O aumento na incidência do carcinoma hepatocelular (CHC) tem sido atribuído ao aumento da obesidade, diabetes e doença hepática gordurosa não alcoólica. (DHGNA), estando ainda por ser melhor esclarecidos vários aspectos histopatológicos e imuno-histoquímicos. O objetivo deste estudo é avaliar aspectos clínicos e patológicos de pacientes com CHC secundário a DHGNA, assim como relacionar a marcadores imuno-histoquímicos de classe proliferativa. Avaliamos 35 espécimes de CHC de 21 pacientes diagnosticados com DHGNA submetidos a ressecção hepática (12 pacientes) ou a transplante hepático (8 pacientes) ou ambos (1 paciente), de 2005 a 2015. Dados demográficos, clínicos e bioquímicos foram relacionados a características histológicas e reatividade imuno-histoquímica para K19, marcando características de células progenitoras e Ki-67, marcando as células em ciclo celular. Um total de 35 nódulos foram detectados em 21 pacientes. A cirrose estava presente em 12 casos (7 F4A x 4F4B x 1F4C de acordo com estadiamento de Laennec) e 9 pacientes não apresentavam cirrose (estadiamento DHGNA: F2: 6pts, F3 = 3pts). A idade variou de 50 a 77 anos e 16 pacientes eram do sexo masculino (76%). Dezesseis pacientes (76%) apresentavam diabetes mellitus, 17 pacientes (81%) apresentavam hipertensão arterial e 19 pacientes (90%) tinham IMC superior a 25 kg / m2. O CHC ocorreu em 8 pacientes CHILD A, 4 CHILD B e em 9 pacientes sem cirrose. O nível de alfa-fetoproteína foi normal em 13 (62%) pacientes. Dentre os critérios histológicos, 25 (70%) nódulos foram diagnosticados como \"CHC esteatohepatítico\". Embora 63% tenham sido pouco diferenciados (G.3/G.4) de acordo com Edmondson & Steiner (1954), apenas 21% apresentaram níveis elevados de Ki-67 ( > 10%). No caso da K19, também 21% dos pacientes apresentaram expressão positiva ( > 5%), e foi associado a maior inflamação intratumoral (G 2/3). Curiosamente, 75% dos pacientes com alta expressão de Ki-67 ( > 10%) não eram cirróticos. Em conclusão: 1. Nesta casuística cirúrgica, o CHC relacionado com DHGNA foi encontrado em não cirróticos em 42% dos casos, com nível normal de alfafetoproteína em 62%. 2. Os marcadores histológicos de \"CHC esteatohepatítico\" estiveram altamente prevalentes. 3. A imunoexpressão positiva de K19 e Ki-67 ocorreu em apenas 21% dos pacientes, o que pode sugerir que o CHC na síndrome metabólica pode ser preferencialmente \"um subtipo inflamatório e não proliferativo de CHC\" / The increase incidence of hepatocellular carcinoma (HCC) has been attributed to the increase in obesity, diabetes and non-alcoholic fatty liver disease. (NAFLD), and several histopathological and immunohistochemical aspects are still to be better clarified. The aim of this study is to assess clinical and pathological aspects of patients with HCC secondary to NAFLD as well as to related to immunohistochemical markers of proliferative class. We evaluated 35 HCC specimens from 21 patients diagnosed with NAFLD undergoing liver resection (12 patients) or liver transplantation (8 patients) or both (1 patient) from 2005 to 2015. Demographic, clinical and biochemical data were related to histological features and immunohistochemical reactivity for K19, marking characteristics of progenitor cells and Ki-67, marking the cells in cell cycle. A total of 35 nodules were detected from 21 patients. Cirrhosis was present in 12 cases (7 F4A x 4F4B x 1F4C according to Laennec Staging) and 9 patients did not have cirrhosis (NAFLD staging: F2: 6pts, F3=3pts). Ages ranged from 50 to 77 years and 16 patients were male (76%). Sixteen patients (76%) had diabetes mellitus, 17 patients (81%) had arterial hypertension and 19 patients (90%) had BMI above 25kg/m2. HCC occurred in 8 patients Child A, 4 Child B and in 9 patients without cirrhosis. Alpha-fetoprotein level was normal in 13 (62%) patients. Among the histological criteria, 25 (70%) nodules were diagnosed as \"steatohepatitic HCC\". Although 63% were poorly differentiated (G.3/ G.4) according to Edmondson & Steiner (1954), only 21% presented high levels of Ki-67 ( > 10%). In the case of K19, 21% of patients presented positive expression (> 5%), and was associated with greater intratumoral inflammation (G 2/3). Interestingly, 75% of the patients with high Ki67 expression ( > 10%) were non-cirrhotic. In conclusion: 1. In this surgical series, HCC related to NAFLD was found in non-cirrhotic patients in 42% of cases, with a normal level of alpha-fetoprotein in 62%. 2. Histological markers of \"steatohepatitic HCC\" were highly prevalent. 3. Positive immunoexpression of K19 and Ki-67 occurred in only 21% of patients, which might suggest that HCC in metabolic syndrome might be preferentially \"an inflammatory, non-proliferative subtype of HCC\"

O aumento na incidência do carcinoma hepatocelular (CHC) tem sido atribuído ao aumento da obesidade, diabetes e doença hepática gordurosa não alcoólica. (DHGNA), estando ainda por ser melhor esclarecidos vários aspectos histopatológicos e imuno-histoquímicos. O objetivo deste estudo é avaliar aspectos clínicos e patológicos de pacientes com CHC secundário a DHGNA, assim como relacionar a marcadores imuno-histoquímicos de classe proliferativa. Avaliamos 35 espécimes de CHC de 21 pacientes diagnosticados com DHGNA submetidos a ressecção hepática (12 pacientes) ou a transplante hepático (8 pacientes) ou ambos (1 paciente), de 2005 a 2015. Dados demográficos, clínicos e bioquímicos foram relacionados a características histológicas e reatividade imuno-histoquímica para K19, marcando características de células progenitoras e Ki-67, marcando as células em ciclo celular. Um total de 35 nódulos foram detectados em 21 pacientes. A cirrose estava presente em 12 casos (7 F4A x 4F4B x 1F4C de acordo com estadiamento de Laennec) e 9 pacientes não apresentavam cirrose (estadiamento DHGNA: F2: 6pts, F3 = 3pts). A idade variou de 50 a 77 anos e 16 pacientes eram do sexo masculino (76%). Dezesseis pacientes (76%) apresentavam diabetes mellitus, 17 pacientes (81%) apresentavam hipertensão arterial e 19 pacientes (90%) tinham IMC superior a 25 kg / m2. O CHC ocorreu em 8 pacientes CHILD A, 4 CHILD B e em 9 pacientes sem cirrose. O nível de alfa-fetoproteína foi normal em 13 (62%) pacientes. Dentre os critérios histológicos, 25 (70%) nódulos foram diagnosticados como \"CHC esteatohepatítico\". Embora 63% tenham sido pouco diferenciados (G.3/G.4) de acordo com Edmondson & Steiner (1954), apenas 21% apresentaram níveis elevados de Ki-67 ( > 10%). No caso da K19, também 21% dos pacientes apresentaram expressão positiva ( > 5%), e foi associado a maior inflamação intratumoral (G 2/3). Curiosamente, 75% dos pacientes com alta expressão de Ki-67 ( > 10%) não eram cirróticos. Em conclusão: 1. Nesta casuística cirúrgica, o CHC relacionado com DHGNA foi encontrado em não cirróticos em 42% dos casos, com nível normal de alfafetoproteína em 62%. 2. Os marcadores histológicos de \"CHC esteatohepatítico\" estiveram altamente prevalentes. 3. A imunoexpressão positiva de K19 e Ki-67 ocorreu em apenas 21% dos pacientes, o que pode sugerir que o CHC na síndrome metabólica pode ser preferencialmente \"um subtipo inflamatório e não proliferativo de CHC\" / The increase incidence of hepatocellular carcinoma (HCC) has been attributed to the increase in obesity, diabetes and non-alcoholic fatty liver disease. (NAFLD), and several histopathological and immunohistochemical aspects are still to be better clarified. The aim of this study is to assess clinical and pathological aspects of patients with HCC secondary to NAFLD as well as to related to immunohistochemical markers of proliferative class. We evaluated 35 HCC specimens from 21 patients diagnosed with NAFLD undergoing liver resection (12 patients) or liver transplantation (8 patients) or both (1 patient) from 2005 to 2015. Demographic, clinical and biochemical data were related to histological features and immunohistochemical reactivity for K19, marking characteristics of progenitor cells and Ki-67, marking the cells in cell cycle. A total of 35 nodules were detected from 21 patients. Cirrhosis was present in 12 cases (7 F4A x 4F4B x 1F4C according to Laennec Staging) and 9 patients did not have cirrhosis (NAFLD staging: F2: 6pts, F3=3pts). Ages ranged from 50 to 77 years and 16 patients were male (76%). Sixteen patients (76%) had diabetes mellitus, 17 patients (81%) had arterial hypertension and 19 patients (90%) had BMI above 25kg/m2. HCC occurred in 8 patients Child A, 4 Child B and in 9 patients without cirrhosis. Alpha-fetoprotein level was normal in 13 (62%) patients. Among the histological criteria, 25 (70%) nodules were diagnosed as \"steatohepatitic HCC\". Although 63% were poorly differentiated (G.3/ G.4) according to Edmondson & Steiner (1954), only 21% presented high levels of Ki-67 ( > 10%). In the case of K19, 21% of patients presented positive expression (> 5%), and was associated with greater intratumoral inflammation (G 2/3). Interestingly, 75% of the patients with high Ki67 expression ( > 10%) were non-cirrhotic. In conclusion: 1. In this surgical series, HCC related to NAFLD was found in non-cirrhotic patients in 42% of cases, with a normal level of alpha-fetoprotein in 62%. 2. Histological markers of \"steatohepatitic HCC\" were highly prevalent. 3. Positive immunoexpression of K19 and Ki-67 occurred in only 21% of patients, which might suggest that HCC in metabolic syndrome might be preferentially \"an inflammatory, non-proliferative subtype of HCC\"

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

O carcinoma hepatocelular (CHC) é a quinta neoplasia maligna mais frequente no mundo, sendo que, em mais de 80% dos casos, seu aparecimento está relacionado à presença de cirrose hepática (CH). A infecção crônica pelo vírus da hepatite C (VHC) é uma das principais causas de cirrose hepática no mundo e, consequentemente, de CHC. Este estudo caso-controle foi baseado na análise de variáveis clínicas, bioquímicas e sorológicas de 31 pacientes cirróticos pelo VHC com CHC de pequeno tamanho (<= 3 cm, tamanho médio = 22 mm) comparando-os com grupo controle de 62 pacientes cirróticos pelo VHC sem CHC, pareados por idade e sexo. Os principais objetivos foram identificar marcadores auxiliares ao diagnóstico de CHC e desenvolver um modelo linear para o diagnóstico presuntivo de CHC de pequeno tamanho. Os dados levantados foram submetidos à análise univariada. Demonstramos diferenças significantes entre os dois grupos com relação à presença de marcadores de infecção pregressa pelo vírus da hepatite B; às dosagens séricas de aspartato aminotransferase, de alanina aminotransferase, de gamaglutamil transpeptidase; à contagem de plaquetas, fibrinogênio plasmático, alfafetoproteína e resposta virológica sustentada ao tratamento com interferon. As variáveis significantes foram submetidas à análise multivariada com procedimento de regressão logística \"stepwise\" para ajustar o modelo linear. Esta análise multivariada selecionou duas variáveis preditoras para o diagnóstico de CHC, a aspartato aminotransferase e a alfa-fetoproteína, com uma probabilidade de 0,26; sensibilidade de 74,2% e especificidade de 66,1%. Entretanto este modelo linear, devido a sua baixa probabilidade, não pode ser empregado para o diagnóstico de CHC, porém potencializa a identificação de um grupo de pacientes com maior risco para o seu desenvolvimento, merecendo assim um programa de rastreamento mais cuidadoso / Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer worldwilde. In 80% of cases HCC develop in cirrhotic livers. Hepatitis C virus infection is considered one of the main causes of liver cirrhosis and HCC. We conducted a case-control study involved 31 small HCC cases (<= 3 cm, mean size = 22 mm) and 62 age and gender matched control HCV liver cirrhosis subjects. All included patients (case and controls) were HCV positive (confirmed by RT-PCR). The present study was based on the comparison of clinical, biochemical and serological data in these patients. Our aims were to identify auxiliar parameters for the diagnosis of HCC and develop a linear model that predict the diagnosis of HCC. Data were submitted to an univariate analysis. Significant differences between the groups were observed in relation to markers of hepatitis B past infection, aspartate aminotransferase, alanine aminotransferase, gammaglutamyltransferase, platelet count, fibrinogen, alpha-fetoprotein and sustained response after Interferon therapy. The significant variables were submitted to a multivariate analysis with stepwise logistic regression procedure to adjust a linear model to estimate the probability of diagnosis of HCC. Two significant variables to HCC prediction were found, the aspartate aminotransferase and alpha-fetoprotein adjusted a linear model that allows HCC diagnosis with 0,26 probability, 74,2% sensibility and 66% specificity. However, because this model has low probability, it cannot make the HCC diagnosis but allows identify the potential group of patients with major risk to rise HCC, so that deserves more accurate surveillance strategy

CONTEXTO E OBJETIVO: O manejo do carcinoma hepatocelular (HCC) é desafiador devido a agressividade tumoral e cirrose associada. Faltam opções locais efetivas após falha à quimioembolização transarterial (TACE). Nosso objetivo foi testar através de estudo prospectivo fase II, a eficácia e segurança da Radioterapia Estereotáctica Corpórea (SBRT) em pacientes com HCC e resposta parcial ou contraindicação à TACE. MÉTODO: Pacientes com até 5 lesões de HCC restritas ao fígado realizaram SBRT na dose de 30 a 50 Gy em 5 frações. O desfecho primário foi sobrevida livre de progressão das lesões tratadas. Os desfechos secundários foram sobrevida livre de progressão hepática, sobrevida livre de progressão a distância, sobrevida global e toxicidade. Este estudo está registrado em clinicaltrials.gov sob o número NCT02221778. RESULTADO: De novembro de 2014 a junho de 2018, 19 pacientes receberam SBRT na dose mediana de 40 Gy (range 30 - 50 Gy). Todos tinham escore de Child Pugh A. A idade mediana foi de 67 anos (range 42-84 anos). A doença de base foi hepatite C em 42%, hepatite B em 26% e álcool em 26%. TACE prévia foi realizada em 84% dos pacientes, com mediana de duas TACEs (range 0-5). O número mediano de lesões foi dois (range 1-4), com tamanho mediano de 4 cm (1,5-10 cm). 32% dos pacientes tinham trombose tumoral; a AFP mediana pré-tratamento foi de 142,5 ng/ml (range 4,2 - 5 494 ng/ml). A sobrevida livre de progressão local em 1 ano foi de 80% (IC95%, 50% a 93%). A sobrevida livre de progressão hepática, sobrevida livre de progressão a distância e sobrevida global em 1 ano foram, respectivamente, de 52%, 82% e 84%. Não houve toxicidades clínicas grau 3 ou 4. Toxicidades laboratoriais até grau 3 ocorreram em 3 pacientes (16%). Resposta radiológica completa foi atingida em 53% dos pacientes, 42% tiveram resposta parcial. O tempo mediano para melhor resposta foi de 3,4 meses (range 2,4-12,6 meses). CONCLUSÃO: A SBRT é uma opção eficaz, segura e não invasiva em pacientes com HCC e resposta parcial ou contraindicação à quimioembolização / BACKGROUND AND PURPOUSE: Management of hepatocellular carcinoma (HCC) is challenging due to tumor aggressiveness and associated cirrhosis. There is paucity of effective local options after failure of transarterial chemoembolization (TACE). Our objective was to test in the setting of a phase II prospective study, the efficacy and safety of Stereotactic Body Radiation Therapy (SBRT) in patients with partial response or unsuitable for TACE. METHODS: Patients with HCC and up to five liver-only lesions received SBRT 30 to 50 Gy in 5 fractions. Primary endpoint was local progression-free survival. Secondary endpoints were liver progression-free survival, distant progression-free survival, overall survival and toxicity. This study is registered at clinicaltrials.gov NCT02221778. RESULTS: From Nov 2014 through Jun 2018, 19 patients received SBRT with a median dose of 40Gy (range 30 - 50 Gy). All patients were Child Pugh A. Median age was 67 years old (range 42-84y). Underlying liver disease was hepatitis C in 42% of patients, hepatitis B in 26% and alcohol-related in 26%. 84% received previous TACE, with a median of two TACEs (range 0-5). Patients had a median of two lesions (range 1-4), with median size of 4 cm (1.5-10 cm). 32% had tumor vascular thrombosis; median pretreatment AFP was 142.5 ng/ml (range 4.2 - 5,494 ng/ml). 1y local progression-free survival was 80% (95% CI, 50% to 93%). 1y liver progression-free survival, distant progression-free survival and overall survival were, respectively, 52%, 82% and 84%. No patient had clinical grade 3 or 4 toxicities. Laboratory toxicities up to grade 3 occurred in three patients (16%). Complete radiological response was seen in 53% of patients, 42% had partial response. Median time for best response was 3.4 months (range 2.4-12.6 months). CONCLUSION: SBRT is an effective, safe and noninvasive option in HCC patients with partial response or unsuitable for TACE

INTRODUÇÃO E OBJETIVOS: A doença hepática gordurosa não alcoólica (DHGNA) tem sido associada ao carcinoma hepatocelular (CHC), muitas vezes em paciente com hepatopatia avançada. Este estudo objetivou avaliar o efeito do sorafenibe no modelo experimental de CHC avançado secundário à DHGNA, padronizar o PET com 18F-FDG para avaliar o CHC neste modelo e avaliar se há relação entre o grau de avidez pelo 18F-FDG e o grau de diferenciação tumoral do CHC. METODOLOGIA: Estudo foi aprovado pela Comissão de Ética no Uso de Animais. Foram utilizados trinta ratos Sprague-Dawley, machos, com 3 meses de vida, pesando entre 300-400g. O CHC secundário à DHGNA foi induzido pela combinação de dieta hiperlipídica deficiente em colina e dietilnitrosamina na dose de 100 mg/ L na água de beber ad libitum por 16 semanas. Após este período foram suspensos os estímulos carcinogênicos, realizou-se ultrassonografia abdominal para caracterização dos nódulos hepáticos maiores que 2 mm, e foi feita a divisão dos dois grupos segundo randomização e iniciada a administração diária do fármaco por gavagem durante 3 semanas: Controle (n=10) - 1 mL salina, Sorafenibe (n=20): 5mg/ kg/ dia. Ao término do tratamento, os animais realizaram PET (Gamma Medica-Ideas, USA) com 18F-FDG (média de 18F-FDG injetada de 1,02 ± 0,17 mCi ou 37,7 ± 6,29 MBq). Três dias após o PET, os animais foram anestesiados e foi feita a eutanásia, quando foi coletado material hepático. As lâminas foram avaliadas, por patologista veterinário experiente, na coloração de hematoxilina-eosina e imunohistoquímica para glutamina sintetase, antígeno específico de hepatócitos 1 e citoqueratina-19. RESULTADOS: A mortalidade nos dois grupos foi de 60% (p=0,07). Os achados ultrassonográficos mostraram grupos homogêneos com média de nódulos por animal: 4,88 ± 2,75 no controle e 4,95 ± 3,11 no sorafenibe (p=0,48). Na 19ª semana, viu-se que a média de lesões hipercaptantes por animal no PET foi de 4,37 ± 1,59 no grupo sorafenibe e 8,5 ± 3,7 no controle (p=0,006). A avidez máxima do 18F-FDG (SUVmáx) foi diferente entre os grupos estudados: 2,4 ± 1,98 no sorafenibe e 3,8 ± 1,74 no controle (p=0,01). Houve correlação direta entre o CHC pouco diferenciado/indiferenciado e os maiores valores de SUVmed (R2 = 0,34, p=0,04), SUVmax (R2 = 0,44, p=0,01), relação Tumor SUVmax/Fígado SUVmax (R2 = 0,42, p=0,02) e relação Tumor SUVmax/ Músculo SUVmax (R2 = 0,54, p=0,006). A média por animal de CHC confirmado pela histologia foi menor no grupo sorafenibe que no controle (5,5 ± 1,5 vs 3,3 ± 0,48, p=0,01). E o grupo tratado com sorafenibe apresentou mais CHC bem diferenciado que o controle (39% vs 5%, respectivamente, p=0,01), bem como, menor presença de CHC pouco diferenciado que o grupo controle (52% vs 81%, p-0,003). CONCLUSÃO: O sorafenibe reduziu o número médio de CHC, a agressividade dos CHC e menor SUVmax dos tumores. A metodologia do PET foi padronizada para este modelo animal específico. O PET 18F-FDG pode ser utilizado para avaliar não invasivamente o grau de diferenciação histológica do CHC, pois valores maiores de SUVmed, SUVmax, Tumor SUVmax/Fígado SUVmax e Tumor SUVmax/ Músculo SUVmax foram correlacionados com CHC pouco diferenciado / BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) has been linked to hepatocellular carcinoma (HCC), often in patients with advanced liver disease. This study aimed to: assess the effect of sorafenib in the experimental model of NAFLD related HCC, standardize PET 18F-FDG to be an assessment tool of HCC in this model and to assess if there is a correlation between the degree of avidity for 18F-FDG and the degree of tumor differentiation. METHODS: The ethics committee on animal use approved this study. Thirty male sprague-dawley rats were used, weighing between 300-400g. NAFLD related HCC was induced by the combination of fat and choline deficient diet with diethylnitrosamine (100 mg/L) in drinking water for 16 weeks. After this period these carcinogenic stimuli were suspended, and liver nodules were identified by abdominal ultrasound. Two groups were randomized: control (n=10) and treatment (n=20). Rats received daily gavage administration of 1 mL saline in the control group and sorafenib (5mg/kg/day) in the treatment group. After treatment, animals performed PET (Gamma Medica-Ideas, USA) with 18F-FDG (average of 18F-FDG injected 1.02 ± 0.17 mCi or 37.7 ± 6.29 MBq). Three days after the PET, the animals were anesthetized and euthanized. Histological aspect was evaluated by experienced veterinary pathologist. RESULTS: The mortality in both groups was 60% (p = 0.07). The sonographic findings showed homogeneous groups with average nodules per animal of: 4.88 ± 2.75 in control and 4.95 ± 3.11 in sorafenib (p = 0.48). On the 19th week, it was observed that the average hypercaptant lesion per animal in PET was 4.37 ± 1.59 in the sorafenib group and 8.5 ± 3.7 in control group (p = 0.006). Average SUVmax was different between groups: 2.4 ± 1.98 in the sorafenib group and 3.8 ± 1.74 in the control group (p = 0.01). A direct correlation was found between the poorly differentiated HCC and larger values of: SUVmed (R2 = 0.34, p = 0.04), SUVmax (R2 = 0.44, p = 0.01) tumorSUVmax / LiverSUVmax ratio (R2 = 0.42, p = 0.02) and tumorSUVmax / MuscleSUVmax ratio (R2 = 0.54, p = 0.006). HCC average per animal was lower in the sorafenib group than in the control group (5.5 ± 1.5 vs. 3.3 ± 0.48; p = 0.01). And sorafenib group had more well differentiated HCC (39% vs 5%, respectively, p = 0.01) and lower presence of poorly differentiated HCC (52% vs 81%, p -0.003) than the control group. CONCLUSION: Sorafenib reduced the average number of HCC, the aggressiveness of HCC and lowered values of tumors SUVmax. The methodology of PET was standardized for this particular animal model. PET 18F-FDG can be used noninvasively to assess the degree of histological differentiation of HCC, as higher values of SUVmed, SUVmax, tumorSUVmax/ LiverSUVmax ratio and tumorSUVmax / MuscleSUVmax ratio were correlated with poorly differentiated HCC

Cheung Sao Fong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves xxiv-xxxvii). / Abstracts in English and Chinese. / Examination Committee List --- p.i / Declaration --- p.ii / Acknowledgements --- p.iii / Abstract --- p.v / Abstract in Chinese --- p.viii / List of Figures and Tables --- p.x / List of Abbreviations --- p.xix / Table of Content --- p.xxiii / Chapter Chapter 1 --- INTRODUCTION --- p.1 / Chapter 1.1 --- Human Liver Cancer --- p.1 / Chapter 1.1.1 --- Incidence of Hepatocellular Carcinoma --- p.1 / Chapter 1.1.2 --- Causes and Symptoms of Hepatocellular Carcinoma --- p.4 / Chapter 1.1.3 --- Treatment Options for Hepatocellular Carcinoma --- p.4 / Chapter 1.1.4 --- Multi-drug Resistance --- p.5 / Chapter 1.1.4.1 --- Mechanisms of Multi-drug Resistance --- p.5 / Chapter 1.2 --- Traditional Chinese Medicine --- p.10 / Chapter 1.2.1 --- Sophora flavescens and Radix Sophorae --- p.10 / Chapter 1.2.2 --- Flavonoid and its Sub-classification --- p.13 / Chapter 1.2.3 --- Flavonoid and Human Health --- p.15 / Chapter 1.3 --- Cell Death --- p.17 / Chapter 1.3.1 --- Necrosis --- p.17 / Chapter 1.3.2 --- Apoptosis --- p.17 / Chapter 1.3.3 --- Signaling Pathways in Apoptosis --- p.18 / Chapter 1.3.3.1 --- Extrinsic (Death Receptor-mediated) Pathway --- p.20 / Chapter 1.3.3.2 --- Intrinsic (Mitochondrial) Pathway --- p.21 / Chapter 1.3.3.3 --- Cysteine Aspartatic Acid Proteases --- p.21 / Chapter 1.4 --- Research Objective (s) --- p.22 / Chapter Chapter 2 --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Cell Lines --- p.23 / Chapter 2.1.1.1 --- HepG2 --- p.24 / Chapter 2.1.1.2 --- RHepG2 --- p.24 / Chapter 2.1.1.3 --- WRL-68 --- p.25 / Chapter 2.1.2 --- Culture Media --- p.26 / Chapter 2.1.2.1 --- Rosewell Park Memorial Institute( RPMl) 1640 Medium --- p.26 / Chapter 2.1.2.2 --- Dulbecco's Modified Eagle's Medium (DMEM) --- p.26 / Chapter 2.1.3 --- Animals --- p.27 / Chapter 2.2 --- Traditional Chinese Medicines and Conventional Anti-cancer Drugs --- p.27 / Chapter 2.3 --- Antibodies --- p.29 / Chapter 2.4 --- Chemicals --- p.30 / Chapter 2.5 --- Reagents and Buffers --- p.34 / Chapter 2.5.1 --- Reagents for Silica Gel Column Chromatography --- p.34 / Chapter 2.5.2 --- Buffers for Common Use --- p.34 / Chapter 2.5.3 --- Reagents for Cell Viability Assay --- p.35 / Chapter 2.5.4 --- Reagents and Buffers for Typical Apoptosis Experiments --- p.35 / Chapter 2.5.4.1 --- Cell Cycle Analysis --- p.35 / Chapter 2.5.4.2 --- Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) Assay --- p.35 / Chapter 2.5.4.3 --- DNA Fragmentation Detection --- p.35 / Chapter 2.5.5 --- Reagents and Buffers for Western Blot Study --- p.36 / Chapter 2.5.5.1 --- Whole-cell Protein Extraction --- p.38 / Chapter 2.5.5.2 --- Mitochondrial and Cytosolic Fraction Protein Extraction --- p.38 / Chapter 2.5.6 --- Reagents and Buffers for Mitochondrial Transmembrane Potential Depolarization Measurement --- p.39 / Chapter 2.5.7 --- Reagents and Buffers for in vivo Animal Study --- p.39 / Chapter 2.5.8 --- Reagents and Buffers for Two-Dimensional Gel Electrophoresis --- p.40 / Chapter 2.5.8.1 --- Sample Preparation --- p.40 / Chapter 2.5.8.2 --- First Dimension Gel Electrophoresis - Isoelectric Focusing (IEF) --- p.40 / Chapter 2.5.8.3 --- Second Dimension Gel 日ectrophoresis - SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.40 / Chapter 2.5.8.4 --- Silver Staining --- p.41 / Chapter 2.5.9 --- Reagents for Mass Spectrometry Preparation --- p.42 / Chapter 2.5.9.1 --- Destaining --- p.42 / Chapter 2.5.9.2 --- Trypsin Digestion --- p.42 / Chapter 2.5.9.3 --- Desalting of Peptide Mixture --- p.43 / Chapter 2.5.10 --- Reagents and Buffers for Real-Time PCR --- p.43 / Chapter 2.6 --- Methods --- p.44 / Chapter 2.6.1 --- Isolation of Bioactive Constituents by Silica Gel Column Chromatography --- p.44 / Chapter 2.6.2 --- Cell Viability Assay --- p.45 / Chapter 2.6.3 --- Typical Apoptosis Experiments --- p.45 / Chapter 2.6.3.1 --- Cell Cycle Analysis --- p.46 / Chapter 2.6.3.2 --- Annexin V-FITC/ PI Staining Experiment --- p.47 / Chapter 2.6.3.3 --- Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) Assay --- p.48 / Chapter 2.6.3.4 --- DNA Fragmentation Reaction --- p.48 / Chapter 2.6.4 --- Western Blot Study --- p.49 / Chapter 2.6.4.1 --- Whole-cell Protein Extraction --- p.49 / Chapter 2.6.4.2 --- Mitochondrial and Cytosolic Fraction Protein Extraction --- p.50 / Chapter 2.6.5 --- Caspase Activity Determination --- p.54 / Chapter 2.6.6 --- Mitochondrial Transmembrane Potential Depolarization Measurement --- p.55 / Chapter 2.6.7 --- in vivo Animal Study --- p.56 / Chapter 2.6.8 --- Two-Dimensional Gel Electrophoresis --- p.58 / Chapter 2.6.8.1 --- Sample Preparation --- p.58 / Chapter 2.6.8.2 --- First Dimension Electrophoresis - Isoelectric Focusing (IEF) --- p.59 / Chapter 2.6.8.3 --- Second Dimension Electrophoresis - SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.60 / Chapter 2.6.8.4 --- Silver Staining --- p.61 / Chapter 2.6.9 --- Mass Spectrometry Preparation --- p.63 / Chapter 2.6.9.1 --- Destaining and Trypsin Digestion --- p.63 / Chapter 2.6.9.2 --- Peptide Extraction --- p.63 / Chapter 2.6.9.3 --- Desalting of Peptide Mixture --- p.64 / Chapter 2.6.10 --- Real-Time PCR --- p.65 / Chapter 2.6.11 --- Cellular Glutathione Level Detection --- p.69 / Chapter 2.7 --- Statistical Analysis --- p.70 / Chapter Chapter 3 --- RESULTS AND DISCUSSIONS - CYTOTOXICITY OF FLAVONOIDS ISOLATED FROM RADIX SOPHORAE --- p.72 / Chapter 3.1 --- Screening of Cytotoxic Flavonoids from Radix Sophorae --- p.72 / Chapter 3.2 --- Cytotoxicity of Leachianone A on Human Hepatoma Cell Lines --- p.74 / Chapter 3.3 --- Cytotoxicity of Leachianone A on Human Normal Liver Cell Line --- p.77 / Chapter 3.4 --- Cytotoxicity of Sophoraflavone J on Human Hepatoma Cell Line --- p.79 / Chapter 3.5 --- Cytotoxicity of Sophoraflavone J on Human Normal Liver Cell Line --- p.79 / Chapter 3.6 --- Cytotoxicities of Cisplatin and Taxol on Human Hepatoma as well as Normal Liver Cell Lines --- p.81 / Chapter 3.7 --- Conclusion --- p.86 / Chapter Chapter 4 --- "RESULTS AND DISCUSSIONS - MECHANISTIC STUDY OF LEACHIANONE A-INDUCED CELL DEATH IN HEPATOMA CELLS, HepG2 and RHepG2" --- p.88 / Chapter 4.1 --- Promotion of Cell Cycle Arrest --- p.88 / Chapter 4.2 --- Induction of Apoptosis as Evidenced by Phosphatidylserine Externalization and DNA Fragmentation --- p.93 / Chapter 4.2.1 --- Occurrence of Phosphatidylserine Externalization --- p.94 / Chapter 4.2.2 --- DNA Fragmentation Detection --- p.99 / Chapter 4.2.2.1 --- Terminal Deoxynucleotidyl Transferase(TdT)-mediated dUTP Nick End Labeling (TUNEL) Assay --- p.99 / Chapter 4.2.2.2 --- DNA Laddering Pattern in Agarose Gel Electrophoresis --- p.103 / Chapter 4.3 --- Recruitment of Multiple Signaling Pathways in Leachianone A-induced Apoptosis --- p.105 / Chapter 4.3.1 --- "Activation of Caspases-3, -8, and -9" --- p.105 / Chapter 4.3.2 --- Altered Expressions of Bcl-2 Family Proteins --- p.112 / Chapter 4.3.3 --- Loss of Mitochondrial Membrane Potential --- p.115 / Chapter 4.4 --- in vivo Tumor Growth Inhibition in HepG2-bearing Nude Mice --- p.121 / Chapter 4.5 --- Conclusion --- p.127 / Chapter Chapter 5 --- RESULTS AND DISCUSSIONS - MECHANISTIC STUDY OF SOPHORAFLAVONE J-INDUCED CELL DEATH IN HEPATOMA CELLS HepG2 --- p.132 / Chapter 5.1 --- Execution of Cellular Apoptosis --- p.133 / Chapter 5.2 --- Involvement of Multiple Signaling Pathways in Sophoraflavone J-induced Apoptosis --- p.138 / Chapter 5.3 --- Differential Proteomes of Control and Sophoraflavone J-treated HepG2 Cells --- p.148 / Chapter 5.4 --- Conclusion --- p.167 / Chapter Chapter 6 --- OVERALL CONCLUSION AND FUTURE PERSPECTIVES --- p.169 / References --- p.xxiv

Made available in DSpace on 2016-01-26T12:51:38Z (GMT). No. of bitstreams: 1 raphaelraphe_dissert.pdf: 1654822 bytes, checksum: 678de193b930e82d2dafeef1d384be12 (MD5) Previous issue date: 2012-09-24 / Introduction: Hepatocellular carcinoma (HCC) is the most common primary neoplasm of the liver. It constitutes an important cause of cancer mortality in cirrhotic patients. Objective: To evaluate the epidemiological aspects relating them to the risk factors of HCC in a closed population. Methods: We conducted a retrospective cross-sectional study with chart review of patients treated from November 1998 to May 2011 in the Departments of Liver Transplantation, Gastroenterology and Pathology and the Cancer Institute of the Hospital de Base, São José do Rio Preto. The study was approved by the Ethics and Research. Results: A total of 272 patients with HCC, 229(84.2%) were male and 43 (15.8%) were female. The mean age was 57.1 years (SD 10.9 years) and predominantly caucasian (91.5%). Cirrhosis present in 98.2% with Child-Turcotte-Pugh class B in 104 patients (38.9%). The etiology, the most frequent was infection with hepatitis C virus in 145(55.1%) patients being single cause in 88(33.4%). The results suggest that sex is associated with the principal factors in the etiology of HCC, but age was not associated with disease etiology. In 220 patients, the largest nodule, ranged from 6mm to 260mm in diameter with a mean of 61.4mm (SD 41.5mm). In 145(64.2%) patients revealed the presence of a nodule and 46(20.3%) patients multifocal. It was found that 8(3.6%) only nodules had a diameter smaller than 20mm and 74(33.5%) diameters of between twenty and fifty millimeters. Of the 214 patients classified according to staging, 70(32.7%) were early stage and 97 among more advanced stage and terminal (30.4% and 14.9% respectively). Thirty(11%) patients were incidental findings. Regarding the diagnosis of HCC, 175 patients (68.1%) were diagnosed by an imaging study. The time between diagnosis and initial treatment, a total of 224 patients, 86(38.4%) was started in the first month and 46(20.5%) between 30 and 60 days. The average start of treatment was 70.7 days (SD 86.1 days), specific treatment was conducted in 236(86.8%) patients in which chemoembolization 127(46.7%) and liver transplantation in 72(26.5%), of whom 33(45.8%) received chemoembolization as a "bridge" to transplant. Thirty-four patients (12.5%) received only supportive therapy. Level of &#945;-fetoprotein was measured in 209 patients with 29.2% less than 20 ng/ml and 34.9% at above 400 ng/ml. In patients with thyroid nodules diameter greater than or equal to 10 cm, were 67.8% &#945;-fetoprotein levels greater than 400 ng / ml. In 144 patients, histological analysis showed that in 94 (65.3%) nodules were moderately differentiated. Conclusion: Prevalence of male and involvement in the 5th decade of life. Hepatic cirrhosis present in most patients. Infection with hepatitis C followed by alcoholic liver disease were more common etiologies. Diagnosis was delayed or advanced stages, and dosage levels of &#945;-fetoprotein was not good tool diagnostic. Treatment of HCC showed a predominance of non-curative therapies due to late diagnosis. / Introdução: Carcinoma hepatocelular (CHC) é a neoplasia primária mais comum do fígado. Constitui-se em importante causa de mortalidade por câncer em pacientes cirróticos. Objetivo: Avaliar os aspectos epidemiológicos relacionando-os com os fatores de risco do CHC em uma população fechada. Casuística e Métodos: Foi realizado um estudo transversal retrospectivo, com revisão de prontuários de pacientes atendidos de novembro de 1998 a maio de 2011 nos Serviços de Transplante de Fígado, Gastroenterologia, Anatomia Patológica e do Instituto do Câncer do Hospital de Base de São José do Rio Preto. O estudo foi aprovado pelo Comitê de Ética e Pesquisa. Resultados: Do total de 272 pacientes com CHC, 229(84,2%) eram do sexo masculino e 43(15,8%) do sexo feminino. A idade média foi de 57,1 anos (desvio padrão de 10,9 anos) e predomínio da raça branca (91,5%). Cirrose hepática presente em 98,2% com classificação de Child-Turcotte-Pugh classe B em 104 pacientes (38,9%). Quanto à etiologia, a mais frequente foi infecção por vírus da hepatite C em 145(55,1%) pacientes, sendo causa isolada em 88(33,4%) pacientes. Os resultados sugerem que o sexo esteja associado aos principais fatores da etiologia do CHC, mas a idade não apresentou associação com a etiologia da doença. Em 220 pacientes, o maior nódulo, variou de 6mm a 260mm de diâmetro com média de 61,4mm (desvio padrão de 41,5mm). Em 145 pacientes (64,2%), observou-se presença de um nódulo, dois nódulos em 26(11,5%), três nódulos em 9(4%) e 46 pacientes (20,3%) multifocal. Verificou-se que 8(3,6%) nódulos únicos apresentavam diâmetro menor que 20mm e 74(33,5%) diametros entre dois e cinco cm. Dos 214 pacientes classificados quanto ao estadiamento de acordo com o Barcelona Clinic Liver Cancer (BCLC), 70(32,7%) eram estadio precoce e 97 entre estadios avancado e terminal (30,4% e 14,9% respectivamente). Trinta pacientes (11%) foram achados incidentais. Quanto ao diagnostico de CHC, 175 pacientes (68,1%) foram diagnosticados por meio de um exame de imagem. Quanto ao tempo entre o diagnostico e o primeiro tratamento, de um total de 224 pacientes, 86 (38,4%) foi iniciado no primeiro mes e 46(20,5%) entre 30 e 60 dias. A media para o inicio do tratamento foi de 70,7 dias (desvio padrao de 86,1 dias), Tratamento especifico foi realizado em 236(86,8%) pacientes sendo quimioembolizacao exclusiva em 127(46,7%) e transplante de figado em 72 (26,5%) e destes, 33(45,8%) receberam quimioembolizacao como gponte h para o transplante. Trinta e quatro pacientes (12,5%) receberam apenas terapia de suporte. Nivel de ¿-fetoproteina foi dosado em 209 pacientes, com 29,2% menor que 20 ng/ml e 34,9% niveis superiores a 400 ng/ml. Em pacientes com nodulos de diametro maior ou igual a 100 mm, 67,8% apresentaram ¿-fetoproteina com niveis superiores a 400 ng/ml. Em 144 pacientes, a analise histologica apontou que em 94(65,3%) os nodulos eram moderadamente diferenciados. Conclusao: Predominio do sexo masculino e acometimento na 5a decada de vida. A cirrose hepatica foi o principal fator de risco para CHC. Infeccao do virus da hepatite C seguida de doenca hepatica alcoolica foram etiologias mais frequentes. Diagnostico ocorreu nas fases tardia ou avancada na maioria dos pacientes e a dosagem dos niveis de ¿-fetoproteina nao se mostrou boa ferramenta diagnóstica. Tratamento do CHC apresentou predomínio de terapias não curativas devido ao diagnóstico tardio.

A doença hepática associada a sobrecarga de ferro pode ocorrer devido a causas genéticas ou secundárias. Esse estudo avaliou pacientes com hepatopatia crônica com sobrecarga de ferro submetidos à pesquisa das mutações HFE no período de 2007-2009 e classificou como portadores de hemocromatose hereditária HFE (HH-HFE) aqueles que apresentavam as mutações C282Y/C282Y ou C282Y/H63D e como sobrecarga de ferro não HFE aqueles que apresentavam outras mutações no gene HFE como C282Y/-, H63D/- e H63D/H63D ou pacientes sem qualquer uma dessas mutações mencionadas. Os objetivos do estudo foram 1) analisar e correlacionar os aspectos fenotípicos e genotípicos de grupo de indivíduos com doença hepática crônica e sobrecarga de ferro; 2) caracterizar o quadro clínico, laboratorial e anatomopatológico, em busca de achados compatíveis com o fenótipo de hemocromatose; 3) Correlacionar o quadro clínico com as mutações no gene HFE. Foram analisados 108 indivíduos portadores de hepatopatia crônica selecionados a partir de saturação de transferrina (ST) > 45% e ferritina sérica > 350 ng/mL. Foram estudados e comparados 16 pacientes no grupo HH-HFE com 92 no grupo sobrecarga de ferro não HFE. Da casuística geral, a idade média ao diagnóstico da doença foi de 46,69 anos (16-77), com 70,73% constituída por indivíduos de cor branca, 77,57% do sexo masculino e 64,8% tinham cirrose hepática. A frequência de cirrose hepática não diferiu entre os grupos, entretanto, artropatia, carcinoma hepatocelular, diabetes e osteoporose foram mais frequentes no grupo HH- HFE (53,8% x 15,9%, 31,2% x 7,06%, 56,2% x 30%, 72,7% x 32,1%, respectivamente, p < 0,05). Os pacientes com mutações HFE diagnósticas de HH apresentaram maior chance de ter carcinoma hepatocelular (OR= 5,0, p= 0,032) quando comparados com os portadores de outros genótipos HFE e aqueles sem mutação. Os níveis de ST, ferro e ferritina também foram maiores naquele grupo, bem como os graus de siderose 3 e 4 (p= 0,026). A ST foi a variável que se correlacionou independentemente com o diagnóstico das mutações C282Y/C282Y e C282Y/H63D. A frequência de fatores de risco para sobrecarga de ferro não diferiu entre os grupos. Observou-se, entretanto, que no grupo HH-HFE havia maior número de pacientes sem qualquer fator de risco detectado (p= 0,019). Níveis de ST > 82% apresentaram maior valor preditivo negativo para o diagnóstico de HH-HFE do que os de ferritina, ferro, capacidade total de ligação de ferro e de transferrina. Concluímos que os portadores de HH-HFE têm maiores graus de sobrecarga de ferro quando comparados ao grupo de sobrecarga de ferro não-HFE; em indivíduos com doença hepática crônica. ST > 82% tem maior acurácia para diagnóstico de HH-HFE; portadores de mutações C282Y em homozigose ou em heterozigose composta com H63D têm maior chance de apresentar carcinoma hepatocelular do que os portadores de outras mutações no gene HFE e pacientes sem mutação / Chronic liver disease related to iron overload may occur due to genetic or secondary causes. This study analyzed patients with chronic liver diseases and iron overload who were tested for HFE mutations from 2007 to 2009. Patients with C282Y/C282Y or C282Y/H63D mutations were diagnosed with HFE hereditary hemochromatosis (HFE-HH) and those with other HFE genotypes (C282Y/-, H63D/- or H63D/H63D) or individuals without HFE mutations (wild type) were designed as non-HFE iron overload. The aims of this study were: 1) to analyze and to establish correlations between phenotypic and genotypic aspects of individuals with chronic liver disease and with iron overload; 2) to charachterize the clinical manifestations, laboratory and histological findings consistent with the phenotype of hemochromatosis; 3) to verify associations between clinical manifestations and HFE mutations. One hundred and eight patients with chronic liver diseases and with iron overload, defined as transferrin saturation (TS) > 45% and serum ferritin levels > 350 ng/mL were included. Sixteen patients had HH-HFE and were compared with 92 patients with non-HFE iron overload group. The average of age at diagnosis was 46.69 years (16-77), 70.73% were Caucasians, 77.57% were male and 64.8% had hepatic cirrhosis. The proportion of hepatic cirrhosis was similar in both groups, nevertheless arthropathy, hepatocellular carcinoma, diabetes and osteoporosis were more frequent in the HFE-HH group (53,8% x 15,9%, 31,2% x 7,06%, 56,2% x 30%, 72,7% x 32,1%, respectively, p < 0,05). The HFE C282Y/C282Y or C282Y/ H63D genotypes had a higher chance to develop hepatocellular carcinoma (OR= 5.0, p= 0.032) when compared with the other HFE genotypes and with those wild type. The levels of TS, serum iron and ferritin were greater in HFE-HH group, as well as hepatic siderosis grade 3 and 4 (p= 0.026). TS was the biochemical marker of iron overload with the higher independent correlation with the presence of C282Y/C282Y and C282Y/H63D mutations. The frequency of risk factors for iron overload was not different between the groups, however, in HFE-HH group a greater number of patients without any risk factor was detected (p= 0.019). TS > 82% had a higher predictive negative value for diagnosing HFE-HH when compared to the levels of ferritin, serum iron, total iron binding capacity and transferrin. We concluded that the HFE-HH patients had a greater iron overload than patients with chronic liver diseases with non-HFE iron overload. TS > 82% had more accuracy to diagnose HFE-HH. The carriers of C282Y/C282Y or C282Y/H63D mutations had a higher probability to develop hepatocellular carcinoma, when compared to the patients with HFE genotypes and patients wild type

Yu, Wai Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 79-88). / Abstracts in English and Chinese. / Acknowledgements --- p.IV / Abstract --- p.V / 論文摘要 --- p.VII / Table of Contents --- p.IX / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cancer --- p.1 / Chapter 1.2 --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.2.1 --- Risk factors causing HCC --- p.3 / Chapter 1.2.2 --- Molecular mechanism of HCC --- p.7 / Chapter 1.2.3 --- Treatment of HCC --- p.8 / Chapter 1.3 --- Tetrandrine (Tet) - A Natural Compound Derived from Traditional Chinese Medicine (TCM) --- p.10 / Chapter 1.3.1 --- Traditional Chinese Medicine (TCM) --- p.10 / Chapter 1.3.2 --- Tetrandrine (Tet) --- p.12 / Chapter 1.4 --- Molecular View of Apoptosis --- p.14 / Chapter 1.4.1 --- Overview of apoptosis --- p.14 / Chapter 1.4.2 --- Caspase cascade --- p.15 / Chapter 1.4.3 --- Bcl-2 protein family --- p.18 / Chapter 1.4.4 --- The role of mitochondria in apoptosis --- p.20 / Chapter 1.5 --- Anti-cancer Agents Inducing Apoptosis Are New Targets --- p.22 / Chapter 1.6 --- Aim of Study --- p.26 / Chapter Chapter 2 --- Materials and Methods --- p.27 / Chapter 2.1 --- Cell Culture And Treatment --- p.27 / Chapter 2.1.1 --- Cell lines used --- p.27 / Chapter 2.1.2 --- Tetrandrine (Tet) --- p.28 / Chapter 2.1.3 --- Chemicals and reagents 2 --- p.83 / Chapter 2.1.4 --- Solution preparation --- p.29 / Chapter 2.1.5 --- Procedures --- p.30 / Chapter 2.2 --- Cell viability --- p.32 / Chapter 2.2.1 --- Chemicals and reagents . --- p.32 / Chapter 2.2.2 --- Solution preparation --- p.32 / Chapter 2.2.3 --- Procedures --- p.32 / Chapter 2.3 --- Apoptosis detection --- p.34 / Chapter 2.3.1 --- Chemicals and reagents --- p.34 / Chapter 2.3.2 --- Solution preparation --- p.35 / Chapter 2.3.3 --- Procedures --- p.36 / Chapter 2.4 --- Gene expression in tet-induced apoptotic cells --- p.39 / Chapter 2.4.1 --- Chemicals and reagents --- p.39 / Chapter 2.4.2 --- Solution preparation --- p.40 / Chapter 2.4.3 --- Procedures --- p.40 / Chapter 2.5 --- Protein expression in tet-induced apoptotic cells --- p.44 / Chapter 2.5.1 --- Chemicals and reagents --- p.44 / Chapter 2.5.2 --- Solution preparation --- p.45 / Chapter 2.5.3 --- Procedures --- p.48 / Chapter 2.6 --- Cell cycle analysis of tet-treated cells --- p.54 / Chapter 2.5.1 --- Chemicals and reagents --- p.54 / Chapter 2.5.2 --- Solution preparation --- p.54 / Chapter 2.5.3 --- Procedures --- p.54 / Chapter Chapter 3 --- Result --- p.56 / Chapter Chapter 4 --- Discussion --- p.70 / Chapter 4.1 --- Dose- and Time- Dependent Inhibitory Effects of Tet were found on HuH-7 And JHH-4 Cell Lines --- p.70 / Chapter 4.2 --- Tet Is More Selective Towards Liver Cancer Cells --- p.71 / Chapter 4.3 --- The Cell Death in HuH-7 Cells Induced by Tet is Mediated Through Apoptosis --- p.72 / Chapter 4.4 --- Hepatocellular Carcinoma (HCC)Tet Induces G1 Phase Cell Cycle Arrest as Part of Its Mechanism in Inducing Apoptosis in HuH-7 Cells --- p.73 / Chapter 4.5 --- Tet Could Probably Induce G1 Phase Cell Cycle Arrest in JHH-4 Cells --- p.75 / Chapter 4.6 --- "Tet-induced Apoptosis Involves the Intrinsic, Caspase-Dependent Pathway in Both the HuH-7 and JHH-4 Cell Lines" --- p.75 / Chapter 4.7 --- Proteins in Bcl-2 Family are Involved in the Inhibitory Mechanism of Tet --- p.77 / Reference --- p.79