INTRODUÇÃO: Não existem estudos preditivos sobre fadiga em doentes com câncer colo-retal, embora fadiga seja descrita como freqüente na população oncológica. OBJETIVO: Identificar os fatores de risco e preditivos independentes de fadiga em doentes com câncer colo-retal. MÉTODO: Estudo preditivo que envolveu amostra não-probabilística de 157 pacientes adultos ambulatoriais com tumor primário de cólon ou reto (idade média 60±11,7 anos; 54% homens; média de anos de escolaridade 10,7±5,4 anos; estádio IV 44,8%), atendidos em quatro serviços de oncologia do município de São Paulo, Brasil (julho/2006 a julho/2007). Os pacientes preencheram a Ficha de Identificação, a Escala de Fadiga de Piper-Revisada (0-10; ponto de corte: >4; ?=0,94), o Inventário de Depressão de Beck (0-63; ponto de corte: >13; ?=0,83), a Escala de Karnofky (0%-100%; ponto de corte: <80), a Escala de Prejuízo do Sono (0-10; ponto de corte: >5) e a Escala de Dor (0-10; ponto de corte: >6). Os pontos de corte foram estabelecidos pela análise da curva ROC (Receiver Operating Characteristic), com exceção do ponto de corte de fadiga, que foi estabelecido após análise da distribuição dos escores em percentis e do critério proposto pela National Comprehensive Cancer Network. RESULTADOS: Fadiga foi referida por 26,8% dos doentes. Os fatores de risco para fadiga foram os seguintes: serviço de saúde público, dor, prejuízo do sono, depressão e capacidade funcional prejudicada (p<0,05). A análise de regressão logística identificou três fatores preditivos: depressão, capacidade funcional e prejuízo do sono. A depressão aumentou em 4 vezes a chance de ocorrer fadiga (OR: 4,2; IC95% 1,68-10,39), a capacidade funcional aumentou em 3 vezes (OR: 3,2; IC95% 1,37-7,51) e o prejuízo do sono também em 3 vezes (OR: 3,2; IC95% 1,30-8,09). Quando os três fatores preditivos estiveram presentes, a probabilidade de ocorrer fadiga foi de 80%, o que indicou boa capacidade de predição. Quando os três fatores preditivos estiveram ausentes, a probabilidade de ocorrer fadiga foi de 8%. A especificidade e sensibilidade do modelo foram de 81,9% e 58,6%, respectivamente, indicando baixa chance de falsos positivos e alta chance de falsos negativos. CONCLUSÕES: Depressão, capacidade funcional e prejuízo do sono foram preditores de fadiga. O estudo disponibiliza tabela de probabilidade de predição de fadiga e propõe que, por meio da avaliação da depressão, da capacidade funcional e do prejuízo do sono, é possível conhecer a probabilidade do paciente ter fadiga, o que é inédito nessa população e de grande utilidade na clínica / INTRODUCTION: There are no studies that identify the predictive factors of fatigue among colo-rectal cancer patients, although fatigue is described as a frequent problem in the oncology setting. AIM: Identify risk factors and independent predictors of fatigue in colo-rectal cancer patients. METHOD: Predictive study that involved non-probabilistic sample of 157 adult outpatients with primary colon or rectal (mean age 60±11.7 years; 54% male; educational level 10.7±5.4 years; cancer stage IV 44.8%), recruited from 4 oncology clinics in Sao Paulo, Brazil (July/2006 to July/2007). Patients filled out an Identification Profile, Piper Fatigue Scale-revised (0-10; cut-score: >4, ?=0,94), Beck Depression Inventory (0-63; cut-score: >13, ?=0,83), Karnofsky Scale (0%-100%; cut-score: <80%), Sleep disturbance scale (0-10; cut-score: >5), and Pain scale (0-10; cut-score: >6). The cut scores were established by ROC (Receiver Operating Characteristic) curves, except for fatigue cut-score, which was established after an analysis of the distribution of the scores in percentiles and of the criteria proposed by National Comprehensive Cancer Network. RESULTS: Fatigue was identified by 26.8% of the patients. The risk factors for fatigue were: public oncology service, pain, sleep disturbance, poor performance status, and depression (p<0.05). Logistic regression identified three predictors: depression, sleep disturbance, and performance status. Depression increased the chance for fatigue to occur by four times (OR: 4.2; 95%CI 1.68-10.39), performance status increased by three times (OR: 3.2; 95%CI 1.37-7.51), and sleep disturbance also increased the chance by three times (OR: 3.2; 95%CI 1.30-8.09). When the three factors were present in concomitance, the probability that patients had fatigue was 80%, which was considered a good predictive capacity. When none of the factors were present, the probability that patients had fatigue was 8%. The specificity and sensibility of this model were 81.9% and 58.6%, respectively, indicating that there is low chance of false positive and high chance of false negatives. CONCLUSIONS: Depression, performance status, and sleep disturbance were predictive factors of fatigue. The study presents a prediction table and proposes that by assessing depression, performance status and sleep disturbance, it is possible to know the probability that a patient will have fatigue. This finding is original and applicable in clinical practice

INTRODUÇÃO: O câncer colorretal é um dos cânceres mais incidentes no mundo. As maiores incidências são descritas em homens e aumentam com a idade. Porém, estudos recentes reportaram aumento da incidência entre os mais jovens. Apesar das maiores incidências serem descritas em países desenvolvidos, estes apresentam tendência de estabilidade ou declínio. Em países com tendência de aumento, a mudança no padrão alimentar é a principal hipótese para este aumento. A mortalidade apresenta tendência de queda, principalmente em países que investiram em diagnóstico precoce e tratamento oportunos. OBJETIVO: Estudar as desigualdades na incidência e mortalidade do câncer colorretal no Município de São Paulo e Brasil. MÉTODOS: Esta tese está dividida em três manuscritos. MANUSCRITO 1: Simulação e comparação de técnicas de correção de dados incompletos de idade para o cálculo de taxas de incidência. A partir de seis bases com diferentes proporções de dados incompletos para idade (5 até 50%), foram comparados dois métodos de correção. Verificou-se que bases com 5% ou mais de dados incompletos para idade apresentaram taxas de incidência subestimadas. O fator de correção retificou as subestimativas das taxas padronizadas, entretanto, esta técnica não permitiu corrigir taxas específicas por idade. A imputação múltipla foi útil na correção das taxas padronizadas e específicas em bancos com até 30% de dados incompletos. Bases com 5% ou mais de dados incompletos necessitam de aplicação de correção. A imputação múltipla mostrou-se superior ao fator de correção, pois permitiu corrigir as taxas de incidência específicas. MANUSCRITO 2: Descrição e análise das taxas de mortalidade por câncer de colorretal no Brasil, segundo sexo e Unidades da Federação e indicadores socioeconômicos. As tendências das taxas de mortalidade foram estimadas por modelo de regressão linear. No Brasil,ocorreu aumento da tendência da taxa de mortalidade em todos os estados para o sexo masculino e em 21 estados para o sexo feminino. Ao ajustar por taxa de mortalidade por causas mal definidas, Produto Interno Bruto e Coeficiente de Gini, a tendência de aumento manteve-se significativa (p<0,05) no Brasil e em sete estados em homens e em nove estados em mulheres. O aumento da taxa de mortalidade por câncer colorretal em algumas Unidades Federativas e no Brasil pode estar relacionada ao aumento da incidência, ao atraso no diagnóstico e no tratamento. MANUSCRITO 3: descrição e análise das taxas de incidência e mortalidade por câncer colorretal no município de São Paulo, segundo sexo e faixa etária. Foram calculadas as razões das taxas padronizadas de incidência e mortalidade, estimada a mudança percentual anual média (AAPC) e o efeito idade-período-coorte. As razões das taxas de incidência e mortalidade foram superiores a 1 em homens a partir dos 50 anos e para o total da população. Ao comparar com indivíduos de 30 a 39 anos, em ambos os sexos, as razões das taxas foram maiores que 1 após 40 anos. Verificou-se tendência de aumento da incidência em mulheres (AAPC 0,9%) e da mortalidade em homens (AAPC 1,0%) e em mulheres (AAPC 0,2%). O modelo idade-período-coorte apresentou melhor ajuste para a incidência, em ambos os sexos (p<0,001). A mortalidade em homens foi explicada pelo modelo idade-drift (p<0,001). No Munícipio de São Paulo, a tendência crescente da incidência pode estar relacionada à alimentação inadequada e ao aumento do diagnóstico e a da mortalidade por diagnóstico e tratamento inoportunos. / INTRODUCTION: Colorectal cancer is one of the most common cancers worldwide. The highest incidence rates are described in men and with increasing age. However, recent studies have reported an increase in incidence among younger people. Although the highest incidence are described in developed countries, they show a tendency of stability or decline. In countries with increasing trends, the change in dietary pattern is the main hypothesis for this increase. Mortality is declining, especially in countries that have invested in diagnosis and treatment. OBJECTIVE: To study the inequalities in the incidence and mortality from colorectal cancer in the city of São Paulo and Brazil. METHODS: This thesis is divided into three manuscripts. MANUSCRIPT 1: Simulation and comparison of incomplete data correction techniques for the calculation of incidence rates. From six databases with different proportions of incomplete data for age (5 to 50%), two correction methods were compared. Databases with 5% or more of incomplete data for age were found to have underestimated rates. The correction factor mitigated the underestimation of the standardized rates; however, this technique did not allow the correction of age-specific rates. Multiple imputation was useful in correcting standardized and age-specific rates in datasets with up to 30% incomplete data. Databases with 5% or more of incomplete data need to be corrected. Multiple imputation technique was a superior method in comparison to the correction factor. MANUSCRIPT 2: description and analysis of mortality rates from colorectal cancer in Brazil, according to sex and States, according to socioeconomic indicators. For the trend analysis, the linear regression models were chosen through linear regression In Brazil, there was an increase in the mortality rate in all states for males and in 21 states for females. When the mortality rate was controlled for ill-defined causes, Gross Domestic Product and Gini Index, the increasing trend of mortality remained significant (p-value <0.05) in Brazil and in seven states in men and in nine states in women. The increase in the colorectal cancer mortality rate in some States and in Brazil may be related to the possible increase in incidence delayed diagnosis and treatment. MANUSCRIPT 3: description and analysis of incidence and mortality rates for colorectal cancer in the city of São Paulo, according to sex and age group. The incidence and mortality rate ratios were estimated. Joinpoint regression was performed to obtain average annual percent change (AAPC) and age-period-cohort analysis was used to examine the incidence trends. The incidence and mortality rates ratio were higher than 1 in men aged 50 and over. When comparing individuals aged 30 to 39 in both sexes, the rates ratios were higher than 1 after 40 years. There was an upward trend in incidence in women only (AAPC 0.9%) and mortality in men (AAPC 1.0%) and women (AAPC 0.2%). The age-period-cohort model provided the best fit to incidence in both sexes (p <0.001). Mortality in men was explained by the age-drift model (p <0.001). In the city of São Paulo, the increasing trend in incidence may be related to inadequate diet and increased diagnosis and mortality due to untimely diagnosis and treatment.

A síndrome do câncer de colo-retal hereditário não relacionado a polipose (HNPCC) foi caracterizada na década de 60 e por volta dos anos 90 os critérios de Amsterdam para o diagnóstico clínico de HNPCC foram estabelecidos, envolvendo história familiar em duas gerações sucessivas, com três indivíduos apresentando acometimento do cólon e/ou reto e um dos indivíduos diagnosticados abaixo dos 45 anos. A instabilidade de microssatélites (MSI) começou a ser descrita em 1993 em pacientes portadores do HNPCC, mas é comum em outras neoplasias esporádicas. Em 1996 os critérios de Bethesda, que envolvem o acometimento preferencial do cólon proximal, histologia indiferenciada ou mucosecretora, presença de tumores sincrônicos e metacrônicos ou associação a tumores extracólicos, além da história familiar, foram propostos pelo Grupo Internacional Colaborativo para o estudo do HNPCC (ICG-HNPCC), com o intuito de rastrear maior número de pacientes suspeitos de serem portadores da síndrome. A MSI é caracterizada pela perda ou ganho de unidades repetitivas em regiões do DNA contendo mono, di, tri ou tetranucleotídeos dispostos seguidamente no genoma. No presente estudo, nosso objetivo foi analisar a freqüência de instabilidade de microssatélites (MSI) pela análise de 7 regiões microssatélites (BAT25, BAT26, D5S346, D2S123, D17S250, BAT40, TP53), correlacionando os dados obtidos com algumas características clinicopatológicas dos pacientes. O DNA de 106 pares de amostras de tecido normal e tumor provenientes de portadores de carcinoma colo-retal (CCR), foi estudado mediante a realização da reação em cadeia pela polimerase (PCR), seguida pela análise em gel de poliacrilamida desnaturante. Os pacientes foram divididos em dois grupos, um com 75 pacientes com pelo menos um dos critérios de Bethesda (tumor no cólon proximal, histologia pouco diferenciada, padrão indiferenciado ou mucinoso, idade igual ou inferior a 45 anos, sincronicidade ou metacronicidade dos tumores) e 31 pacientes sem nenhum desses critérios. Dos 106 casos, 14,1% (15/106) apresentaram MSI em 30% ou mais dos marcadores analisados, sendo classificados com instabilidade de microssatélite de alto grau (MSI-H), 17,9% (19/106) dos casos foram classificados com instabilidade de microssatélite de baixo grau (MSI-L) e 67,9% (72/106) com estabilidade de microssatélites (MSS). Se considerarmos apenas a análise do grupo com pelo menos um dos critérios de Bethesda considerados, observamos que 18,6% (14/75) dos casos apresentaram MSI-H, 18,6% (14/75) foram MSI-L e 62,6% (47/75) MSS. No grupo sem nenhum dos critérios de Bethesda analisados, 3,2% (1/31) apresentaram MSI-H, enquanto 16,1% (5/31) e 80,6% (25/31) foram MSI-L e MSS respectivamente. Quando consideramos os marcadores propostos pelo Grupo Colaborativo Internacional para o estudo do HNPCC - ICG-HNPCC (BAT25, BAT26, D5S346, D2S123, D17S250) observamos que no grupo sem critérios de Bethesda passamos a ter apenas casos classificados como MSI-L e MSS. No grupo com pelo menos um dos critérios passamos a ter 21 ,3% (16/75) dos casos classificados com MSI-H e 14,7% (11/75) de casos MSI-L. Os marcadores mais sensíveis para a análise de MSI-H foram BAT25 e BAT26, enquanto os marcadores D17S250 e TP53 foram os mais freqüentes no casos MSI-L. Ao considerarmos os casos com pelo menos um dos critérios de Bethesda e apenas o painel proposto pelo ICG-HNPCC, o marcador BAT25 identificou os casos MSI-H com 100% de sensibilidade e 96,6% de especificidade, enquanto o BAT26 apresentou 93,7% e 98,9% de sensibilidade e especificidade respectivamente. A avaliação dos critérios de Bethesda considerados neste estudo mostrou que a localização proximal do tumor se correlacionou com os casos MSI-H. / The Hereditary Non-Polyposis Colorectal Cancer Syndrome (HNPCC) was characterized in the 60\'s, and the Amsterdam criteria for the clinical HNPCC diagnosis were established in the 90\'s involving the family history of two successive generations with three individuals having their colon and/or rectum affected, one of them having been diagnosed when under 45 years of age. The microsatellite instability (MSI) was first described in 1993 in bearers of HNPCC, but it is common in other sporadic tumors. In 1996, the Bethesda criteria, which include the preferential involvement of the proximal colon, undifferentiated or mucosecretory histology, presence of synchronic and metachronic tumors or association to extracolonic tumors, besides family history, were proposed by the International Colaborative Group for the study of HNPCC (ICG-HNPCC) with the purpose of tracking a higher number of bearers of that syndrome. The MSI is characterized by the loss or gain of repetitive units in DNA regions containing mono-, di-, tri- or tetranucleotides sequentially arranged in the genome. The aim of this paper was to evaluate the frequency MSI by analyzing 7 microsatellite regions (BAT25, BAT26 , D5S346, D2S123, D17S250, BAT40, TP53), and correlating the data so obtained with clinicopathologic characteristics of the patients. The DNA of 106 pairs of samples taken from normal tissues and colorectal carcinomas (CRC) was studied through the polimerase chain reaction (PCR) followed by analysis in denaturing poliacrylamide gel. The patients were separated into two groups, one consisting of 75 patients with at least one of the Bethesda criteria (tumor in the proximal colon, poorly differentiated histology, undifferentiated or mucinous pattern, age equal to or below 45 years, tumor synchronicity or metachronicity) and 31 patients without any of such criteria. From the 106 cases, 14.1% (15/106) exhibited MSI in 30% or more of the markers, then classified as having high microsatellite instability (MSI-H); 17.9% (19/106) were classified as low microsatellite instability (MSI-L) and 67.9% (72/106) as having microsatellite stability (MSS). If we consider only the analysis of the group with at least one of the Bethesda criteria, we observe that 18.6% (14/75) of the cases showed MSI-H, 18.6% were MSI-L and 62.6% (47/75) were MSS. In the group without any of the Bethesda criteria, 3.2% (1/31) showed MSI-H, while 16.1% (5/31) and 80.6% (25/31) were respectively MSI-L and MSS. When we consider the markers proposed by the Intenational Colaborative Group for the study of HNPCC - ICG-HNPCC (BAT25, BAT26, D5S346, D2S123, D17S250), we observe, in the group without Bethesda criteria, only cases which were classified as MSI-L and MSS. In the group with at least one of the Bethesda criteria we find 21.3% (16/75) of the cases classified as MSI-H and 14.7% (11/75) as MSI-L. The most sensitive markers for MSI-H were BAT25 and BAT26, whereas the markers D17S250 and TP53 were the most frequent in MSI-L cases. When the cases with at least one of the Bethesda criteria, were evalueted only with the panel propose by ICG-HNPCC, the BAT25 marker identified MSI-H cases with 100% sensitivity and 96.6% specificity, while the BAT26 exhibited respectively 93,7% and 98,9%. The evaluation of the Bethesda criteria considered in this paper showed that the proximal location of the tumor correlated significantly with the cases of MSI-H.

A População brasileira apresenta elevada diversidade genética devido à multietnicidade, que têm implicações clínicas/genéticas importantes. O estudo de genes polimórficos pode auxiliar na detecção de pessoas com maior risco de desenvolver câncer, caracterização de evolução diferenciada, resposta distinta ao tratamento quimioterápico ou radioterápico e prognóstico. A ciclooxigenase-2 (COX-2) é induzida em resposta ao fator de crescimento e citocinas, sendo expressa nas doenças inflamatórias, lesões pré-malignas e tumores colorretais. Este trabalho teve como objetivos avaliar a influência dos polimorfismos 1195A > G e 8473T > C do gene COX-2 como fatores de risco para o desenvolvimento de câncer colorretal e investigar o impacto dos polimorfismos na progressão e sobrevida de pacientes submetidos ao tratamento cirúrgico por câncer colorretal. Avaliaram-se SNPs (polimorfismos de nucleotídeo único) em 230 pacientes submetidos à ressecção cirúrgica no Hospital das Clínicas (SP), seguidos por 5 anos e 196 controles, operados por doença benigna na mesma instituição, pareados quanto ao sexo e idade, sem histórico individual ou familial de câncer. Isolou-se o DNA dos leucócitos utilizando-se do kit de extração e purificação PureLink DNA Minikit, seguido de amplificação pela reação em cadeia da polimerase (PCR). Utilizou-se a análise do PCR em Tempo Real para determinar os genoótipos os polimorfismos através dos ensaios TaqMan ® SNP Genotyping Assay. Os resultados encontrados foram associados aos dados epidemiológicos e clinicopatológicos dos pacientes. Determinaram-se as frequências genotípicas, alélicas e estimaram-se as frequências de haplótipos dos polimorfismos do COX-2 -1195A > G e 8473T > C. As populações estão em equilíbrio de Hardy-Weinberg, a exceção do grupo controle para o polimorfismo 8473T > C (p=0,02). As frequências foram similares nos grupos caso e controle para genótipos e haplótipos, portanto, não há associação entre esses polimorfismos e risco de CCR. Em relação às variáveis epidemiológicas e anatomopatológicas do grupo caso, demonstraram-se associação das mesmas com alguns perfis genotípicos. Encontrou-se frequência elevada do genótipo polimórfico -1195GG na população oriental, grupo constituído em sua maioria por japoneses, e dos genótipos 8473TC e CC em afrodescendentes (p < 0,05). Neste grupo, o genótipo -1195GG é ausente. Foi encontrada. Encontrou-se associação entre invasão angiolinfática e genótipo polimórfico 8473 CC (p < 0,05). Na análise de sobrevida, houve associação, no modelo codominante e dominante, do genótipo COX-2 -1195GG com menor sobrevida global (AAxGG: RR = 2,78; IC95% = 1,13-6,84; p < 0,020 e AA/AG x GG: RR = 2,59; IC95% = 1,07-6,27; p < 0,04), utilizando o modelo de regressão múltipla, ajustado para as variáveis de confusão. Assim, pode-se concluir que as variantes -1195A > G e 8473T > C não participam da suscetibilidade genética ao CCR na população brasileira. O polimorfismo -1195A > G, associado à menor sobrevida, pode atuar como marcador prognóstico nestes pacientes / Brazilian population displays very high levels of genomic diversity due to the multi-ethnicity, which have important clinical/genomic implications. Polymorphic genes\' study may aid in the detection of people at higher risk of developing cancer, characterization of differentiated outcome, distinctive response to chemotherapy or radiotherapy and prognosis. Cyclooxygenase-2 (COX-2) is induced in response to growth factor and cytokines, and it is expressed in inflammatory diseases, precancerous lesions and colorectal tumors. This study aimed to evaluate the influence of COX-2 -1195A> G and 8473T> C gene polymorphisms as a risk factor for developing colorectal cancer and to investigate the impact of polymorphisms on progression and survival in patients who have undergone surgical treatment for colorectal cancer. We evaluated SNPs (Single nucleotide Polymorphism) of 230 colorectal cancer resected patients admitted at the Hospital das Clinicas (SP), followed by 5 years, and 196 controls, operated for benign disease at the same institution, matched for age and sex, and no individual or familial history of cancer. DNA was isolated from leukocyte using PureLink (TM) Genomic DNA Mini Kit, followed by amplification by polymerase chain reaction (PCR). Real-time analysis was used for genotyping of polymorphisms, through the TaqMan ® SNP Genotyping Assay. The results of the polymorphisms were associated to epidemiological, clinicopathological and immunohistochemical features of the patients. Were determined genotype and allelic frequencies, and were estimated the haplotype frequencies of COX-2 -1195A > G and 8473T > C polymorphisms. The populations are in Hardy-Weinberg equilibrium, except for the control group to the 8473T > C polymorphism (p = 0,02). The frequencies were similar in case and control groups for genotypes and haplotypes, therefore, there is no association between these polymorphisms and risk of CCR. Regarding the epidemiological and pathological variables in the case group, we demonstrate their association with some genotypic profiles. A high frequency of the polymorphic genotype -1195GG was found in an Asiatic population, group composed in its majority by Japaneses, and 8473TC and CC genotypes in African descent (p < 0,05). In this group, the 1195GG genotype is absent. Association was found between angiolymphatic invasion and polymorphic genotype 8473 CC (p < 0,05). In survival analysis, there was an association, in co-dominant and dominant model, of the COX-2 genotype -1195GG with decreased overall survival (AAxGG: RR = 2,78, 95% CI 1,13-6,84; p < 0,020 and AA / AG x GG: RR = 2,59, 95% CI 1,07-6,27; p < 0,04), using the multiple regression model, adjusted for confounding variables. Therefore, -1195A variants > G and 8473T > C does not appear participate in genetic susceptibility to CCR in the Brazilian population, but the polymorphism -1195A > G, associated with decreased survival, may act as a prognostic marker in these patients

INTRODUÇÃO: O câncer colorretal (CCR) é doença de grande importância, com elevada incidência e mortalidade. Restrito às camadas mucosa e submucosa, é denominado CCR precoce, sendo passível de tratamento endoscópico curativo na ausência de fatores de risco para metástases linfonodais. Entretanto, as técnicas para tal não são universalmente disponíveis, e muitos destes pacientes são tratados por ressecções cirúrgicas. OBJETIVO: Buscar estudos com os resultados do tratamento cirúrgico e endoscópico do CCR precoce e compará-los por meio de metanálise. MÉTODOS: Foi efetuada busca nas bases MEDLINE, EMBASE, LILACS, CENTRAL COCHRANE e EBSCO por estudos comparativos que incluíram pacientes com CCR precoce, submetidos a tratamento endoscópico ou cirúrgico. Dados de mortalidade, recidiva a longo prazo, taxas de ressecção em bloco e ressecção curativa, tempo de procedimento, taxas de complicações e complicações graves foram extraídos. Viés foi individualmente avaliado pela escala de Newcastle-Ottawa. Foram efetuados cálculos do risco absoluto (RA), seu aumento (ARA) ou redução (RAA), e dos números necessários para causar dano (NNH) ou tratar (NNT), com intervalo de confiança (IC) de 95% para significância estatística. Para metanálise dos dados, foi utilizado o programa RevMan 5, com uso do teste de Mantel-Haenszel para calcular um IC de 95%, e expressão dos resultados em gráficos de forest e funnel plot. A consistência entre os estudos foi relatada por I2. RESULTADOS: Foram encontradas 12.905 entradas via PUBMED e 12.818 via outras bases. Após remoção de duplicatas e aplicação dos critérios de inclusão e exclusão, restaram quatro estudos de coorte retrospectiva. Não foi observada diferença para mortalidade (339 pacientes) ou recorrência a longo prazo (577 pacientes). Foi observada superioridade do tratamento cirúrgico em 11% (-0,11 [IC 95% -0,14; -0,09, P < 0,05]) para taxa de ressecção em bloco (1.085 pacientes), e em 11% (-0,11 [IC 95% -0,21; -0,02, P < 0,05]) para taxa de ressecção curativa (1.112 pacientes). O tratamento endoscópico demonstrou superioridade de 7% (-0,07 [IC 95% -0,10; -0,03]) para taxa de complicações (1.085 pacientes), de 8% (-0,08 [IC 95% -0,15; -0,01]) para complicações graves (827 pacientes), e de 130 minutos (-130,34 [IC 95% -190,5; -70,17, P < 0,05) para tempo de procedimento (827 pacientes). CONCLUSÃO: Conforme os dados disponíveis na literatura, o tratamento do CCR precoce apresenta melhores resultados pela técnica cirúrgica quanto às taxas de ressecção em bloco e curativa, e menos complicações e menor tempo de procedimento pela técnica endoscópica. Não há diferença quanto às taxas de mortalidade e recidiva a longo prazo / INTRODUCTION: Colorectal cancer (CRC) represents an important disease, with high incidence and mortality. When restricted to the mucosa and submucosa it is called early CRC, with curative endoscopic treatment in the absence of histopathological conditions associated with high risk of linfonodal metastasis. However, such treatment techniques are not widely available, and many of these patients undergo surgical resection. OBJECTIVES: To search for studies of endoscopic and surgical treatment modalities for early CRC, and compare these results through meta-analysis. METHODS: MEDLINE, EMBASE, LILACS, CENTRAL COCHRANE, and EBSCO databases were searched for comparative studies, which included early CRC patients subjected to surgical or endoscopic treatments. Data regarding mortality, recurrence, en bloc resection rate, curative resection rate, procedure time, complications rate, and severe complications rate were extracted. Study bias was evaluated using the Newcastle-Ottawa score. Absolute Risk (AR), AR reduction (ARR), AR increase (ARI), and the number necessary to treat (NNT) or to harm (NNH) were computed, using a 95% confidence interval (CI) as statistically significant. RevMan 5 software was used for meta-analysis, applying Mantel-Haenszel tests to achieve a 95% CI; results were represented by forest and funnel plots. Consistency among studies was reported via I2. RESULTS: 12,905 entries were found via PUBMED, and 12,818 via other databases. After removal of duplicates and observation of inclusion and exclusion criteria, 4 retrospective cohort studies were considered for analysis. No differences in mortality (339 patients) and long-term recurrence (577) were observed between the two groups. Surgical treatment had higher rates of en bloc resection (-0.11 [95% CI -0.14, -0.09, P < 0.05] - 1,085 patients) and curative resection (-0.11 [IC 95% CI -0.21, - 0.02, P < 0.05] - 1,112 patients) when compared to endoscopic treatment. Endoscopic treatment had lower total complication rate (0.07 [IC 95% -0.10; - 0.03] - 1,085 patients), lower severe complication rate (-0.08 [IC 95% -0.15; - 0.01] - 827 patients), and shorter procedure time than the surgical treatment (-130.34 min [IC 95% -190.5; -70.17, P < 0.05] - 827 patients). CONCLUSION: According to available data, surgical treatment of early CRC yields better results regarding en bloc resection and curative resection rates; endoscopic treatment of early CRC results in lower complication rate and shorter procedure time. There are no differences in mortality and long-term recurrence rates

INTRODUÇÃO: Não existem estudos preditivos sobre fadiga em doentes com câncer colo-retal, embora fadiga seja descrita como freqüente na população oncológica. OBJETIVO: Identificar os fatores de risco e preditivos independentes de fadiga em doentes com câncer colo-retal. MÉTODO: Estudo preditivo que envolveu amostra não-probabilística de 157 pacientes adultos ambulatoriais com tumor primário de cólon ou reto (idade média 60±11,7 anos; 54% homens; média de anos de escolaridade 10,7±5,4 anos; estádio IV 44,8%), atendidos em quatro serviços de oncologia do município de São Paulo, Brasil (julho/2006 a julho/2007). Os pacientes preencheram a Ficha de Identificação, a Escala de Fadiga de Piper-Revisada (0-10; ponto de corte: >4; ?=0,94), o Inventário de Depressão de Beck (0-63; ponto de corte: >13; ?=0,83), a Escala de Karnofky (0%-100%; ponto de corte: <80), a Escala de Prejuízo do Sono (0-10; ponto de corte: >5) e a Escala de Dor (0-10; ponto de corte: >6). Os pontos de corte foram estabelecidos pela análise da curva ROC (Receiver Operating Characteristic), com exceção do ponto de corte de fadiga, que foi estabelecido após análise da distribuição dos escores em percentis e do critério proposto pela National Comprehensive Cancer Network. RESULTADOS: Fadiga foi referida por 26,8% dos doentes. Os fatores de risco para fadiga foram os seguintes: serviço de saúde público, dor, prejuízo do sono, depressão e capacidade funcional prejudicada (p<0,05). A análise de regressão logística identificou três fatores preditivos: depressão, capacidade funcional e prejuízo do sono. A depressão aumentou em 4 vezes a chance de ocorrer fadiga (OR: 4,2; IC95% 1,68-10,39), a capacidade funcional aumentou em 3 vezes (OR: 3,2; IC95% 1,37-7,51) e o prejuízo do sono também em 3 vezes (OR: 3,2; IC95% 1,30-8,09). Quando os três fatores preditivos estiveram presentes, a probabilidade de ocorrer fadiga foi de 80%, o que indicou boa capacidade de predição. Quando os três fatores preditivos estiveram ausentes, a probabilidade de ocorrer fadiga foi de 8%. A especificidade e sensibilidade do modelo foram de 81,9% e 58,6%, respectivamente, indicando baixa chance de falsos positivos e alta chance de falsos negativos. CONCLUSÕES: Depressão, capacidade funcional e prejuízo do sono foram preditores de fadiga. O estudo disponibiliza tabela de probabilidade de predição de fadiga e propõe que, por meio da avaliação da depressão, da capacidade funcional e do prejuízo do sono, é possível conhecer a probabilidade do paciente ter fadiga, o que é inédito nessa população e de grande utilidade na clínica / INTRODUCTION: There are no studies that identify the predictive factors of fatigue among colo-rectal cancer patients, although fatigue is described as a frequent problem in the oncology setting. AIM: Identify risk factors and independent predictors of fatigue in colo-rectal cancer patients. METHOD: Predictive study that involved non-probabilistic sample of 157 adult outpatients with primary colon or rectal (mean age 60±11.7 years; 54% male; educational level 10.7±5.4 years; cancer stage IV 44.8%), recruited from 4 oncology clinics in Sao Paulo, Brazil (July/2006 to July/2007). Patients filled out an Identification Profile, Piper Fatigue Scale-revised (0-10; cut-score: >4, ?=0,94), Beck Depression Inventory (0-63; cut-score: >13, ?=0,83), Karnofsky Scale (0%-100%; cut-score: <80%), Sleep disturbance scale (0-10; cut-score: >5), and Pain scale (0-10; cut-score: >6). The cut scores were established by ROC (Receiver Operating Characteristic) curves, except for fatigue cut-score, which was established after an analysis of the distribution of the scores in percentiles and of the criteria proposed by National Comprehensive Cancer Network. RESULTS: Fatigue was identified by 26.8% of the patients. The risk factors for fatigue were: public oncology service, pain, sleep disturbance, poor performance status, and depression (p<0.05). Logistic regression identified three predictors: depression, sleep disturbance, and performance status. Depression increased the chance for fatigue to occur by four times (OR: 4.2; 95%CI 1.68-10.39), performance status increased by three times (OR: 3.2; 95%CI 1.37-7.51), and sleep disturbance also increased the chance by three times (OR: 3.2; 95%CI 1.30-8.09). When the three factors were present in concomitance, the probability that patients had fatigue was 80%, which was considered a good predictive capacity. When none of the factors were present, the probability that patients had fatigue was 8%. The specificity and sensibility of this model were 81.9% and 58.6%, respectively, indicating that there is low chance of false positive and high chance of false negatives. CONCLUSIONS: Depression, performance status, and sleep disturbance were predictive factors of fatigue. The study presents a prediction table and proposes that by assessing depression, performance status and sleep disturbance, it is possible to know the probability that a patient will have fatigue. This finding is original and applicable in clinical practice

Estudos de polimorfismos podem auxiliar na detecção de pessoas com maior risco de desenvolver câncer, caracterização de evolução diferenciada e resposta distinta ao tratamento quimioterápico ou radioterápico. O conhecimento de como estão distribuídas as frequências genotípicas é relevante quando se estuda uma população específica. Neste trabalho foi analisado os polimorfismos da enzima metilenotetrahidrofolatoredutase (MTHFR) em pacientes com Câncer Colorretal (CCR). A MTHFR tem papel importante no metabolismo do folato, metilação e síntese do DNA. A metilação do DNA desempenha um papel crítico no controle da atividade gênica. As vias de metilação e variações do gene MTHFR podem afetar o desenvolvimento do câncer e prognósticos de doenças, com isso seus efeitos precisam ser monitorados de perto no tratamento do câncer. Os genótipos variantes dos polimorfismos MTHFR677 C >T e MTHFR1298 A>C do gene da MTHFR estão associados à diminuição importante da atividade desta enzima. Este trabalho teve como objetivo verificar a frequência dos polimorfismos MTFR (677C > T e 1298A > C) em pacientes com adenocarcinomacolorretal e analisar estas frequências com os dados clinicopatológicos, incluindo-se: sexo, idade, localização tumoral, tipo histológico, antecedentes de tabagismo e alcoolismo e sobrevida dos pacientes. Duzentos e vinte cinco pacientes com o diagnóstico de adenocarcinomacolorretal, histologicamente confirmado, admitidos no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo formam o grupo caso. Utilizou-se a análise do PCR em Tempo Real para determinar os genótipos os polimorfismos através dos ensaios TaqMan ® SNP GenotypingAssay. Os resultados encontrados foram associados aos dados epidemiológicos e clinicopatológicos dos pacientes. As populações estão em equilíbrio de Hardy-Weinberg. Determinaram-se as frequências dos polimorfismos MTFR (677C > T e 1298A > C) em pacientes com adenocarcinomacolorretal onde foram levantadas e agrupadas por genótipos assim como a significância. Na análise das razões de risco de óbito por CCR na presença dos genótipos estudados não foram encontrada associações estatisticamente significativas. A curva de sobrevida comparando os genótipos no teste de Long Rank para os SNPs MTHFR 677C > T e 1298A > C não mostraram diferenças significativas na sobrevida global para pacientes com CCR. Conclui-se que as frequências dos polimorfismos MTFR (677C > T e 1298A > C) em pacientes com adenocarcinoma colorretal foram levantadas e metade dos indivíduos apresentaram frequências genotípicas de homozigotos selvagens nos dois polimorfismos estudados (CC e AA), após as associações dos polimorfismos mencionados com os dados clinicopatológicos, sexo, idade, localização tumoral, tipo histológico, antecedentes de tabagismo e alcoolismo e sobrevida não houve associações estatisticamente significativas / Polymorphism studies may help to detect people at higher risk of developing cancer, characterization of differentiated evolution, distinct response to chemotherapeutic or radiotherapeutic treatment, knowledge of how genotype frequencies are distributed becomes necessary for any work with a specific population. In this work, the polymorphisms of the enzyme methylenetetrahydrofolatoreductase (MTHFR) were analyzed in patients with Colorectal Cancer (CRC). MTHFR plays an important role in folate metabolism, methylation and DNA synthesis. DNA methylation plays a critical role in the control of gene activity. Methylation pathways and variations of the MTHFR gene may affect the development of cancer and prognosis of diseases, so their effects need to be closely monitored in the treatment of cancer. The variant genotypes of the MTHFR677 C > T and MTHFR1298 A > C polymorphisms of the MTHFR gene are associated with a significant decrease in the activity of this enzyme. The aim of this study was to verify the frequency of MTFR polymorphisms (677C > T and 1298A > C) in patients with adenocarcinomes and to analyze these frequencies with clinicopathological data, including: sex, age, tumor location, histological type, history of smoking and alcoholism and patient survival. Two hundred and twenty five patients with the diagnosis of histologically confirmed adenocarcinomes were admitted to the Hospital das Clínicas of the Faculty of Medicine of the University of São Paulo, forming the case group. Real-time PCR analysis was used to determine the genotype polymorphisms through the TaqMan ® SNP Genotyping Assay assays. The results were associated with the epidemiological and clinicopathological data of the patients. The populations are in Hardy-Weinberg equilibrium. The frequencies of the MTFR polymorphisms (677C > T and 1298A > C) were determined in patients with adenocarcinoma-choroid where they were raised and grouped by genotypes as well as significance. The analysis of death risk ratios by RCC in the presence of the studied genotypes, there was no statistically significant associations were found. The survival curve comparing the genotypes in the Long Rank test for MTHFR 677C > T and 1298A > C SNPs did not show significant differences in overall survival for CRC patients. It was concluded that the frequencies of MTFR polymorphisms (677C > T and 1298A > C) in patients with colorectal adenocarcinoma were raised and half of the individuals presented genotype frequencies of wild homozygotes in the two polymorphisms studied (CC and AA), after associations of polymorphisms mentioned, there was no statistically significant association between these polymorphisms and the variables studied: sex, age, tumor location, histological type, history of smoking and alcoholism and survival

INTRODUÇÃO: Aproximadamente 50% dos pacientes com tumor colorretal apresentam metástase hepática e a hepatectomia é o procedimento terapêutico de escolha. Discutem-se diversos fatores prognósticos, entre eles a margem cirúrgica é um fator sempre recorrente, pois não existe consenso da distância mínima necessária entre o nódulo metastático e a linha de secção hepática. Alguns autores identificaram que a margem cirúrgica maior que 1cm é um fator de melhor prognóstico com maior sobrevida e menor recidiva. Contudo, outros estudos demonstram resultados semelhantes entre pacientes com margens cirúrgicas maiores que 1cm, exíguas e até mesmo microscopicamente acometidas. Essas controvérsias conduzem à idéia de que outros fatores biológicos possam estar envolvidos na fisiopatologia de recorrência. Assim sendo, é de grande importância a avaliação da relação das margens cirúrgicas de ressecções hepáticas de metástases de câncer colorretal com a sobrevida e recidiva da doença. OBJETIVOS: Avaliar as margens cirúrgicas e o tipo de borda tumoral nas ressecções de metástases hepáticas de câncer colorretal e sua correlação com recidiva local e sobrevida. MÉTODOS: Estudo retrospectivo, baseado na revisão dos prontuários de 91 pacientes submetidos à ressecção de metástases hepáticas de neoplasia colorretal, durante o período compreendido entre janeiro de 2000 e dezembro de 2009. Revisão histopatológica prospectiva de todos os casos com aferição da menor margem cirúrgica e classificação das bordas tumorais como expansiva ou infiltrativa. RESULTADOS: Não houve diferença estatística nas taxas de recidiva e no tempo de sobrevivência global entre as margens livres e acometidas, assim como não houve diferença entre as margens subcentimétricas e maiores de 1cm. A sobrevida livre de doença dos pacientes com margens microscopicamente acometidas foi significativamente menor do que os pacientes com margens livres (p=0,002). A análise multivariada identificou o tipo de borda infiltrativa como fator de risco para recidiva (0,05). A sobrevida livre de doença foi significativamente menor nos pacientes com borda infiltrativa em comparação com os tumores com bordas expansivas (p=0,05). CONCLUSÕES: As ressecções de metástase hepática com margens livres de doença, independentemente da distância da margem, não influencia na recidiva tumoral (hepática ou extra-hepática) ou sobrevida do paciente. A borda tumoral do tipo infiltrativa foi fator de risco para recidiva / INTRODUCTION: Approximately 50% of patients with colorectal cancer have liver metastases and hepatectomy is the therapeutic procedure of choice. Surgical margin is an ever-recurring discussed prognostic factor, because there is no consensus of the minimum required distance between the metastatic nodule and the liver section line. Some authors reported surgical margin larger than 1 cm as a better prognosis factor ensuring longer survival rates and lower recurrence. However, other studies showed similar outcomes among patients with surgical margins larger than 1 cm, narrow margins and even microscopically affected ones. These controversies led the idea that other biological factors may be involved in the pathophysiology of recurrence. Therefore, it is valuable to assess the relationship between surgical margins of liver resection for colorectal cancer metastases with survival and recurrence. OBJECTIVES: To evaluate the surgical margins size and tumors growth pattern of colorectal liver metastases and its correlation with local recurrence and survival. METHODS: A retrospective study based on review of medical records of 91 patients undergoing resection of colorectal liver metastases during the period between January 2000 and December 2009. In addition, we undertook a detailed pathologic analysis of each pathological specimen with record of the closest surgical margins and tumors growth pattern classification as pushing or infiltrative. RESULTS: There was no statistical difference in recurrence rates and overall survival time between positive or negative margins, as well as no difference between the margins of 1cm width or more with subcentimeter margins. The disease-free survival of patients with microscopically positive margins was significantly lower than patients with negative margins (p = 0.002). Multivariate analysis identified infiltrative tumor growth pattern as a risk factor for recurrence (p=0.05). Disease-free survival was significantly lower in patients with infiltrative growth pattern compared to tumors with expansive margins (p = 0.05). CONCLUSIONS: Colorectal liver metastases resection with negative margins, regardless of width, has no influence on recurrence (hepatic or extrahepatic), neither on patient survival. The infiltrative tumor growth pattern type was a risk factor for recurrence

TCP-1是一种新型的定向于肿瘤血管的多肽，通过小鼠体内的噬菌体展示技术筛选得到。在之前的研究中，我们已证明TCP-1具有定向于肿瘤血管并有效靶向运输抗肿瘤药物和显像剂的特性。本研究的目的是进一步研究在原位结肠癌模型中定向运输抗肿瘤药物肿瘤坏死因子(TNFα)，以及在结肠癌临床样本中运输显像剂异硫氰酸荧光素(FITC)的能力。并对TCP-1与肿瘤坏死因子的融合蛋白TCP-1/TNFα的抗肿瘤机制进行阐述。 / 本研究中，我们首先尝试用TCP-1作为载体，将增强绿色荧光蛋白靶向运输至肿瘤血管。结果证明TCP-1可以成功将蛋白运输到在肿瘤血管而非其它正常的组织器官上。TCP-1还可以靶向运输肿瘤坏死因子并增强其抗肿瘤作用。和肿瘤坏死因子比较，融合蛋白TCP-1/TNFα处理组的凋亡细胞数量增多，肿瘤微血管数目减少，并且无明显毒副作用。与结肠癌的一线化疗药物5-氟尿嘧啶(5-FU)联合给药后，与TNFα与5-FU联合给药相比较，融合蛋白TCP-1/TNFα联合5-FU在以下方面具有更明显的作用：抑制肿瘤生长，增加肿瘤细胞凋亡和抑制肿瘤细胞增殖，促进肿瘤血管正常化，升高瘤内免疫细胞以及减轻骨髓和脾内的免疫抑制反应。经检测TCP-1的靶向运输增加了瘤内的TNFα以及5-FU的浓度。这些都表明TCP-1不但可以靶向运输TCP-1/TNFα至肿瘤血管，还可以增加CD8+细胞的浸润增加瘤内免疫反应以及增加血管对抗肿瘤药物的通透性。以上都对抗肿瘤起到重要作用。 / 在临床的结肠癌样本中，TCP-1对肿瘤血管的结合能力也得到了证实。48.98%的结肠癌样本对TCP-1的结合为阳性。统计学分析显示TCP-1的结合与结肠癌的分期和肿瘤位置有关，对于N2期，位于乙状结肠的肿瘤的结合尤为明显。本研究的主要目的是将分离鉴定出的TCP-1发展成为结肠癌的生物标记，并且作为运输抗肿瘤药物和显像剂的载体应用于结肠癌的诊断和治疗中。鉴于TCP-1的靶向运输特点，将会有机会研发更多的抗肿瘤药物，同时增强传统化疗药的抗肿瘤作用。这些都可以优化肿瘤治疗的方案。综上所述，TCP-1是一种在结肠癌治疗诊断中具有广阔前景的多肽。 / TCP-1 is a novel vasculature-targeting peptide. It was discovered through the in vivo phage library selection in mice. It was demonstrated that TCP-1 peptide exhibited a homing ability to the neovasculature of colon tumors and was capable of efficiently delivering imaging agents and chemotherapeutic drugs to this target site. The current study is to further investigate the targeting ability of TCP-1 to deliver a known immunomodulator, tumor necrosis factor α (TNFα) as an example of anti-cancer drug in an orthotopic colorectal cancer (CRC) model and fluorescein isothiocyanate (FITC) as imaging agent for testing the binding capacity for tumors in colorectal cancer patients. The mechanisms for the action of this novel biologic TCP-1/TNFα in the treatment of colon cancer in mice were also defined. / In this study, we observed that TCP-1 peptide delivered enhanced green fluorescent protein (EGFP) only to tumor blood vessel other than normal organs after TCP- 1/EGFP injection. This was not observed after EGFP injection. This finding showed that TCP-1 can deliver biologic protein to the tumor blood vessels. Furthermore, results from TNFα or TCP-1/TNFα targeted delivery experiments showed that TCP- 1/TNFα displayed stronger anti-cancer effects than TNFα alone on the induction of apoptosis and reduction in number of microvessels in the tumors, without significant effect in systemic toxicity. In the combined therapy with 5-fluorouracil (5-FU), a standard drug for colon cancer treatment, pretreatment with low dose (1 ng TNFα /mouse) of TNFα or TCP-1/TNFα potentiated the anti-cancer action of 5-FU. In this regard, TCP-1/TNFα could significantly reduce tumor size and weight, increase number of apoptotic cells, inhibit tumor cell proliferation, normalize tumor blood vessels, facilitate infiltration of immune cells to tumor mass and attenuate immunosuppression in bone marrow and spleen. Moreover, TCP-1 could significantly increase intratumoral levels of TNFα and 5-FU. It was also suggested that TCP-1 could selectively deliver TNFα to the tumor blood vessels and modulate the immune response by increasing CD8+ cells infiltration to tumors and increase vascular permeability to 5-FU. These observations may be the key actions to reduce tumor growth. / The binding ability of TCP-1 was also detected in clinical samples from colorectal cancer patients in which 24/49 (48.98%) tumor tissues were positive with TCP-1 binding signal. Statistical analysis showed that TCP-1 had a strong and significant binding with colorectal cancer at the N2 stage among the different colorectal cancer stages (P=0.028) and location in the colon at the sigmoid (P<0.001). / Our study also focused on the isolation and identification of the binding molecule of TCP-1 in order to develop it into a biomarker for CRC and using TCP-1 as a carrier in delivering anti-cancer drugs and imaging agents to colon tumors for cancer therapy and diagnosis. With the homing property of TCP-1 on colon tumor blood vessels, new types of anti-cancer drugs will be developed and their combinations with conventional chemotherapy drugs will optimize the therapeutic outcome and improve regimen of treatment for CRC. Taken together, TCP-1 peptide appears to be a promising agent in molecular imaging and drug delivery for CRC diagnosis and therapy. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Lu, Lan. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 157-177). / Abstracts also in Chinese. / Lu, Lan.

CFTR基因突變或者功能缺失是否導致包括胃腸道在內的各種組織惡性腫瘤的發生風險增加目前仍然是一個充滿爭議的問題。同時，眾所周知，緊密連接分子在腫瘤發生和轉移的過程發揮了關鍵的作用。本論文首次發現了CFTR基因與一種緊密連接分子AF-6/afadin的在人類結直腸腫瘤中的表達水平呈高度相關，并研究了CFTR和AF-6/afadin之間潛在的相互作用及其在結直腸腫瘤轉移中的功能。 / 論文的第一部份首先用實時定量PCR和免疫組織化學的方法比較了CFTR在結直腸腫瘤和正常組織的表達情況，發現CFTR表達水平在腫瘤組織中有顯著的下降。令人感興趣的是，我們同時發現CFTR和AF-6/afadin在腫瘤組織中的表達呈高度正相關，并由此展開了後續的體外實驗，研究對CFTR與AF-6/afadin之間可能的相互聯繫。利用免疫螢光染色和免疫共沉澱的方法，我們發現了這兩種蛋白分子共表達在結直腸腫瘤細胞的接觸面，并存在相互作用。用CFTR突變蛋白的免疫共沉澱實驗進一步發現，這種相互作用需要CFTR分子在細胞膜表面的正確定位及其PDZ結構域結合位點。實驗還發現與CFTR的相互作用加強了AF-6/afadin與細胞骨架蛋白系統的結合。在結直腸腫瘤細胞中CFTR基因敲减导致了AF-6/afadin蛋白定位混亂，從細胞連接位點轉移到細胞漿內，并因此破壞了上皮細胞的緊密性。極性生長細胞的跨上皮電阻降低而滲透性增強的實驗結果證實了CFTR基因敲減導致的上皮細胞緊密性的破壞。同時，AF-6/afadin蛋白水平也隨著CFTR基因敲減而降低，但mRNA水平未發生明顯的改變。蛋白降解系統的抑製劑逆轉了CFTR基因敲減細胞中AF-6/afadin蛋白的減少，提示CFTR基因敲減增加了AF-6/afadin的蛋白降解。這些實驗結果揭示了通過與細胞連接分子AF-6/afadin的相互作用以及調節，CFTR可能在上皮細胞極性的調節以及腫瘤發展過程中起重要作用。 / 論文的第二部份研究了CFTR和AF-6/afadin在結直腸腫瘤細胞上皮細胞間充質化（EMT）和轉移過程中的功能及機制。我們之前的工作已經揭示抑制CFTR的功能可以誘導結直腸腫瘤LIM1863細胞的EMT過程。本研究在另外三株不同的結直腸腫瘤細胞（SW480，SW1116和HRT-18）中進一步證實了抑制CFTR誘導的EMT過程。細胞形態轉變，上皮細胞標誌物的下調，間充質細胞標誌物的上調以及受損的上皮細胞緊密性均證實了對CFTR的抑制可以在這三種細胞中成功誘導EMT的發生。我們發現在以上所有細胞EMT的過程中，AF-6/afadin的蛋白表達水平都發生了顯著的下調。在HRT-18細胞中過表達AF-6/afadin，可以逆轉由CFTR抑製劑誘導的上皮細胞標誌分子的下調和間充質標誌分子的上調，表明抑制CFTR誘導的EMT過程是由AF-6/afadin參與介導的。此外，CFTR基因敲減導致結直腸腫瘤細胞的惡性表型強化，包括減弱的細胞粘附性，增強的貼壁依賴性生長、侵襲和遷移。另外，CFTR基因敲減激活了ERK的磷酸化，過表達AF-6/afadin可以阻斷ERK途徑的激活。CFTR基因敲減而增強的細胞侵襲性也可以被外源性AF-6/afadin或者ERK途徑的抑製劑U0126完全逆轉，提示作為AF-6/afadin的下游靶信號，ERK介導了CFTR在腫瘤侵襲中的作用。更重要的是，我們分析了CFTR和AF-6/afadin的表達水平與結直腸癌病人腫瘤進展的關係，發現在嚴重TNM腫瘤分期或者有腫瘤遠處轉移的病人中CFTR的表達水平顯著低於輕型分期或未发生转移的病人中的水平，而且CFTR和/或AF-6/afadin低表達的病人的預後更差。這些實驗結果顯示CFTR的缺失可能通過抑制AF-6/afadin和激活ERK通路而與EMT和結直腸癌癥轉移的過程高度相關。 / 綜上所述，本研究揭示了以往未報道過的CFTR在結直腸腫瘤發病機理中的功能，提示CFTR可以用作一種新的腫瘤的潛在預後指標。 / The question whether mutation or dysfunction of CFTR increases the risk of malignancies in various tissues, including the gastrointestinal tract, remains highly controversial. Meanwhile, it is well-known that adherens junctions play critical roles in the process of cancer development and metastasis. In this thesis we found for the first time a highly correlation between expression levels of CFTR and an adherens junction molecule AF-6/afadin in human colorectal tumours, and investigated the potential interaction between CFTR and AF-6/afadin and their functional roles in the metastasis of colorectal cancer. / In the first section of this thesis, we started our studies with comparing the expression of CFTR between human colorectal tumours and normal colorectal tissues. Real time quantitative PCR and immunohistochemistry results revealed a dramatically reduced CFTR level in the cancer tissues. Intriguingly, we noticed a highly positive correlation between CFTR and AF-6/afadin expression in tumours, which prompted the further in vitro investigation of possible interaction between CFTR and AF-6/afadin. Using immunofluoresent staining and co-immunoprecipitation, we found that the two proteins were colocalized at cell-cell junctions and interacted with each other in colorectal cancer cell lines. Further Co-IP experiments performed with CFTR mutations revealed that this protein interaction requires the proper localization of CFTR in cell membrane and its PDZ-interacting domain. Moreover the interaction with CFTR strengthens the binding of AF-6/afadin to the cytoskeleton system. Knockdown of CFTR in colorectal cancer cells resulted in the disorganized localization of AF-6/afadin protein from junctional sites to the cytoplasm and impaired epithelial tightness, which was confirmed by significantly reduced transepithelial resistance and increased permeability of polarized cells. Meanwhile, the protein level of AF-6/afadin was down-regulated in CFTR-knockdown cells, while no significant changes were detected at the mRNA level. Protein degradation inhibitor reversed the repression of AF-6/afadin protein in CFTR knockdown cells, suggesting the protein degradation of AF-6/afadin was increased by CFTR knockdown. These data revealed that CFTR interacts with and regulates the cell adhesion molecular AF-6/afadin in colorectal cells, which may be important in the regulation of epithelial cell polarity and cancer development. / In the second section of this thesis, we studied the functional roles and mechanisms of CFTR and AF-6/afadin in the epithelial-mesenchymal transition (EMT) and metastasis of human colorectal cancer cells. Our previous work has revealed inhibition of CFTR can induce EMT in a colorectal cancer cell line, LIM1863. This study further confirmed the induction of EMT by inhibiting CFTR in several other colorectal cancer cell lines (SW480, SW1116 and HRT-18), which was evaluated by morphological changes, down-regulation of epithelial markers or up-regulation of mesenchymal markers, and impaired epithelial cell tightness. In all these cell lines, we found that the protein levels of AF-6/afadin were significantly reduced. Over-expression of AF-6/afadin in HRT-18 cells reversed the down-regulated epithelial markers and up-regulated mesenchymal markers induced by CFTR inhibition, indicating that the CFTR inhibition-induced EMT is mediated by AF-6/afadin. Moreover, knockdown of CFTR in HRT-18 or RKO cells resulted in enhanced malignant phenotypes, including decreased cell adhesion, increased anchorage-independent cell growth, invasion, and migration. In addition, extracellular signal-regulated kinase (ERK) phosphorylation was activated by CFTR knockdown, which was abolished by over-expression of AF-6/afadin. The enhanced invasiveness of CFTR knockdown cells was also completely inhibited by either exogenous AF-6/afadin or ERK inhibitor, U0126, suggesting that ERK, the downstream target of AF-6/afadin, is involved in mediating the effect of CFTR in cancer invasion. More importantly, we analyzed the association of CFTR and AF-6/afadin expression levels with tumour progression of patients with colorectal cancer, and revealed that CFTR expression was significantly lower in patients with more severe TNM stage or with metastasis to distant organs than those with milder stage or with no metastasis. The prognosis was poorer in patients with lower expression of CFTR and/or AF-6/afadin than those with higher expressions. These data showed that dysfunction of CFTR is highly associated with EMT and colorectal cancer metastasis, probably via repression of AF-6/afadin and activation of ERK pathways. / In summary, the present study has revealed a previously undefined role of CFTR in the pathogenesis of colorectal cancer and indicated its potential as a new prognostic indicator. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Sun, Tingting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 113-127). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 中文摘要 --- p.iv / Publications --- p.vi / Conference Abstract --- p.vii / Declaration --- p.viii / Acknowledgements --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / List of Abbreviations --- p.xiv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1. --- Colorectal Cancer --- p.1 / Chapter 1.1.1. --- Structure of Human Normal Colon and Rectum Epithelium --- p.1 / Chapter 1.1.2. --- Staging of Colorectal Cancer --- p.3 / Chapter 1.1.3. --- Metastasis of Colorectal Cancer --- p.3 / Chapter 1.1.4. --- K-Ras mutation and It Downstream Pathways in Colorectal Cancer Metastasis --- p.11 / Chapter 1.1.5. --- Prognosis of Colorectal Cancer --- p.14 / Chapter 1.2. --- Epithelial Cell Junctional Complexes --- p.14 / Chapter 1.2.1. --- Junctional Complexes and Epithelial Cell Polarity --- p.15 / Chapter 1.2.2. --- Classic Cadherin-catenin Complex --- p.17 / Chapter 1.2.3. --- Novel Nectin-afadin Complex --- p.19 / Chapter 1.2.4. --- Cell Polarity and Cancer Progression --- p.23 / Chapter 1.3. --- Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) --- p.24 / Chapter 1.3.1. --- Structure of CFTR --- p.24 / Chapter 1.3.2. --- Mutations of CFTR --- p.24 / Chapter 1.3.3. --- Functions of CFTR --- p.26 / Chapter 1.3.4. --- Cancer Risk of CF Patients --- p.33 / Chapter 1.4. --- Hypothesis and Aims --- p.34 / Chapter Chapter 2 --- Materials and Methods --- p.35 / Chapter 2.1. --- Materials --- p.35 / Chapter 2.1.1. --- Reagents and Chemicals --- p.35 / Chapter 2.1.2. --- Antibodies --- p.35 / Chapter 2.1.3. --- Primers --- p.35 / Chapter 2.1.4. --- Solutions and Buffers --- p.35 / Chapter 2.1.5. --- Human Specimens --- p.36 / Chapter 2.2. --- Methods --- p.36 / Chapter 2.2.1. --- Cell Culture --- p.36 / Chapter 2.2.2. --- Transfection --- p.36 / Chapter 2.2.3. --- Selection of Stable Clones --- p.40 / Chapter 2.2.4. --- RNA Extraction and RT-PCR --- p.40 / Chapter 2.2.5. --- Quantitative Real Time PCR --- p.41 / Chapter 2.2.6. --- Protein Extraction and Western Blotting --- p.42 / Chapter 2.2.7. --- Immunostaining --- p.45 / Chapter 2.2.8. --- In vitro Cell Functional Assays --- p.46 / Chapter 2.2.9. --- Epithelial Tightness Measurement --- p.48 / Chapter 2.2.10. --- Statistical Analysis --- p.49 / Chapter Chapter 3 --- Interaction of CFTR with AF-6/afadin and Its Importance in Maintaining Colorectal Epithelial Cell Polarity --- p.50 / Chapter 3.1. --- Introduction --- p.50 / Chapter 3.2. --- Objectives --- p.53 / Chapter 3.3. --- Experimental plan --- p.54 / Chapter 3.4. --- Results --- p.55 / Chapter 3.4.1. --- The expression of CFTR and AF-6/afadin is decreased and positively correlated in human colorectal cancer --- p.55 / Chapter 3.4.2. --- CFTR colocalizes and interacts with AF-6/afadin in human colorectal cancer cells --- p.58 / Chapter 3.4.3. --- PDZ binding motif and membrane localization of CFTR are necessary for the interaction between CFTR and AF-6/afadin --- p.64 / Chapter 3.4.4. --- Knockdown of CFTR interferes with cell junction formation in colorectal cancer cells --- p.66 / Chapter 3.5. --- Discussion --- p.71 / Chapter Chapter 4 --- CFTR as a Suppressor and Prognosis Indicator of Metastasis in Human Colorectal Cancer --- p.77 / Chapter 4.1. --- Introduction --- p.77 / Chapter 4.2. --- Objectives --- p.80 / Chapter 4.3. --- Experimental plan --- p.81 / Chapter 4.4. --- Results --- p.82 / Chapter 4.4.1. --- CFTR inhibition-induced EMT in colorectal cancer cells involves AF-6/afadin --- p.82 / Chapter 4.4.2. --- Knockdown of CFTR aggravates malignant phenotype of colorectal cancer cells --- p.86 / Chapter 4.4.3. --- AF-6/afadin mediates the effect of CFTR on cell invasion in colon cancer through ERK --- p.91 / Chapter 4.4.4. --- CFTR and AF-6/afadin expression is correlated with the prognosis of colorectal cancer --- p.97 / Chapter 4.5. --- Discussion --- p.100 / Chapter Chapter 5 --- General Discussion and Conclusion --- p.105 / Chapter 5.1. --- The diversified roles of CFTR in epithelial cells --- p.105 / Chapter 5.2. --- The unfolding relationship between CFTR and cancer development --- p.107 / Chapter 5.3. --- Future studies --- p.109 / Chapter 5.4. --- Conclusions --- p.112 / Reference List --- p.113 / Chapter Appendix A --- Reagents and Chemicals --- p.128 / Chapter Appendix B --- Antibody List --- p.131 / Chapter Appendix C --- Primer List --- p.132 / Chapter Appendix D --- Solution Recipe --- p.133