic chemotherapy associated with cell therapy also showed significant results in reducing the tumor mass and mitigation of systemic toxicity effects. The extracted tumor cells of liver tumors are preferably in the sub-G1 phase in groups of animals treated with MSC in association with gemcitabine (29%) and doxorubicin (21%) chemotherapy, whereas the single treatment with was 22% doxorubicin and 15% for gemcitabine in addition to significantly induce reduction of phases S and G2 / M. In the group of animals treated with MTC in combination with gemcitabine chemotherapy there is an increased expression of caspase 3 and 8 and IL-1R and CD44. The group of animals treated with MSCs showed increased markers expression of CD90 and CD14. The results obtained in the treatment of orthotopic HCC model with MSC associated with the chemotherapeutic doxorubicin and gemcitabine showed efficacy in reducing tumor burden and attenuation of the side effects caused by systemic chemotherapy, as well its potential immunomodulatory effect

INTRODUCTION: Hepatocellular carcinoma (HCC) is a deadly cancer with increasing incidence in western countries. Diagnosis of HCC is based on imaging exams and most Cancer Centers do not recommend core-biopsies for definitive histopathological confirmation. Moreover, only patients with early disease stage are eligible to curative-intent treatments including surgical resection and liver transplantation. Hence, access to tissue samples in HCC patients with intermediate and advanced disease is limited, which further precluded the evaluation of morphological features and molecular drivers of HCC dissemination, particularly in relation to metastatic spread. Evaluation of autopsy specimens with adequate representation of primary and metastatic disease can overcome such limitations and provide insights on the mechanisms and patterns of distant dissemination in HCC. METHODS: This study included 88 autopsy specimens from patients with HCC, including 20 with distant metastases. Tissue microarrays (TMA) were generated from 194 hepatic and 36 extra-hepatic nodules histologically available from these patients. All nodules were assessed for multiple histological features including degree of differentiation, nuclear, nucleolar and architectural grades, and cellular crowding. Immunohistochemistry (IHC) was performed for markers of hepatocyte differentiation (HepPar1, Arginase and CD10), CTNNB1 mutation status (beta-catenin and Glutamine Synthetase), HCC stem-like features (Keratin 19, CD44 and EpCam), and epithelial-mesenchyme transition (Vimentin and Claudin 1). Phenotypic heterogeneity in the primary disease was assessed in 50 patients with multiple hepatic nodules and heterogeneity in the metastatic disease was evaluated in 12 patients with multiple extra-hepatic nodules. Mutations in the TERT-promoter region was evaluated in six patients with multi-nodular primary and metastatic disease. RESULTS: Metastatic sites included lungs (16/20, 80%), peritoneum (4/20, 20%) lymph nodes (4/20, 20%) and adrenal gland (3/20, 15%). Subclinical micro-metastases, undetected in imaging and macroscopic examination, were identified in 30% of the patients with disseminated disease to the lung. AFP serum concentration >= 100 ng/mL, dominant nodule >= 5.0 cm, multi-nodularity, macrovascular invasion, high histological, nuclear and architectural grades, cellular-crowding, and expression of Keratin 19 and EpCam in the primary disease were associated with the presence of distant metastases in HCC. Histological and IHC analyses showed that all HCC metastatic nodules could be traced back to the primary disease. Phenotypic inter-nodular heterogeneity was detected in 27/50 (54%) patients with multinodular hepatic disease. Heterogeneity was less pronounced in extra-hepatic nodules, being present in only 2/12 (17%) patients with multiple metastatic tumors. These results were further validated by the limited mutation heterogeneity of the TERT-promoter region in metastatic compared to primary nodules. CONCLUSIONS: HCC shows a strong hematogenous tropism and predilection for lung dissemination. Metastatic HCC nodules are enriched in histological features of aggressive behavior and in markers of stem-like properties. The limited phenotypic internodular heterogeneity within the primary compared to metastatic nodules suggests evolutionary constrains in HCC extra-hepatic dissemination / INTRODUÇÃO: Carcinoma hepatocelular (CHC) é um câncer de alta mortalidade e incidência crescente em países ocidentais. O diagnóstico de CHC é realizado através de exames de imagem e a grande maioria dos Centros de Tratamento de Câncer não recomendam biópsias incisionais para confirmação histopatológica do diagnóstico de CHC. Além disso, apenas os pacientes com CHC em estágios precoces são submetidos a tratamentos com intenção curativa como ressecção cirúrgica e transplante hepático. Dessa forma, o acesso a amostras teciduais em pacientes com CHC em estágios intermediários e avançados é bastante limitada, dificultando a avalição de achados morfológicos e eventos moleculares associados a progressão tumoral, e em especial relacionados a disseminação extra-hepática. A avalição de amostras de autópsia com adequada representação da doença primária e metastática pode superar tais limitações e sugerir mecanismos e padrões de disseminação a distância em CHC. MÉTODOS: O presente estudo incluiu 88 autópsias em pacientes com CHC, abrangendo 20 pacientes com metástase à distância. Micro matrizes teciduais (TMA) foram construídas com 194 nódulos hepáticos e 36 nódulos extra-hepáticos desses pacientes. A avaliação dos nódulos incluiu múltiplos critérios histológicos como diferenciação tumoral; graus nuclear, nucleolar e arquitetural, e celularidade. Imuno-histoquímica (IHQ) foi realizada para marcadores de diferenciação hepatocitária (HepPar1, Arginase e CD10), status de mutação de CTNNB1 (Beta-catenina e Glutamina Sintetase), propriedades biológicas de células progenitoras em CHC (Queratina 19, CD44 e EpCam), e marcadores de transição epitélio-mesênquima (Vimentina e Claudina 1). A heterogeneidade fenotípica na doença primaria foi avaliada em 50 pacientes com múltiplos nódulos hepáticos e, na doença metastática, em 12 pacientes com múltiplos nódulos extra-hepáticos. Mutações na região promotora de TERT foram avaliadas em seis pacientes com doença multi-nodular primária e metastática. RESULTADOS: Foram observadas metástases para os pulmões (16/20, 80%), peritônio (4/20, 20%), linfonodos (4/20, 20%) e glândula adrenal (3/20, 15%). Metástases subclínicas, não detectadas em exames de imagem e avaliação macroscópica, foram identificadas em 30% dos pacientes com comprometimento pulmonar. Concentração sérica de alfa-feto-proteína >= 100 ng/mL, nódulo dominante >= 5.0 cm, multi-nodularidade, invasão macrovascular alto grau histológico, nuclear e arquitetural, celularidade e expressão IHQ de Queratina 19 e EpCam na doença primária mostraram associação com a presença de metástases em CHC. Todos os nódulos metastáticos reproduziram os achados histológicos e IHQ da doença primária correspondente. Heterogeneidade fenotípica inter-nodular foi detectada em 27/50 (54%) pacientes com múltiplos nódulos hepáticos. A heterogeneidade extra-hepática foi menos expressiva, presente em apenas 2/12 (17%) pacientes com múltiplos nódulos metastáticos. Também houve limitada heterogeneidade para mutações na região promotora de TERT na doença metastática comparada à doença primária. CONCLUSÕES: CHC tem forte tropismo hematogênico e predileção por metástases pulmonares. O CHC metastático possui alta prevalência de altos graus histológicos e de marcadores de células progenitoras. A restrita heterogeneidade fenotípica da doença metastática comparada à doença primária sugere restrições evolutivas e seleção de clones tumorais na disseminação extra-hepática de CHC

INTRODUÇÃO: Enquanto a análise de sobrevida tradicional estima inadequadamente o prognóstico futuro dada alguma sobrevida inicial, a sobrevida condicional ajusta a sobrevida futura pela já observada, permitindo a compreensão da distribuição temporal do impacto dos preditores. OBJETIVOS: Estimar e analisar as sobrevidas global e livre de doença até o décimo ano pós-operatório; identificar preditores independentes destes desfechos; estimar e analisar as sobrevidas condicionais global e livre de doença de cinco anos dada a sobrevida até o quinto ano pós-operatório; analisar o comportamento dos preditores dos desfechos ao longo do tempo. MÉTODOS: Estudo retrospectivo envolvendo 13 centros brasileiros. Dados clínicos, radiológicos e anatomopatológicos foram considerados. Utilizou-se o método de Kaplan-Meier com o teste log-rank para comparar fatores e a regressão de Cox obteve a razão de riscos. A sobrevida condicional foi calculada a partir das tábuas de sobrevida e a diferença padronizada reavaliou as variáveis consideradas significativas. RESULTADOS: 1157 pacientes foram incluídos. A sobrevida global de 1, 3, 5, 7 e 10 anos foi 78,6%, 72,3%, 66,0%, 61,3% e 59,4%, respectivamente. Foram preditoras de sobrevida global: idade [HR 1,04 (IC 95% 1,02-1,06), p 0.000], sexo feminino [HR 1,35 (IC 95% 1,02-1,79), p 0.038], recidiva pós-operatória do CHC [HR 1,35 (IC 95% 1,08-1,79), p 0.003], diâmetro do maior nódulo viável no explante [HR 1,01 (IC95% 1,01-1,02), p 0.006], invasão vascular não discriminada [HR 3,18 (IC95% 1,48-6,85), p 0.004], invasão micro [HR 1,65 (IC 95% 1,27-2,15), p 0.001] e macrovascular [HR 2,25 (IC 95% 1,30-3,89), p 0.000]. A sobrevida condicional global de 5 anos ao final do 1°, 3° e 5° anos foi 79,5%, 82,2% e 90,0%, respectivamente. As variáveis preditoras na análise univariada tiveram comportamento errático ao longo do tempo. A sobrevida atuarial livre de doença em 1, 3, 5, 7 e 10 anos foi 94,2%, 90,1%, 89,8%, 87,5% e 87,5%, respectivamente. Foram preditoras de sobrevida livre de doença: nível sérico de alfa-fetoproteína no diagnóstico [HR 1,0 (IC 95% 1,01-1,02), p 0.000], CHC dentro do critério de Milão no diagnóstico [HR 0,42 (IC 95% 0,22-0,80), p 0.008], explante dentro do critério de Milão [HR 0,34 (IC 95% 0,17-0,68), p 0.002], explante com neoplasia pouco diferenciada ou hepatocolangiocarcinoma [HR 3,04 (IC 95% 1,75-5,30), p 0.000], invasão vascular não discriminada [HR 15,72 (IC 95% 3,44-71,83), p 0.000], invasão micro [HR 3,40 (IC 95% 1,83-6,28), p 0.000] e macrovascular [HR 11,96 (IC 95% 5,20-27,47), p 0.000]. A sobrevida condicional livre de doença de 5 anos ao final do 1°, 3° e 5° anos foi 94,1%, 97,1% e 97,4%, respectivamente. Variáveis preditoras na análise univariada em geral tem maior impacto no primeiro ou segundo ano. CONCLUSÕES: Os resultados do transplante no Brasil foram comparáveis àqueles observados nos EUA e Europa. Considerando-se as perdas precoces, as curvas de sobrevida pelo método Kaplan-Meier foram pessimistas e a análise de sobrevida condicional fornece outra perspectiva para estes dados. O comportamento das variáveis determinantes de prognóstico não é uniforme ao longo do tempo / INTRODUCTION: Traditional survival analysis provides inadequate estimates of the future prognosis for patients with accrued survival. Conversely, conditional survival adjusts future survival by the already accrued survival. It provides insights into the temporal distribution of the effect of predictors. OBJECTIVES: To estimate and to analyse overall and disease free survival until the 10th post-operative year; to identify independent predictors of these outcomes; to estimate and to analyse 5-year overall and disease free conditional survival until the 5th post-operative year; to analyse the behaviour of the predictors of outcomes during follow-up. METHODS: Retrospective cohort from 13 Brazilian transplantation centers. Clinical, radiological, and anatomopathological data were considered. The Kaplan-Meier method with the longrank test for the comparison of factors was applied and the Cox proportional hazards model provided the hazard ratios. Conditional survival was calculated through life tables, while differences between significative variables were reassessed by the standardized difference. RESULTS: 1157 patients were included. Overall survival in 1, 3, 5, 7 and 10 years was 78.6%, 72.3%, 66.0%, 61.3%, and 59.4%, respectively. 350 (30.3%) deaths were observed, 240 (68.6%) in the 1st year. Overall survival was independently predicted by age [HR 1.04 (95% CI 1.02-1.06), p 0.000], female sex [HR 1.35 (95% CI 1.02-1.79), p 0.038], post-operative HCC recurrence [HR 1.35 (95% CI 1.08-1.79), p 0.003], diameter of the largest viable nodule on the explant [HR 1.01 (95% CI 1.01-1.02), p 0.006], non-discriminated vascular invasion [HR 3.18 (95% CI 1.48-6.85), p 0.004], micro [HR 1.65 (95% CI 1.27-2.15), p 0.001] and macrovascular invasion [HR 2.25 (95% CI 1.30-3.89), p 0.000]. 5-year overall conditional survival at the end of the 1st, 3rd and 5th post-operative years was 79.5%, 82.2%, and 90.0%, respectively. Predictors of overall survival identified on univariate analysis presented an erratic behaviour over time. Disease free survival in 1, 3, 5, 7 and 10 years was 94.2%, 90.1%, 89.8%, 87.5%, and 87.5%, respectively. 97 (8.4%) reccurrences occurred. Disease free survival was independently predicted by serum alpha-fetoprotein upon diagnosis [HR 1.0 (95% CI 1.01-1.02), p 0.000], HCC within the Milan criteria upon diagnosis [HR 0.42 (95% CI 0.22-0.80), p 0.008], explant within the Milan criteria [HR 0.34 (95% CI 0.17-0.68), p 0.002], undifferentiated tumor or hepatocholangiocarcinoma on the explant [HR 3.04 (95% CI 1.75-5.30), p 0.000], non-discriminated vascular invasion [HR 15.72 (95% CI 3.44-71.83), p 0.000], micro [HR 3.40 (95% CI 1.83-6.28), p 0.000], and macrovascular invasion [HR 11.96 (95% CI 5.20-27.47), p 0.000]. 5-year disease free conditional survival at the end of the 1st, 3rd and 5th post-operative years was 94.1%, 97.1%, and 97.4%, respectively. Predictors of recurrence on the univariate analysis usually presented with greater impact during the 1st or 2nd post-operative year. CONCLUSIONS: Outcomes of liver transplantation in Brazil were comparable to those from the US and Europe. Survival estimates through the Kaplan-Meier method were pessimistic due to greater early losses. Conditional survival offers a different perspective for the same data. The behaviour of predictive values varies over time

O objetivo principal deste estudo foi avaliar fatores virais associados com a evolução para o carcinoma hepatocelular (CHC) em pacientes com hepatite B crônica. Para tanto caracterizamos os subgenótipos do HBV, investigamos a ocorrência de mutações nos genes pré-core/core do HBV associadas à presença de CHC avaliamos por análise filogenética a associação de linhagens virais com a ocorrência de CHC e por fim a associação de outros fatores de risco com o desenvolvimento de CHC. Foram incluídos 119 amostras de soro de pacientes com infecção crônica pelo HBV, destas amostras 60 pertencem ao grupo 1 (CHC), que são pacientes com diagnóstico confirmado de carcinoma hepatocelular e 59 amostras pertencem ao grupo 2 (sem CHC) que são pacientes com hepatite crônica sem detecção prévia de nódulos hepáticos. Foram obtidas informações acerca da idade, sexo e naturalidade. Além disso, os pacientes responderam a um questionário sobre fatores de riscos associados ao desenvolvimento de CHC. Foram realizados exames bioquímicos, sorológicos, determinação da carga viral, e amplificação por nested PCR e sequenciamento das regiões S/polimerase e pré-core/core do genoma viral para posterior caracterização dos genótipos/subgenótipos do HBV e pesquisa de mutações associadas com evolução da doença hepática. Em relação à idade e sexo não houve grande variação entre os grupos. Quanto à naturalidade a maioria era procedente da região sudeste, seguido pela região nordeste; e por fim seis pacientes eram procedentes de outros países. Com base no sobrenome dos pacientes avaliou-se também a frequência de etnia oriental na casuística estudada, que foi similar nos 2 grupos. O perfil sorológico HBeAg negativo foi o mais frequente nos dois grupos de pacientes, assim como níveis de carga viral abaixo de 2.000 UI/mL. Em relação aos exames bioquímicos foram observadas diferenças estatisticamente significantes nos níveis séricos de AFP (p= 0,0013), FA (p= 0,0003) e GGT (p= 0,005). Dentre os fatores de risco analisados neste estudo, o consumo de amendoim foi o único que apresentou significância estatística (p= 0,003). A região S/pol foi amplificada e sequenciada com sucesso em 58 amostras (28 do grupo 1 e 30 do grupo 2). Entre as 58 amostras analisadas 4 genótipos e 8 subgenótipos do HBV foram identificados, sendo o subgenótipo A1 o mais frequente nos dois grupos. Não se observou diferença estatisticamente significante na distribuição dos subgenótipos entre os dois grupos de pacientes. Na topologia da árvore filogenética construída com sequências do HBV isoladas dos pacientes incluídos neste estudo e sequências disponíveis no GenBank não se observou padrões de agrupamento associados com o perfil clinico do paciente (com e sem CHC). Foram obtidas sequências de boa qualidade da região précore/ core em 44 amostras, sendo 20 amostras do grupo 1 e 24 do grupo 2. Diversas das mutações investigadas foram identificadas na região précore/ core, as quais foram avaliadas estatisticamente para verificar a existência de diferença na frequência das mesmas entre os grupos de pacientes estudados. Entre as mutações identificadas se destacaram com significância estatística as seguintes mutações: T1768A (p= 0,006), a combinação das mutações C1766T + T1768A (p= 0,043) e G1888H (p= 0,05). Na análise de regressão logística simples foi possível identificar que a chance de um paciente do grupo 2 desenvolver CHC aumenta 14,7 vezes na presença de infecção por cepas do HBV com a mutação T1768A, enquanto que a infecção com cepas do HBV que albergam a mutação G1888H reduz tal chance 2,5 vezes / The aim of this study was to evaluate viral factors associated with the progression to hepatocellular carcinoma (HCC) in patients with chronic hepatitis B. For this goal, we characterized HBV subgenotypes, investigated the occurrence of mutations in pre-core/core genes associated with progression to HCC, characterized HBV strains through phylogenetic analyzes and evaluated risk factors associated with HCC. Were included 119 serum samples from patients with chronic HBV infection that were classified in 2 groups: 60 patients with confirmed HCC diagnosis (group 1) and 59 patients with advanced hepatitis B liver disease without the detection of nodular liver lesions and without HCC (group 2). Data about the age, sex and geographic precedence were obtained from medical records. The patients also answered a questionnaire on risk factors for developing HCC. Biochemical, serological and viral load testing were performed in all samples. Moreover, S/polymerase and precore /core regions of HBV DNA were amplified by nested PCR and sequenced by Sanger method. Sequences were analyzed to identify HBV genotypes and subgenotypes and to detect mutations in the precore/core gene. Patient\'s age and sex did not differ between the two groups. Most of the patients came from the Southeast region, followed by the Northeast region; and six patients were from other countries. Based on the patient\'s surname, they were evaluated concerning Eastern ethnicity, which was similar in the 2 groups. Most of the patients included in this study were HBeAg negative and showed viral load bellow 2,000 IU/mL. Concerning the biochemistry assays, statistically significant differences in serum levels of AFP (p = 0.0013), AP (p = 0.0003) and GGT (p = 0.005) were found. Among the risk factors analyzed in this study, peanut consumption was the only one statistically significant (p = 0.003). The S/pol region was successfully amplified and sequenced in 58 samples (28 from Group 1 and 30 from Group 2). Among the 58 samples analyzed, 4 genotypes and 8 subgenotypes were identified, subgenotype A1 was the most frequent in both groups and there was no statistically significant difference in the distribution of them between the two groups. In the phylogenetic tree topology built with HBV sequences isolated from patients included in this study and sequences available in GenBank, it was not observed any clustering associated with the clinical profile of the patients (with or without HCC). Sequences of good quality from pre-core/core region were obtained from 44 samples, 20 from group 1 and 24 from group 2. These sequences were analyzed and several mutations were found among which stood out with statistical significance: T1768A (p = 0.006) C1766T + T1768A (p = 0.043) and G1888H (p = 0.05). In addition to the comparative analysis, the changes were subjected to a simple logistic regression analysis which found that the chance of a patient in group 2 developed HCC increases 14.7 times in the presence of HBV infection strains with the T1768A mutation, while infection with HBV strains harboring the mutation G1888H reduces this chance by 2.5 times

INTRODUÇÃO: A cirrose hepática é fator de risco para o Carcinoma hepatocelular (CHC) e no Brasil, a etiologia de doença hepática crônica mais frequente associada ao CHC é a infecção crônica pelo vírus da hepatite C (VHC). Angiogênese e deficiência de folato podem estar associados ao risco de CHC. Deste modo, os polimorfismos de genes relacionados com esses processos o VEGF e MTHFR em doentes com hepatite C crônica e CHC podem conduzir a marcadores genéticos para CHC na infecção por HCV.OBJETIVOS: Avaliar os polimorfismos G634C (rs2010963), C460T (rs833061), C936T (rs3025039) do gene do VEGF e polimorfismos C677T (rs1801133) e C1298A (1801131) da MTHFR, em pacientes brasileiros com CHC e infecção crônica pelo VHC . PACIENTES E MÉTODOS: Foram incluídos 119 pacientes com seguimento de CHC e VHC na clínica de hepatologia do HUOC / UPE e do IFP-PE, Brasil. Este estudo teve aprovação do comitê de ética e os pacientes assinaram o formulário de consentimento. Todos os pacientes tiveram diagnóstico de VHC por PCR e CHC por ressonância magnética e / ou tomografia.RESULTADOS: Dentre os indivíduos com CHC, 70,59% foram homens. A média de idade dos participantes foi de 62,3 anos. De acordo com a classificação de Child-Pugh, ficaram assim distribuídos: A 60,9%; B 36,5%; C 2,6%. O tamanho do tumor médio foi de 4,4 cm. Sendo que 52,10% foi multinodular. Foi feita uma análise de associação com os genes MTHFR e VEGF e características do tumor nos pacientes com CHC infectados cronicamente com VHC. Os alelos C da MTHFR (rs1801131) e da VEGF (rs2010963) foram associados com a proteção para o desenvolvimento da forma multinodular, e o alelo T da MTHFR (rs1801133) foi associado a fator de risco para desenvolvimento da forma multinodular p=0,04 OR 1,835 IC (1,0223,297). Foram realizadas análises multivariadas, incluindo-se no modelo o sexo masculino e a idade como potenciais fatores de confusão, o gene VEGF genótipo CC (rs2010963) foi um fator independente associado à proteção ao desenvolvimento de tumores multinodulares. Foi também realizada uma análise de sobrevivência estratificada pelos genótipos MTHFR (rs1801131), MTHFR (rs1801133) e VEGF (rs2010963), mas não foram observados resultados significativos. CONCLUSÕES: Foi observado que os genes MTHFR e VEGF tem influência na forma de apresentação uni ou multinodular do tumor / BACKGROUND: Hepatic cirrhosis is a risk factor for hepatocellular carcinoma (HCC) and in Brazil, the most frequent chronic liver disease associated with HCC is chronic hepatitis C virus (HCV) infection. Angiogenesis and folate deficiency may be associated with the risk of HCC. Thus, gene polymorphisms related to such processes VEGF and MTHFR in patients with chronic hepatitis C and HCC may lead to genetic markers for HCC in HCV infection. OBJECTIVES: To evaluate the polymorphisms G634C (rs2010963), C460T (rs833061), C936T (rs3025039) of the VEGF gene and polymorphisms C677T (rs1801133) and C1298A (1801131) of MTHFR, in Brazilian patients with HCC and chronic HCV infection. PATIENTS AND METHODS: A total of 119 patients with HCC and HCV follow-up were included in the HUOC / UPE and IFP-PE hepatology clinic in Brazil. This study was approved by the ethics committee and the patients signed the consent form. All patients had a diagnosis of HCV by PCR and HCC by magnetic resonance and / or tomography. RESULTS: Among the individuals with HCC, 70.59% were men. The mean age of participants was 62.3 years. According to the Child-Pugh classification, the following were distributed: At 60.9%; B 36.5%; C 2.6%. The mean tumor size was 4.4 cm. Being that 52.10% was multinodular. An association analysis was made with the MTHFR and VEGF genes and tumor characteristics in HCC patients chronically infected with HCV. The C alleles of MTHFR (rs1801131) and VEGF (rs2010963) were associated with developmental protection of the multinodular form, and the MTHFR T allele (rs1801133) was associated with a risk factor for development of the multinodular form p = 0, 04 OR 1,835 IC (1,0223,297). Multivariate analyzes, including male gender and age as potential confounders, were performed in the VEGF gene genotype CC (rs2010963), which was an independent factor associated with the protection of the development of multinodular tumors. A survival analysis stratified by the MTHFR (rs1801131), MTHFR (rs1801133) and VEGF (rs2010963) genotypes was also performed, but no significant results were observed. CONCLUSIONS: It was observed that the MTHFR and VEGF genes have influence on the uni or multinodular presentation of the tumor