Zhu, Ranxu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 123-142). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 6 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2009. / Härtill 4 uppsatser.

Diss. (sammanfattning) Göteborg : Göteborgs universtiet, 2008. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 92-109).

The distinction between cavernous haemangiomas of the liver (which are the second most common hepatic mass lesions) from malignant lesions, is often difficult. An incorrect diagnosis of cavernous haemangioma, in a patient with malignancy, may adversely influence the outcome of subsequent treatment in these patients, due to delay in therapy. Although previous studies have suggested that ⁹⁹ᵐTc erythrocyte blood pool scintigraphy is both highly sensitive and specific for haemangiomas, a basic flaw in all previous studies has been the small number of control patients studied. Bayesian analysis clearly shows that specificity for a test is dependant on the pre-test probability of the lesion being present. Thus all the studies done to date, may reflect an inappropriately high specificity for ⁹⁹ᵐTc scintigraphy, in diagnosing cavernous haemangiomas, because they have mainly studied patients with haemangiomas and relatively few patients with other lesions. This study was thus undertaken to clarify the true accuracy of this technique, in distinguishing haemangiomas from other hepatic mass lesions, by studying a large number of patients with haemangiomas and other hepatic mass lesions.

Yau Mei-sze. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 96-112). / Abstracts in English and Chinese. / Abstract / Abstract (Chinese Version) / Acknowledgements / List of Abbreviations / Table of Contents / List of Tables / List of Figures / Chapter Chapter 1 --- Introduction / Chapter Chapter 2 --- Literature Review / Chapter 2.1 --- Hepatocellular Carcinoma / Chapter 2.2 --- Epidermal Growth Factor Receptor / Chapter 2.2.1 --- Activation of Epidermal Growth Factor Receptor / Chapter 2.2.2 --- Epidermal Growth Factor Receptor Signaling Pathways / Chapter 2.2.3 --- Expression Level and Patient Survival / Chapter 2.2.4 --- Epidermal Growth Factor Receptor Activity and Tumor Cell Growth / Chapter 2.2.5 --- Epidermal Growth Factor Receptor Activity and Radiation / Chapter 2.3 --- "Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor, ZD1839" / Chapter 2.3.1 --- Tumor Cell Growth Control Activities of ZD1839 / Chapter 2.3.2 --- Factors Affecting the Tumor Cell Growth Control Activities of ZD1839 / Chapter 2.3.3 --- Radiosensitization Activities of ZD1839 / Chapter 2.3.4 --- Factors Affecting the Radiosensitization Activities of ZD1839 / Chapter 2.4 --- Study Objectives / Chapter Chapter 3 --- Materials and Methods / Chapter 3.1 --- ZD1839 / Chapter 3.2 --- Cell lines and Cell Culture / Chapter 3.3 --- Immunoblot Analysis / Chapter 3.3.1 --- Total Protein Extraction / Chapter 3.3.2 --- Protein Amount Determination / Chapter 3.3.3 --- Protein Separation / Chapter 3.3.4 --- Blotting / Chapter 3.3.5 --- Antibody Labeling / Chapter 3.3.6 --- Detection of Antibody Binding / Chapter 3.4 --- Cytotoxicity Assay / Chapter 3.5 --- Nucleotide sequence analysis / Chapter 3.5.1 --- Total RNA Extraction / Chapter 3.5.2 --- RNA Amount Determination / Chapter 3.5.3 --- Reverse Transcription - Polymerase Chain Reaction (RT-PCR) / Chapter 3.5.3.1 --- Reverse Transcription / Chapter 3.5.3.2 --- High Fidelity Polymerase Chain Reaction / Chapter 3.5.4 --- Purification of PCR Product / Chapter 3.5.5 --- Cycle Sequencing Reaction / Chapter 3.5.6 --- DNA Precipitation and Sequencing / Chapter 3.6 --- Clonogenic Assay / Chapter 3.7 --- Immunohistochemical Analysis / Chapter Chapter 4 --- Results / Chapter 4.1 --- Immunoblot Analysis / Chapter 4.2 --- Cytotoxicity Assay / Chapter 4.2.1 --- Effect of ZD 1839 on cell morphology / Chapter 4.2.2 --- Effect of ZD 1839 on cell growth / Chapter 4.3 --- Nucleotide sequence analysis / Chapter 4.3.1 --- RNA Concentration of HCC cells / Chapter 4.3.2 --- Sequencing of TK domain within EGFR / Chapter 4.3.3 --- Sequencing of TK domain within HER2 / Chapter 4.4 --- Clonogenic assay / Chapter 4.4.1 --- Effects of ZD 1839 pre-treatment on radiation response / Chapter 4.4.2 --- Effects of ZD 1839 continuous treatment on radiation response / Chapter 4.5 --- Immunohistochemical Analysis / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- Important Findings / Chapter 5.2 --- EGFR Expression of HCC Cells / Chapter 5.3 --- Cytotoxicity of ZD1839 on HCC Cell Lines / Chapter 5.4 --- Factors Affecting the Cytotoxicity of ZD1839 / Chapter 5.4.1 --- Effect of EGFR Expression on ZD1839 Cytotoxicity / Chapter 5.4.2 --- Effect of EGFR Mutations on ZD 1839 Cytotoxicity / Chapter 5.4.3 --- Effect of HER2 Expression on ZD1839 Cytotoxicity / Chapter 5.4.4 --- Effect of HER2 Mutations on ZD 1839 Cytotoxicity / Chapter 5.5 --- Radiation Response ofHCC Cell Lines upon ZD1839 Treatment / Chapter 5.6 --- Factors Affecting Radiation Response of ZD1839-treated HCC Cell Lines / Chapter 5.6.1 --- Effect of Growth Arrest on Radiation Response of HCC Cell Lines / Chapter 5.6.2 --- Other Factors Affecting Radiation Response of HCC Cell Lines / Chapter Chapter 6 --- Conclusion / References

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

A infecção pelo vírus da hepatite C (HCV) pode evoluir gradualmente para hepatite crônica, cirrose e carcinoma hepatocelular (CHC) ao longo de 20 a 30 anos [1-3]. O carcinoma hepatocelular é a quinta neoplasia mais comum em todo o mundo, sendo responsável por mais de 600.000 mortes por ano. Atualmente, cerca de 170 milhões de indivíduos estão infectados pelo HCV, o que corresponde a aproximadamente 3% da população do mundo. A hepatocarcinogênese é um processo complexo, com várias etapas que envolvem alterações genéticas e epigenéticas. Estudos relatam que substituições de aminoácidos (aa) na posição 70 e 91 da região core do HCV podem estar relacionados ao desenvolvimento de CHC. O conhecimento sobre os mecanismos da carcinogênese que envolvem o HCV são importantes para a descoberta de biomarcadores e potenciais alvos terapêuticos do CHC. Neste estudo, foram analisados os genótipos virais e a presença de mutações na região core do HCV, em 94 pacientes com CHC e em 79 pacientes cirróticos (sem CHC). As sequências da região core do HCV foram obtidas pelo método de sequenciamento populacional baseado na metodologia de Sanger. Características demográficas, bioquímicas e sorológicas também foram avaliadas. A idade dos pacientes com CHC foi significativamente maior do que a dos pacientes sem CHC (63 vs 60,5 anos, P=0,025). Uma proporção maior de homens foi observada no grupo CHC (64,4% vs 54%, P=0,329), qual apresentou nível de alfafetoproteína significativamente mais elevado (P=0,003) e menores níveis de albumina em relação ao grupo sem CHC (P=0,012). Elevada variabilidade genética do HCV foi observada. Ao todo, quatro genótipos e sete subtipos foram encontrados. O subtipo 1 b foi o mais frequente em ambos os grupos. Os subtipos encontrados no grupo CHC e cirróticos foram, 1a (13,6%), 1 b (45,7%), 3a (28,8%), 2b (6,8%), 2a (1,7%), 2c (1,7%), 5a (1,7%); e 1a (30%), 1 b (44%), 3a (22%), 2b (2%) e 5a (2%). As mutações R70Q e UC91 M foram observadas principalmente no HCV genótipo 1 b. Não houve associação entre mutações nas posições 70 e 91 na região core do HCV e o desenvolvimento de CHC / Hepatitis C virus (HCV) infection is often persistent and gradually advances from chronic hepatitis (CH) to liver cirrhosis, and hepatocellular carcinoma (HCC) over 20 to 30 years [1-3]. Worldwide, hepatocellular carcinoma is the fifth most common neoplasm and is responsible for more than 600,000 deaths annually due to very poor prognosis. There are about 170 million individuais infected with HCV, corresponding to approximately 3% of world population. Hepatocarcinogenesis is a complex process involving genetic and epigenetic modifications. Studies have reported that amino acid substitutions (a a) at position 70 and 91 of HCV core region may be related to development of HCC. Understanding the pathogenesis of HCV-induced hepatocarcinogenesis is important to identify novel biomarkers and potential therapeutic targets. In this study, the viral genotypes and the presence of mutations in HCV core region were analyzed in 94 patients with HCC, and also in 79 cirrhotic patients (without HCC). HCV core sequences were obtained using population sequencing based on Sanger method. Demographic, biochemical and serological characteristics were also evaluated. The age of patients with HCC were significantly higher than in patients without HCC (63 vs. 60.5 years, P=0.025). High proportion of men was observed in HCC group (64.4% vs 54%, P=0.329). Alpha-fetoprotein levei was significantly higher in HCC group compared to cirrhotic group (P=0.003), and low rates of albumin was observed in cirrhotic group (P=0.012). High genetic variability of HCV was observed, in HCC group, however genotype 1 b was the most common in both groups. Other genotypes were found in HCC group: 1a (13.6%), 1 b (45.7%), 3a (28.8%) 2b (6.8%), 2a (1.7%), 2c (1.7%) and 5a (1.7%). In cirrhotic group was found genotypes 1 a (30%), 1 b (44%), 3a (22%), 2b (2%) and 5a (2%). Mutations R70Q and LlC91 M were mainly observed in individuais infected with HCV genotype 1 b. In the present study, no association between mutations at positions 70 and 91 of HCV