Mit der palliativen transarteriellen Chemoembolisation (TACE) läßt sich eine Verkleinerung von malignen Lebertumoren und eine Verlängerung des Überlebens erreichen. Im Verlauf des Therapiezyklus diente die Phosphor-Magnetresonanzspektroskopie mittels Chemical-Shift-Imaging einem nichtinvasiven Einblick in den zellulären Energie- und Membranstoffwechsel. Eine Differenzierung zwischen primären und sekundären Lebertumoren war anhand der 31P-Spektren nicht möglich. Posttherapeutisch zeigten die Patienten einen Anstieg des anorganischen Phosphats bei Abfall des ß-NTP-Signals, die sich während des sechswöchigen Intervalls vor der nächsten Intervention wieder normalisierten. Therapieansprechen und -versagen zeigten während des Therapiezyklus unterschiedliche spektroskopische Quotientenverläufe. Eine deutliche Volumenreduktion als auch eine homogene und dauerhafte Lipiodoleinlagerung im Tumor gehen zuverlässig mit einem spektroskopischen Therapieansprechen einher. Dies zeigte sich bei der ersten Patientengruppe während der Untersuchung direkt nach der Embolisation und bei der zweiten Gruppe erst vor der nächsten Intervention. Therapieversagen, Tumorprogression und geringe Lipiodoleinlagerung bzw. rasche Entspeicherung im Verlauf ließen sich spektroskopisch nach-weisen. Schwierig bleibt die Zuordnung der Patienten mit uneinheitlichen Quotienten-verläufen. Die MRS kann früher als die klinische Routinediagnostik eine Aussage über das Therapieansprechen treffen. Für den Patienten ergibt sich die Vermeidung einer ineffektiven Therapie. Bei nachgewiesenem Ansprechen hingegen profitiert der Patient durch die wiederholte Anwendung der TACE. Der hohe personelle, technische und zeitliche Aufwand für die Phosphorspektroskopie verhindert bisher den klinischen Routineeinsatz dieses Verfahrens. Im Rahmen der klinischen Forschung, insbe-sondere in der onkologischen Therapiekontrolle, hat die MRS als nichtinvasive Methode heute einen großen Stellenwert. / Using palliative transarterial chemoembolization (TACE) a reduction of malignant liver neoplasms and a prolongation of survival time can be achieved. During the course of therapy phosphorous magnetic resonance spectroscopy by means of chemical-shift-imaging was used for noninvasive examination of the cellular metabolism of energy and membranes. Differentiation between primary and secondary liver tumors was not feasible using 31P-spectra. After therapy patients had shown increased inorganic phosphate signal and reduction of the ß-NTP-signals, which normalized during six week intervall before next intervention. Response and non-response to therapy showed different courses of spectroscopic quotients during therapy cycle. Prominent reduction of volume as well as homogeneous and continous retention of lipiodol within the tumor are reliable combined with spectroscopic response to therapy. The first group of patients showed these signs at the examination immediately after embolization whereas patients of the second group showed these signs not before next intervention. Failure of therapy, progression of tumor and slight retention of lipiodol or rapid elemination during course of therapy were detectable by spectroscopy. Categorization of patients with non-uniform courses of quotients remains difficult. Magnetic resonance spectroscopy is able to determine response to therapy earlier than standard diagnostic methods. As a result ineffective therapy is avoided for the patient. On the other hand the patient profits from repeated administration of TACE. Great expense of personnel, equipment and time so far prevents clinical use of phosphorous spectroscopy as a matter of routine. In conjunction with clinical research MRS is an important non-invasive method especially for oncological therapy monitoring.

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

INTRODUÇÃO: Na maioria dos casos, o carcinoma hepatocelular (CHC) acomete pacientes com cirrose hepática. O algoritmo do Barcelona Clinic Liver Cancer group (BCLC) considera função hepática, características tumorais e, estado geral do paciente para definir o tratamento. Entretanto, a aplicabilidade de um algoritmo terapêutico nem sempre é possível na prática clínica. Este estudo buscou avaliar a aderência às recomendações do BCLC para tratamento dos pacientes com CHC e analisar o impacto sobre a sobrevida nos diferentes estádios. MÉTODOS: Este estudo incluiu todos os pacientes encaminhados para o Instituto do Câncer do Estado de São Paulo para tratamento do CHC entre 2010 e 2012. Os pacientes (n = 364) foram classificados de acordo com as recomendações do BCLC. Se a terapia proposta não foi aplicada, o caso foi considerado como não aderente e as causas foram investigadas. A curva de sobrevida global foi estimada pelo método de sobrevida de Kaplan-Meier e comparada pela regressão de Cox. RESULTADOS: A porcentagem de aderência às recomendações do BCLC foi de 52% e variou entre os estádios: BCLC 0/A, 44%; BCLC B, 78%; BCLC C, 35%; e BCLC D, 67%. A taxa de sobrevida global, em um, dois e três anos, foi de 63, 45 e 33%, respectivamente. Nos pacientes aderentes do estádio BCLC 0/A, a sobrevida global foi significantemente melhor se comparada aos pacientes não aderentes (razão de risco [RR] = 0,19, intervalo de confiança [IC] 95%: 0,09-0,42; p < 0,001). Nos pacientes do estádio BCLC D, a taxa de sobrevida global foi pior em pacientes aderentes comparada aos pacientes não aderentes (RR = 4,0; IC 95%: 1,67-9,88; p < 0,001). Nenhuma diferença foi observada em pacientes do estádio BCLC B ou C classificados como aderentes ou não aderentes. CONCLUSÕES: A porcentagem de aderência às recomendações do BCLC na prática clínica é baixa e varia entre os estádios. A não aderência está associada a pior prognóstico, particularmente em pacientes com estádio precoce / INTRODUCTION: In most cases, hepatocellular carcinoma (HCC) affects patients with liver cirrhosis. Barcelona Clinic Liver Cancer group (BCLC) algorithm takes into consideration liver function, tumor variables and patients general status to guide therapy. However, the application of a therapeutic algorithm is not always feasible in clinical practice. The aim of this study was to assess the adherence of newly diagnosed hepatocellular carcinoma patients to the BCLC treatment guidelines, as well as examine the impact on survival in different stages. METHODS: This study included all patients referred to Instituto do Câncer do Estado de São Paulo for HCC therapy between 2010 and 2012. Patients (n = 364) were classified according to BCLC stage. If the proposed HCC therapy could not be applied, the case was considered to represent deviations from the recommended BCLC guideline. Causes of treatment deviations were investigated. The overall survival (OS) curves were estimated by the Kaplan-Meier survival method and compared by Cox regression. RESULTS: The overall percentage of adherence to BCLC guidelines was 52 % and varied among the disease stages: BCLC 0/A, 44 %; BCLC B, 78 %; BCLC C, 35 %; and BCLC D, 67 %. One-, two-, and three-year OS rates were 63, 45, and 33 %, respectively. In BCLC 0/A, adherent patients presented a significantly better OS compared to non-adherent patients (hazard ratio [HR] = 0.19, 95% confidence interval [CI]: 0.09-0.42; p < 0.001). In BCLC D, the OS rate was worse in adherent patients compared to non-adherent patients (HR = 4.0, 95% CI: 1.67-9.88; p < 0.001), whereas no differences were observed in BCLC stage B or C. CONCLUSIONS: The percentage of adherence to BCLC recommendations in clinical practice is low and varies among the clinical stages. Non-adherence is associated with a worse prognosis, particularly in early stage disease

Grace S.N. Lau. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 335-362). / List of Abbreviations --- p.i / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction and Study Objectives / Chapter 1.1 --- Metabolic activation - role in drug toxicity and carcinogenesis --- p.5 / Chapter 1.2 --- Hepatocellular carcinoma --- p.12 / Chapter 1.2.1 --- Epidemiology --- p.12 / Chapter 1.2.2 --- Aetiological factors --- p.17 / Chapter 1.2.2.1 --- Hepatitis B virus infection --- p.17 / Chapter 1.2.2.2 --- Cirrhosis --- p.24 / Chapter 1.2.2.3 --- Aflatoxins --- p.25 / Chapter 1.2.2.4 --- Other factors --- p.26 / Chapter 1.2.2.5 --- Summary --- p.29 / Chapter 1.3 --- Study objectives --- p.30 / Chapter Chapter 2 --- The Metabolism of Paracetamol in Healthy Subjects andin Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1. --- History of paracetamol --- p.34 / Chapter 2.1.2 --- Pharmacology of paracetamol --- p.37 / Chapter 2.1.3 --- "Absorption, Distribution, Metabolism and Excretion" --- p.38 / Chapter 2.1.3.1 --- Absorption --- p.38 / Chapter 2.1.3.2 --- Distribution --- p.41 / Chapter 2.1.3.3 --- Metabolism --- p.42 / Chapter 2.1.3.4 --- Excretion --- p.57 / Chapter 2.1.4 --- Toxicity and Overdosage --- p.59 / Chapter 2.2 --- Estimation of paracetamol and its metabolites in plasma and urine by high performance liquid chromatography --- p.72 / Chapter 2.2.1 --- Introduction --- p.72 / Chapter 2.2.2 --- Analytical method --- p.76 / Chapter 2.2.2.1 --- Materials --- p.76 / Chapter 2.2.2.2 --- Instrumentation --- p.77 / Chapter 2.2.2.3 --- Collection and storage of samples --- p.79 / Chapter 2.2.2.4 --- Chromatographic conditions --- p.79 / Chapter 2.2.3 --- Urine assay --- p.79 / Chapter 2.2.3.1 --- Preparation of standards and test samples for urine assay --- p.79 / Chapter 2.2.3.2 --- Calculation of results for urine assay --- p.80 / Chapter 2.2.3.3 --- Results of urine assay --- p.81 / Chapter 2.2.3.4 --- Validation of urine assay --- p.81 / Chapter 2.2.4 --- Plasma assay --- p.83 / Chapter 2.2.4.1 --- Preparation of standards and test samples for plasma assay --- p.83 / Chapter 2.2.4.2 --- Calculation of results for plasma assay --- p.91 / Chapter 2.2.4.3 --- Results of plasma assay --- p.91 / Chapter 2.2.4.4 --- Validation of plasma assay --- p.93 / Chapter 2.2.5 --- Summary --- p.99 / Chapter 2.3 --- The pharmacokinetics of paracetamol in healthy subjects --- p.103 / Chapter 2.3.1 --- Introduction --- p.103 / Chapter 2.3.2 --- Study protocol --- p.103 / Chapter 2.3.3 --- Methods --- p.103 / Chapter 2.3.3.1 --- Subjects --- p.103 / Chapter 2.3.3.2 --- Drug administration and sampling --- p.104 / Chapter 2.3.3.3 --- Drug analysis --- p.108 / Chapter 2.3.3.4 --- Calculations --- p.108 / Chapter 2.3.4 --- Pharmacokinetic analysis --- p.109 / Chapter 2.3.5 --- Statistical analysis --- p.113 / Chapter 2.3.6 --- Results --- p.114 / Chapter 2.3.6.1 --- Plasma Results --- p.114 / Chapter 2.3.6.2 --- Urine Results --- p.118 / Chapter 2.3.6.3 --- Pharmacokinetic Results --- p.125 / Chapter 2.3.6.4 --- Statistical Results --- p.134 / Chapter 2.3.7 --- Discussion --- p.145 / Chapter 2.4 --- "The pharmacokinetics of paracetamol in healthy subjects, patients with liver disease and hepatocellular carcinoma" --- p.155 / Chapter 2.4.1 --- Introduction --- p.155 / Chapter 2.4.2 --- Study protocol --- p.156 / Chapter 2.4.3 --- Methods --- p.156 / Chapter 2.4.3.1 --- Subjects --- p.156 / Chapter 2.4.3.2 --- Drug administration and sampling --- p.157 / Chapter 2.4.3.3 --- Drug analysis --- p.160 / Chapter 2.4.3.4 --- Calculations --- p.160 / Chapter 2.4.4 --- Pharmacokinetic analysis --- p.161 / Chapter 2.4.6 --- Results --- p.162 / Chapter 2.4.6.1 --- Plasma Results --- p.162 / Chapter 2.4.6.2 --- Urine Results --- p.162 / Chapter 2.4.6.3 --- Pharmacokinetic Results --- p.179 / Chapter 2.4.7 --- Discussion --- p.194 / Chapter 2.4.8 --- Summary --- p.203 / Chapter Chapter 3 --- Metabolic Activation of Aflatoxin B1 in Healthy Subjects and in Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 3.1 --- General introduction --- p.206 / Chapter 3.1.1 --- Chemical structures and properties --- p.207 / Chapter 3.1.2 --- Contamination of food by aflatoxins --- p.209 / Chapter 3.1.3 --- Metabolism of aflatoxins --- p.210 / Chapter 3.1.4 --- Human diseases possibly related to exposure to aflatoxins --- p.226 / Chapter 3.1.4.1 --- Acute aflatoxicosis --- p.226 / Chapter 3.1.4.2 --- Reye's syndrome --- p.227 / Chapter 3.1.4.3 --- Kwashiorkor --- p.228 / Chapter 3.1.4.4 --- Impaired immune function --- p.229 / Chapter 3.1.4.5 --- Hepatocellular carcinoma --- p.230 / Chapter 3.1.5 --- Biochemical and molecular epidemiology of aflatoxins --- p.232 / Chapter 3.2 --- Development of an ELISA method to monitor AFB1 exposure in human serum --- p.237 / Chapter 3.2.1 --- Introduction --- p.237 / Chapter 3.2.2 --- Preparation of all the components necessary for analysing AFB1-albumin adducts by ELISA --- p.243 / Chapter 3.2.2.1 --- Materials --- p.243 / Chapter 3.2.2.2 --- Preparation of rabbit AFB1 antiserum --- p.244 / Chapter 3.2.2.3 --- Preparation of the rat monoclonal antibody --- p.244 / Chapter 3.2.2.4 --- Concentration of cell culture supernatant by ammonium sulphate precipitation --- p.246 / Chapter 3.2.2.5 --- Preparation of the BSA-AFB1 conjugate --- p.248 / Chapter 3.2.2.6 --- Preparation of the immunoaffinity gel --- p.250 / Chapter 3.2.2.7 --- Preparation of the ELISA plates --- p.251 / Chapter 3.2.3 --- General procedures used in the analysis of AFB1- albumin adducts --- p.252 / Chapter 3.2.3.1 --- Competitive ELISA binding assay --- p.253 / Chapter 3.2.3.2 --- Sep-pak C18 cartridge --- p.254 / Chapter 3.2.3.3 --- Immunoaffinity column --- p.255 / Chapter 3.2.3.4 --- Evaporation process --- p.255 / Chapter 3.2.3.5 --- HPLC --- p.256 / Chapter 3.2.3.6 --- Radioactive counting --- p.256 / Chapter 3.2.3.7 --- Albumin isolation --- p.257 / Chapter 3.2.3.8 --- Digestion of albumin --- p.257 / Chapter 3.2.3.9 --- Animal procedures --- p.258 / Chapter 3.2.4 --- Validations --- p.259 / Chapter 3.2.4.1 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.259 / Chapter 3 2.4.2 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.259 / Chapter 3 2.4.3 --- Elution characteristics and capacity of the immunoaffinity column --- p.261 / Chapter 3.2.4.4 --- Comparison of immunoaffinity gels prepared with different affinity gels --- p.261 / Chapter 3.2.4.5 --- Immunoaffinity column experiment of AFB1-lysine --- p.263 / Chapter 3.2.4.6 --- HPLC Analysis of fractions from immunoaffinity column --- p.263 / Chapter 3.2.4.8 --- HPLC analysis of fractions from Sep- Pak C18 cartridge --- p.264 / Chapter 3.2.4.9 --- Digestion of serum albumin by proteinase K --- p.264 / Chapter 3.2.4.10 --- Effect of ethanol in samples to be loaded onto Sep-Pak C18 cartridge --- p.265 / Chapter 3.2.4.11 --- Effect of drying in a vacuum concentrator on recovery of radioactivity of 3H-AFB1 --- p.266 / Chapter 3.2.4.12 --- Evaluation of the overall procedure for the analysis of serum albumin adducts of AFB1 --- p.267 / Chapter 3.2.4.13 --- HPLC analysis of samples obtained after digestion and all clean-up procedures --- p.268 / Chapter 3.2.5 --- Results and discussion --- p.268 / Chapter 3.2.5.1 --- BSA-AFB1 conjugate --- p.268 / Chapter 3.2.5.2 --- Treatment of experimental animals with 3H-AFB1 --- p.270 / Chapter 3.2.5.3 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.272 / Chapter 3.2.5.4 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.275 / Chapter 3.2.5.5 --- Sep-Pak C18 cartridge - elution characteristics and capacity --- p.279 / Chapter 3.2.5.6 --- Elution characteristics of immunoaffinity columns --- p.282 / Chapter 3.2.5.7 --- Immunoaffinity column experiment of AFB1-lysine --- p.290 / Chapter 3.2.5.8 --- Digestion of serum albumin by proteinase K --- p.295 / Chapter 3.2.5.9 --- Effect of ethanol in samples to be applied onto Sep-Pak C18 cartridges --- p.297 / Chapter 3.2.5.10 --- Recovery of radioactivity after dryingin a vacuum concentrator --- p.300 / Chapter 3.2.5.11 --- Recovery of the overall clean-up procedure for the analysis of serum albumin adducts of AFB1 --- p.300 / Chapter 3.2.5.12 --- HPLC analysis of samples obtained after all clean-up procedures --- p.305 / Chapter 3.2.5.13 --- The use of rabbit anti-AFB1 anti-serum and rat anti-AFB1 monoclonal antibody --- p.308 / Chapter 3.2.6 --- Summary --- p.309 / Chapter 3.3 --- Monitoring of AFBralbumin adducts in plasma of patients with liver disease and hepatocellular carcinoma --- p.311 / Chapter 3.3.1 --- Introduction --- p.311 / Chapter 3.3.2 --- Material and methods --- p.314 / Chapter 3.3.2.1 --- Subject --- p.314 / Chapter 3.3.2.2 --- Sample collections --- p.315 / Chapter 3.3.2.4 --- Assay for AFB1-albumin adducts --- p.315 / Chapter 3.3.2.5 --- Statistical analysis --- p.318 / Chapter 3.3.3 --- Results and discussion --- p.318 / Chapter Chapter 4 --- Summary and Ideas for Further Studies --- p.330 / Acknowledgements --- p.333 / References --- p.335 / Appendices --- p.364

by Koo Po Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 120-130). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abstract (Chinese version) --- p.iv / Content --- p.vii / List of Abbreviations --- p.xi / List of Figures and Tables --- p.xiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Saponins --- p.1 / Chapter 1.2 --- Structure of Saponin --- p.2 / Chapter 1.2.1 --- Triterpene Class --- p.2 / Chapter 1.2.2 --- Steroid Class --- p.3 / Chapter 1.2.2.1 --- Spirostanol Glycoside --- p.4 / Chapter 1.2.2.2 --- Furostanol Glycoside --- p.4 / Chapter 1.2.3 --- Steroid Alkaloid Class --- p.5 / Chapter 1.3 --- Steroidal Saponin as Anti-Tumor Drug --- p.5 / Chapter 1.4 --- Possible Anti-Tumor Action Mechanisms of Steroid Saponin --- p.6 / Chapter 1.4.1 --- Direct Cytotoxic and Growth Inhibitory Effects --- p.7 / Chapter 1.4.2 --- Immune-Modulatory Effects --- p.8 / Chapter 1.5 --- Possible Anti-Carcinogenicity Action Mechanism of Saponin --- p.9 / Chapter 1.5.1 --- Saponin Binding to Bile Acids --- p.9 / Chapter 1.6 --- Saponin as Cardioactive Drug --- p.9 / Chapter 1.7 --- Liver Cancer --- p.10 / Chapter 1.7.1 --- Prevalence of Hepatocellular Carcinoma (HCC) --- p.11 / Chapter 1.8 --- Coronary Heart Disease (CHD) --- p.12 / Chapter 1.8.1 --- Prevalence and Risk Factors of CHD --- p.12 / Chapter 1.9 --- Diosgenin --- p.14 / Chapter 1.10 --- Hong Kong (HK) Products --- p.15 / Chapter 1.10.1 --- HK-18 (Polyphyllin D) --- p.15 / Chapter 1.11 --- DI AO XIN XUE KANG (DI AO) --- p.17 / Chapter 1.12 --- Aims of My Project --- p.20 / Chapter 1.12.1 --- In Vitro Study of the Effect of HK-18 on Human Hepatocellular Carcinoma Cell Line (HepG2) --- p.21 / Chapter 1.12.2 --- In Vivo Study of the Effect of HK-18 by Human Liver Tumor HepG2 Cells-Bearing Nude Mice Model --- p.21 / Chapter 1.12.3 --- In Vitro Study of the Effect of HK-18 on Multidrug- Resistant Human Hepatocellular Carcinoma Cell Line (R-HepG2) --- p.22 / Chapter 1.12.4 --- Myocardial Ischemia-Reperfusion (IR) Injury in Isolated- Perfused Rat Heart Model --- p.23 / Chapter 1.12.5 --- Effect of DI AO Pretreatment on Global IR Injury --- p.26 / Chapter 1.12.6 --- Effect of DI AO Pretreatment on Isoproterenol-Induced Myocardial Injury in Rats --- p.26 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Cell Lines and Culture Medium / Chapter 2.1.1.1 --- Cell Lines --- p.28 / Chapter 2.1.1.2 --- Culture Medium --- p.29 / Chapter 2.1.2 --- Chemicals --- p.30 / Chapter 2.1.3 --- Buffers and Reagents --- p.31 / Chapter 2.2 --- Methods / Chapter 2.2.1 --- In Vitro Studies --- p.33 / Chapter 2.2.1.1 --- In Vitro Cytotoxicity --- p.33 / Chapter 2.2.1.2 --- Cell Cycle Analysis by Flow Cytometry --- p.34 / Chapter 2.2.1.3 --- Maintenance of P-glycoprotein in R-HepG2 cells by Doxorubicin and HK-18 --- p.35 / Chapter 2.2.1.4 --- Assessment of DNA Fragmentation --- p.36 / Chapter 2.2.2 --- In Vivo Assessment of the Anti-Tumor Activity of HK-18 --- p.37 / Chapter 2.2.2.1 --- Animals and Tumor Inoculation --- p.37 / Chapter 2.2.2.2 --- Drug Administration --- p.37 / Chapter 2.2.2.3 --- Assessment of the Tumor Size and Tumor Weight --- p.38 / Chapter 2.2.2.4 --- Plasma Preparation --- p.38 / Chapter 2.2.2.5 --- Measurement of the Plasma Enzyme Activity --- p.39 / Chapter 2.2.3 --- Isoproterenol (ISO)-Induced Myocardial Injury (Rat Model) --- p.40 / Chapter 2.2.3.1 --- Animals --- p.40 / Chapter 2.2.3.2 --- Drug Preparations --- p.40 / Chapter 2.2.3.3 --- Animal Treatment --- p.41 / Chapter 2.2.3.4 --- Preparation of Myocardial Tissue Homogenate --- p.41 / Chapter 2.2.3.5 --- Preparation of Cytosolic Fraction of Heart Homogenates --- p.42 / Chapter 2.2.3.6 --- Myocardial Antioxidant Enzyme Activity --- p.42 / Chapter 2.2.3.6.1 --- Glutathione Reductase (GRD) --- p.42 / Chapter 2.2.3.6.2 --- Glutathione S-Transferases (GST) --- p.43 / Chapter 2.2.3.7 --- Myocardial Antioxidant Capacity --- p.43 / Chapter 2.2.3.7.1 --- Myocardial Malondialdehyde (MDA) Content --- p.43 / Chapter 2.2.3.7.2 --- Myocardial Thiol Content --- p.44 / Chapter 2.2.3.7.3 --- Tert-Butylhydroperoxide (tBHP)-Induced Thiol Depletion --- p.45 / Chapter 2.2.3.7.4 --- TBHP-Induced Thiobarbituric Acid-Reactive Substances (TBARS) Formation --- p.45 / Chapter 2.2.4 --- Myocardial Ischemia-Reperfusion (IR) Injury --- p.46 / Chapter 2.2.4.1 --- Langendorff Isolated Perfused Rat Heart --- p.46 / Chapter 2.2.4.1.1 --- Preparation of Perfusion Buffer --- p.46 / Chapter 2.2.4.1.2 --- Preparation of Isolated Rat Heart --- p.47 / Chapter 2.2.4.1.3 --- Myocardial Global Ischemia-Reperfusion Injury --- p.49 / Chapter 2.2.4.1.4 --- Contractile Force Recovery --- p.49 / Chapter 2.2.5 --- Statistical Analysis --- p.50 / Chapter Chapter 3 --- Study of HK-18 on Anti-Tumor Effect / Chapter 3.1 --- In Vitro Study of HK-18 on Human Hepatoma Carcinoma Cell Line (HepG2) --- p.51 / Chapter 3.1.1 --- The Effect of HK-18 on Cell Proliferation of HepG2 Cells by MTT Assay --- p.52 / Chapter 3.1.2 --- DNA Fragmentation Assay --- p.54 / Chapter 3.1.3 --- The Effect of HK-18 on Cell Cycle Phase Distribution --- p.57 / Chapter 3.2 --- In Vivo Study of HK-18 on HepG2-Inoculated Nude Mice --- p.61 / Chapter 3.2.1 --- Assessment of the Anti-Tumor Activity of HK-18 --- p.61 / Chapter 3.2.2 --- The Effect of HK-18 Towards Heart Tissue --- p.65 / Chapter 3.2.3 --- In Vitro Study of HK-18 on Multidrug Resistant Cell Line (R-HepG2) --- p.68 / Chapter 3.2.4 --- The Comparison of the Cytotoxicity of DOX on the Parental Cells and Resistant Cells of HepG2 --- p.69 / Chapter 3.2.5 --- The Effect of HK-18 on Cell Proliferation of R-HepG2 Cells by MTT Assay --- p.72 / Chapter 3.2.6 --- DNA Fragmentation Assay --- p.74 / Chapter 3.2.7 --- The Effect of HK-18 on Cell Cycle Phase Distribution --- p.77 / Chapter 3.2.8 --- The Relationship Between HK-18 and P-glycoprotein --- p.80 / Chapter Chapter 4 --- Study of the Cardioprotective Effect of DI AO / Chapter 4.1 --- Myocardial Ischemia-Reperfusion (IR) Injury in Isolated- Perfused Rat Heart --- p.82 / Chapter 4.1.1 --- Time Course of Global Ischemia-Reperfusion-Induced LDH Leakage --- p.82 / Chapter 4.1.2 --- Effect of DI AO Pretreatment on Global IR Injury --- p.85 / Chapter 4.1.2.1 --- LDH Leakage --- p.85 / Chapter 4.1.2.2 --- Contractile Force --- p.87 / Chapter 4.2 --- Isoproterenol-Induced Myocardial Injury in Rats --- p.89 / Chapter 4.2.1 --- Effect of DI AO Pretreatment --- p.89 / Chapter 4.2.2 --- Alternations in the Activity of Myocardial Antioxidant Enzymes --- p.91 / Chapter 4.2.3 --- Alternations in Myocardial Antioxidant Capacity --- p.94 / Chapter Chapter 5 --- Discussion / Chapter 5.1 --- The Significance of the Study of Saponin in the Treatment of Liver Cancer and Heart Injury --- p.96 / Chapter 5.2 --- Effect of HK-18 on Human Hepatocellular Carcinoma Cell --- p.101 / Chapter 5.3 --- Mechanism Study of Anti-Tumor Effect of HK-18 --- p.102 / Chapter 5.4 --- Cytotoxicity of HK-18 Toward Normal Tissue --- p.105 / Chapter 5.5 --- Effect of HK-18 on Multidrug Resistant Human Hepatocellular Carcinoma / Chapter 5.6 --- Protective Effect of DI AO Against Isoproterenol (ISO)- Induced Myocardial Injury --- p.110 / Chapter 5.7 --- Cardioprotective Effect of DI AO Against Ischemia- Reperfusion (IR) Injury --- p.111 / Chapter 5.8 --- Effect of DI AO Pretreatment on Myocardial Antioxidant Enzymes Activities and Antioxidant Capacity --- p.113 / Chapter 5.9 --- Conclusion and Future Prospect --- p.117 / Chapter Chapter 6 --- References --- p.121

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

INTRODUÇÃO: Na maioria dos casos, o carcinoma hepatocelular (CHC) acomete pacientes com cirrose hepática. O algoritmo do Barcelona Clinic Liver Cancer group (BCLC) considera função hepática, características tumorais e, estado geral do paciente para definir o tratamento. Entretanto, a aplicabilidade de um algoritmo terapêutico nem sempre é possível na prática clínica. Este estudo buscou avaliar a aderência às recomendações do BCLC para tratamento dos pacientes com CHC e analisar o impacto sobre a sobrevida nos diferentes estádios. MÉTODOS: Este estudo incluiu todos os pacientes encaminhados para o Instituto do Câncer do Estado de São Paulo para tratamento do CHC entre 2010 e 2012. Os pacientes (n = 364) foram classificados de acordo com as recomendações do BCLC. Se a terapia proposta não foi aplicada, o caso foi considerado como não aderente e as causas foram investigadas. A curva de sobrevida global foi estimada pelo método de sobrevida de Kaplan-Meier e comparada pela regressão de Cox. RESULTADOS: A porcentagem de aderência às recomendações do BCLC foi de 52% e variou entre os estádios: BCLC 0/A, 44%; BCLC B, 78%; BCLC C, 35%; e BCLC D, 67%. A taxa de sobrevida global, em um, dois e três anos, foi de 63, 45 e 33%, respectivamente. Nos pacientes aderentes do estádio BCLC 0/A, a sobrevida global foi significantemente melhor se comparada aos pacientes não aderentes (razão de risco [RR] = 0,19, intervalo de confiança [IC] 95%: 0,09-0,42; p < 0,001). Nos pacientes do estádio BCLC D, a taxa de sobrevida global foi pior em pacientes aderentes comparada aos pacientes não aderentes (RR = 4,0; IC 95%: 1,67-9,88; p < 0,001). Nenhuma diferença foi observada em pacientes do estádio BCLC B ou C classificados como aderentes ou não aderentes. CONCLUSÕES: A porcentagem de aderência às recomendações do BCLC na prática clínica é baixa e varia entre os estádios. A não aderência está associada a pior prognóstico, particularmente em pacientes com estádio precoce / INTRODUCTION: In most cases, hepatocellular carcinoma (HCC) affects patients with liver cirrhosis. Barcelona Clinic Liver Cancer group (BCLC) algorithm takes into consideration liver function, tumor variables and patients general status to guide therapy. However, the application of a therapeutic algorithm is not always feasible in clinical practice. The aim of this study was to assess the adherence of newly diagnosed hepatocellular carcinoma patients to the BCLC treatment guidelines, as well as examine the impact on survival in different stages. METHODS: This study included all patients referred to Instituto do Câncer do Estado de São Paulo for HCC therapy between 2010 and 2012. Patients (n = 364) were classified according to BCLC stage. If the proposed HCC therapy could not be applied, the case was considered to represent deviations from the recommended BCLC guideline. Causes of treatment deviations were investigated. The overall survival (OS) curves were estimated by the Kaplan-Meier survival method and compared by Cox regression. RESULTS: The overall percentage of adherence to BCLC guidelines was 52 % and varied among the disease stages: BCLC 0/A, 44 %; BCLC B, 78 %; BCLC C, 35 %; and BCLC D, 67 %. One-, two-, and three-year OS rates were 63, 45, and 33 %, respectively. In BCLC 0/A, adherent patients presented a significantly better OS compared to non-adherent patients (hazard ratio [HR] = 0.19, 95% confidence interval [CI]: 0.09-0.42; p < 0.001). In BCLC D, the OS rate was worse in adherent patients compared to non-adherent patients (HR = 4.0, 95% CI: 1.67-9.88; p < 0.001), whereas no differences were observed in BCLC stage B or C. CONCLUSIONS: The percentage of adherence to BCLC recommendations in clinical practice is low and varies among the clinical stages. Non-adherence is associated with a worse prognosis, particularly in early stage disease

Hepatocellular carcinoma (HCC) is a common malignancy of the liver that is one of the most frequent causes of cancer-related death in the world. Surgical resection and liver transplantation are the only curative options for HCC, and tumor invasion and metastasis render many patients ineligible for these treatments. Identification of the mechanisms that contribute to invasive and metastatic disease may enlighten therapeutic strategies for those not eligible for surgical treatments. In this dissertation, I describe two sets of experiments to elucidate mechanisms underlying HCC dissemination, involving the activities of Krüppel-like factor 6 and a particular p53 point mutation, R172H. Gene expression profiling of migratory HCC subpopulations demonstrated reduced expression of Krüppel-like factor 6 (KLF6) in invasive HCC cells. Knockdown of KLF6 in HCC cells increased cell transformation and migration. Single-copy deletion of Klf6 in a HCC mouse model results in increased tumor formation, increased metastasis to the lungs, and decreased survival, indicating that KLF6 suppresses both tumor formation and metastasis in HCC. To elucidate the mechanism of KLF6-mediated tumor and metastasis suppression, we performed gene expression profiling and ChIP-sequencing to identify direct transcriptional targets of KLF6 in HCC cells. This analysis revealed novel transcriptional targets of KLF6 in HCC including CDC42EP3 and VAV3, both of which are positive regulators of Rho family GTPases. Concordantly, KLF6 knockdown cells demonstrate increased activity of the Rho family GTPases RAC1 and CDC42, and RAC1 is required for migration induced following KLF6 knockdown. Moreover, VAV3 and CDC42EP3 are also required for enhanced cell migration in HCC cells with KLF6 knockdown. Together, this work describes a novel signaling axis through which KLF6-mediated repression of VAV3 and CDC42EP3 inhibits RAC1Gmediated HCC cell migration in culture, and potentially HCC metastasis in vivo. TP53 gene mutations are commonly found in HCC and are associated with poor prognosis. Prior studies have suggested that p53 mutants can display gain-of- function properties in other tumor types. Therefore, I sought to determine if a particular hotspot p53 mutation, p53R172H, provided enhanced, gain-of-function properties compared to p53 loss in HCC. In vitro, soft agar colony formation and cell migration is reduced upon knockdown of p53R172H, indicating that this mutation is required for transformation-associated phenotypes in these cells. However, p53R172H-expressing mice did not have enhanced tumor formation or metastasis compared to p53-null mice. These data suggest that p53R172H and p53 deletion are functionally equivalent in vivo, and that p53R172H is not a gain-of-function mutant in HCC. Inhibition of the related transcription factors p63 and p73 has been suggested as a potential mechanism by which mutant p53 exerts its gain-of-function effects. Analysis of p63 and p73 target genes demonstrated that they are similarly suppressed in p53-null and p53R172H-expressing HCC cell lines, suggesting a potential explanation for the phenotypes I observed in vivo and in vitro. Together, the studies described in this dissertation increase our understanding of the mechanisms underlying HCC progression and metastasis. Specifically, we find and characterize KLF6 as a novel suppressor of HCC metastasis, and determine the contribution of a common p53 point mutation in HCC. This work contributes to ongoing efforts to improve treatment options for HCC patients.

<p>Tumorsko pupljenje (TP) u karcinomu je morfolo&scaron;ki fenomen koji predstavlja pojavu pojedinačnih ili malih grupa dediferentovanih tumorskih ćelija koje se na invazivnom frontu karcinoma odvajaju od glavne tumorske mase. Kod metastatskog kolorektalnog karcinoma (KRK) definitivno ne možemo odrediti pravi doprinos TP. Cilj je bio da se ispita terapijski patohistolo&scaron;ki odgovor na primenjeni hemioterapijski režim, prognostički i nezavisni negativni značaj TP , kao i korelacija TP i terapijskog odgovora histolo&scaron;ke regresije kod R0 reseciranih sinhronih i metahronih jetrenih metastaza KRK, koji su primali polihemioterapije po protokolu Folfox 4, sa i bez VEGF inhibitora &ndash; bevacizumaba (AV).&nbsp; Studija je prospektivno &ndash; retrospektivna i obuhvata 77 bolesnika oba pola, uzrasta preko 18 godina, sa patohistolo&scaron;ki verifikovanim jetrenim metastazama KRK, koji su operisani u Institutu za onkologiju Vojvodine u periodu od 1. maja 2007. do 1. juna 2017. godine. Od ukupno 120 bolesnika, njih 77 je ispunjavalo sledeće kriterijume: da je histolo&scaron;ki dokazan metastatski adenokarcinom kolorektuma sa R0 resekcijom i da su preoperativno dobijali HT sa biolo&scaron;kom terapijom ili bez nje. Bolesnike smo podelili u dve grupe: KRK &ndash; sinhrona metastatska bolest i KRK &ndash; metahrona metastatska bolest. Nakon selekcije bolesnika, rađena je mikroskopska analiza rutinskih histolo&scaron;kih i imunohistohemijskih preparata i određivana je gustina TP, histolo&scaron;ka regresija prema mTRG bodovanju komparirala se sa radiolo&scaron;kim odgovorom po RECIST-u. Događaji od interesa u kliničkom toku bolesti jesu progresija nakon hirur&scaron;kog zahvata jetrenih metastaza i ukupno preživljavanje u periodu od 24 meseca. Nema statistički značajne patohistolo&scaron;ke razlike u učestalosti lo&scaron;ijeg terapijskog odgovora (mTRG 3 &ndash; 5) u odnosu na bolji terapijski odgovor (mTRG 1, 2) između bolesnika sa sinhronom i metahronom metastatskom bole&scaron;ću KRK, koji su lečeni hemioterapijskim protokolom Folfox4: 13 (76,5%) vs. 13 (72,2%); p = 0,774. Kod bolesnika sa sinhronim metastazama KRK, lečenih hemioterapijskim protokolom Folfox 4, postoji statistički značajna razlika u učestalosti preživljavanja tokom dve godine, i to kod bolesnika sa malom u odnosu na one sa velikom gustinom TP: 10 (90,9%) vs. 5 (55,6%); p = 0,049. Kod tih bolesnika, lečenih hemioterapijskim protokolom Folfox4/AV, postoji statistički značajna razlika u učestalosti preživljavanja tokom dve godine, i to kod bolesnika sa malom u odnosu na one sa velikom gustinom TP: 9 (100%) vs. 6 (33,3%); p = 0,048. Kod bolesnika sa metahronim metastazama KRK lečenih hemioterapijskim protokolom Folfox4, sa i bez AV, nema statistički značajne razlike u učestalosti preživljavanja tokom dve godine u odnosu na gustinu TP. Kod bolesnika sa sinhronim i metahronim metastazama KRK nema statistički značajne razlike u učestalosti lo&scaron;ijeg histolo&scaron;kog odgovora na terapiju (mTRG 3 &ndash; 5) kod onih sa malom u odnosu na one sa velikom gustinom (TP): (8 (50%) vs. 15 (78,9%); p = 0,072 i TP: 8 (80%) vs. 13 (72,2%); p = 0,649). Kod bolesnika sa sinhronim metastazama KRK lečenih hemioterapijskim protokolom Folfox4, sa i bez AV, postoji statistički značajna razlika u učestalosti preživljavanja tokom dve godine u odnosu na gustinu TP. Takođe, kod tih bolesnika velika gustina TP je nezavistan negativan faktor prognoze u odnosu na date terapijske režime, &scaron;to se vidi u preživljavanju tokom dve godine.</p> / <p>Tumor budding (TB) in cancer is a morphological phenomenon representing the appearance of single or small groups of dedifferentiated tumor cells that separate from the main tumor mass on the invasive front of cancer. In metastatic colorectal cancer (MCC), the true contribution of TB cannot be determined. The aim was to investigate the therapeutic pathohistological response to the applied chemotherapy, the prognostic and independent negative significance of TB, as well as the correlation of TB and the therapeutic response of histological regression in R0 resectable synchronous and metachronous liver metastases of MCC receiving polychemotherapy according to the Folfox 4 protocol, with and without VEGF inhibitors - bevacizumab (AV). The research was conducted as a prospective &ndash; retrospective study that included 77 patients of both sex, over 18 years of age, with pathohistologically verified MCC liver metastases, who underwent surgery at the Institute of Oncology of Vojvodina from 1st May 2007 until 1st June 2017. From 120 patients, 77 patients met the following criteria: they had histologically proven metastatic colorectal adenocarcinoma with R0 resection and also received preoperative chemotherapy with or without biological therapy. The patients were divided into two groups: MCC - synchronous metastatic disease and MCC - metachronous metastatic disease. After the patient selection, microscopic analysis of routine histological and immunohistochemical preparations was performed, the density of TB was determined, and the histological regression according to mTRG scoring was compared with a radiologic response according to the RECIST. The events of interest in the clinical course of the disease were the progression of hepatic metastases after surgery and overall survival during 24 months. There is no statistically significant pathohistological difference in the incidence of worse therapeutic response (mTRG 3 - 5) compared to the better therapeutic response (mTRG 1, 2) between patients with synchronous and metachronous MCC who were treated with the Folfox4 chemotherapy protocol: 13 (76.5%) vs. 13 (72.2%); p = 0.774. In patients with synchronous MCC metastases treated with the Folfox 4 chemotherapy protocol, there was a statistically significant difference in the survival rates during two years particularly in patients with low versus high TB density: 10 (90.9%) vs. 5 (55.6%); p = 0.049. In those patients who were treated with the Folfox4 / AV chemotherapy protocol, there was a statistically significant difference in survival rates during two years particularly in patients with low TB density in reference to those with high: 9 (100%) vs. 6 (33.3%); p = 0.048. In patients with metachronous MCC metastases who were treated with the Folfox4 chemotherapy protocol, with and without AV, there was no statistically significant difference in survival rate during two years when referring to the TB density. In patients with synchronous and metachronous metastases, MCC has no statistically significant difference in the incidence of worse histological response to therapy (mTRG 3 - 5) in patients with low TB density versus the ones with high density (TB): (8 (50%) vs. 15 (78.9%); p = 0.072 and TP: 8 (80%) vs. 13 (72.2%); p = 0.649). In patients with synchronous MCC metastases who were treated with the Folfox4 chemotherapy protocol, with and without AV, there is a statistically significant difference in survival rates during a two-year follow up when referring to the TB density. Also, the high density of TB is an independent negative prognostic factor in these patients in reference to the given therapeutic regimens, as seen in the two-year survival rate.</p>

Ng, Po Yan. / Thesis submitted in: November 2006. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 109-120). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Table of Contents --- p.iv / List of Figures --- p.viii / List of Tables --- p.xi / List of Abbreviation --- p.xii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- A General Introduction to the Development and Clinical Activities of Platinum Drugs --- p.1 / Chapter 1.1.1 --- Platinum Drugs used in a Clinical Setting --- p.4 / Chapter 1.1.2 --- Platinum Drugs under Clinical Trials --- p.5 / Chapter 1.1.3 --- Platinum Compounds with Dual Mechanisms --- p.7 / Chapter 1.2 --- Platinum Drug Antitumor Mechanism --- p.9 / Chapter 1.3 --- Limitations of Platinum Drugs --- p.12 / Chapter 1.3.1 --- Toxicity --- p.12 / Chapter 1.3.2 --- Drug Resistance or Cross Resistance --- p.15 / Chapter 1.3.2.1 --- Reduced Drug Accumulation or Increased Drug Efflux --- p.16 / Chapter 1.3.2.2 --- Drug Inactivation --- p.18 / Chapter 1.3.2.3 --- Enhanced DNA Repair --- p.19 / Chapter 1.4 --- Why Combinational Therapy? --- p.21 / Chapter 1.4.1 --- Antimetabolites --- p.20 / Chapter 1.4.2 --- Topoisomerase Inhibitors --- p.22 / Chapter 1.4.3 --- Tubulin-Active Antimitotic Agents --- p.24 / Chapter 1.4.4 --- Demethylcantharidin as a potential candidate for drug combination --- p.28 / Chapter 1.5 --- Study Objectives --- p.31 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Cell Lines --- p.33 / Chapter 2.2 --- Cancer Cell Preparation / Chapter 2.2.1 --- Chemicals and Reagents --- p.33 / Chapter 2.2.2 --- Cell Culture Practice --- p.34 / Chapter 2.2.2.1 --- Subcultures --- p.35 / Chapter 2.2.2.2 --- Cryopreservation --- p.37 / Chapter 2.2.2.3 --- Thawing Cryopreservated Cells --- p.38 / Chapter 2.2.3 --- Development of Drug-Resistant Cell Lines --- p.39 / Chapter 2.3 --- Growth Inhibition Assay / Chapter 2.3.1 --- Evaluation of Cytotoxicity in vitro --- p.40 / Chapter 2.3.2 --- Drug Pretreatment --- p.43 / Chapter 2.3.3 --- Drug Pre-sensitization with Concurrent Treatment --- p.44 / Chapter 2.4 --- Calculations for Drug Combinations --- p.46 / Chapter 2.5 --- Statistical Analysis --- p.49 / Chapter Chapter 3 --- Results and Discussions / Chapter 3.1 --- In vitro Cytotoxicity and Evaluation of Drug Resistance --- p.50 / Chapter 3.2 --- Role of Leaving Ligand in a Platinum Complex --- p.58 / Chapter 3.3 --- Priority in Selecting the Most Effective Drug Combination --- p.66 / Chapter 3.4 --- Drug Combination Studies / Chapter 3.4.1 --- Drug Combination Prescreening --- p.68 / Chapter 3.4.1.1 --- Comparison of the effectiveness of the three Drug Combinations --- p.72 / Chapter 3.4.1.2 --- Rationale for Drug Combination Studies presented in Section 3.4.2 & 3.4.3 --- p.73 / Chapter 3.4.2 --- Drug Pre-sensitization Studies in Colorectal Cancer Cell Lines --- p.74 / Chapter 3.4.2.1 --- Comparison of Drug Pre-sensitization Treatment in Sensitive Colorectal Cancer Cell Lines --- p.84 / Chapter 3.4.2.2 --- Comparison of Drug Pre-sensitization Treatment in Sensitive and Oxaliplatin Resistant HCT116 Colorectal Cancer Cell Lines --- p.87 / Chapter 3.4.3 --- Drug Pre-sensitization Studies in Liver Cancer Cell Lines --- p.89 / Chapter 3.4.3.1 --- Comparison of Drug Pre-sensitization Treatment in Sensitive Liver Cancer Cell Lines --- p.99 / Chapter 3.4.3.2 --- Comparison of Drug Pre-sensitization Treatment in Sensitive and Cisplatin Resistant SK-Hepl Liver Cancer Cell Line --- p.101 / Chapter 3.5 --- Possible Explanation to the Observed Drug Combination Effect --- p.103 / Chapter 3.6 --- General Protocols for Drug Combinations --- p.105 / Chapter Chapter 4 --- Conclusions / Reference --- p.109 / Appendices --- p.121 / Chapter I a. --- "Raw Data of Pre-screening for HCT116 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.122 / Chapter I b. --- "Raw Data of Pre-screening for HCT116 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.123 / Chapter II a. --- "Raw Data of Pre-screening for SK-Hepl (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.124 / Chapter II b. --- "Raw Data of Pre-screening for SK-Hepl ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.125 / Chapter III a. i) --- "Isobolograms for HCT116 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.126 / Chapter III a. ii) --- "Raw Data for HCT116 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.127 / Chapter III b. i) --- "Isobolograms for HCT116 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.128 / Chapter III b. ii) --- "Raw Data for HCT116 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.129 / Chapter IV a. i) --- "Isobolograms for HCT1160xaR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.130 / Chapter IV a. ii) --- "Raw Data for HCT1160xaR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.131 / Chapter IV b. i) --- "Isobolograms for HCT1160xaR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.132 / Chapter IV b. ii) --- "Raw Data for HCT1160xaR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.133 / Chapter V a. i) --- "Isobolograms for HT29 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.134 / Chapter V a. ii) --- "Raw Data for HT29 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.135 / Chapter V b. i) --- "Isobolograms for HT29 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.136 / Chapter V b. ii) --- "Raw Data for HT29 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.137 / Chapter VI a. i) --- Isobolograms for Hep G2 (Cisplatin and [Pt(DMC)(NH3)2]) --- p.138 / Chapter VI a. ii) --- Raw Data for Hep G2 (Cisplatin and [Pt(DMC)(NH3)2]) --- p.139 / Chapter VI b. i) --- "Isobolograms for Hep G2 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.140 / Chapter VI b. ii) --- "Raw Data for Hep G2 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.141 / Chapter VII a. i) --- "isobolograms for SK Hep 1 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.142 / Chapter VII a. ii) --- "Raw Data for SK Hep 1 (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.143 / Chapter VII b.i) --- "Isobolograms for SK Hep 1 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.144 / Chapter VII b. ii) --- "Raw Data for SK Hep 1 ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.145 / Chapter VIII a. i) --- "Isobolograms for SK Hep ICisR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.146 / Chapter VIII a. ii) --- "Raw Data for SK Hep ICisR (Cisplatin, [Pt(DMC)(NH3)2] and Pt(DMC)(NH2CH3)2])" --- p.147 / Chapter VIII b. i) --- "Isobolograms for SK Hep ICisR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.148 / Chapter VIII b. ii) --- "Raw Data for SK Hep ICisR ([Pt(DMC)(R,R-DACH)] and Oxaliplatin)" --- p.149