Thesis--Copenhagen. / Summary in Danish. Includes bibliographical references (p. 276-309) and index.

Thesis--Copenhagen. / Summary in Danish. Includes bibliographical references (p. 276-309) and index.

O câncer de laringe é uma das neoplasias mais comuns das vias aéreas superiores. No Brasil a maioria dos casos são diagnosticados nos estádios clínicos III ou IV. Nos tumores T4a os resultados terapêuticos são discordantes na literatura. Alguns estudos referem melhores taxas de sobrevida global com o tratamento cirúrgico e outros demonstraram taxas de sobrevida global semelhantes entre pacientes submetidos a laringectomia total e os submetidos a quimioradioterapia. Concomitante ao surgimento destas novas modalidades terapêuticas, descritas nas últimas décadas, observou-se nos Estados Unidos queda nas taxas de sobrevida dos pacientes com tumores de laringe. O objetivo deste estudo foi avaliar fatores prognósticos em pacientes portadores de carcinoma epidermóide de laringe cT4aN0-3M0 tratados em quatro instituições - em três diferentes países - de acordo com variáveis demográficas, clínicas, terapêuticas e anatomopatológicas. Em relação as variáveis clínicas, foram observadas piores taxas de sobrevida global nos pacientes com mais de 65 anos, nos classificados como ASA III ou IV, nos casos com tumores com extensão para base de língua. Dentre os pacientes submetidos ao tratamento cirúrgico as piores taxas de sobrevida global foram observados nos casos com margens de ressecção comprometidas, nos com invasão vascular presente e os com estádio linfonodal pN2 ou pN3. As taxas de sobrevida global e câncer específica não diferiu entre os pacientes submetidos ao tratamento cirúrgico ou quimioradioterápico. Por outro lado as melhores taxas de sobrevida livre de recorrência local foi evidenciada nos pacientes submetidos ao tratamento cirúrgico. Na análise multivariada, foram fatores prognósticos independentes para o risco de óbito, a classificação pelo ASA (III ou IV) e o estadiamento linfonodal patológico (pN2 ou pN3) entre os pacientes submetidos à cirurgia, a faixa etária superior a 65 anos, a extensão do tumor para base de língua, para partes moles perilaríngeas e a presença de mais de uma estrutura invadida pelo tumor nos pacientes submetidos a quimioradioterapia. Em conclusão, diversas variáveis são potenciais fatores prognósticos relacionados a sobrevida global, no entando somente a classificação como ASA III ou IV e o estádio pN2 ou pN3 foram identificados como fatores prognósticos independente para o risco de óbito nos pacientes submetidos ao tratamento cirúrgico. Nos pacientes submetidos ao tratamento com quimioradioterapia a faixa etária superior a 65 anos, extensão para base de língua, para partes moles perilaríngeas e presença de duas ou mais estruturas invadidas foram os fatores prognósticos independentes para o risco de óbito / Laryngeal cancer is one of the most common neoplasms of the upper airways. In Brazil most cases are diagnosed at stages III or IV. In T4a tumors, there are discordant treatment results in the literature. Some studies refer to improved rates of overall survival with surgical treatment while other studies show similar rates of overall survival between patients submitted to total laryngectomy or chemoradiotherapy. Concomitant to the emergence of these new therapeutic modalities described in recent decades, a decline in survival rates of patients with laryngeal tumors has been registred in the United States. The aim of this study was to assess prognostic factors in patients with clinical stage cT4aN0-3M0 laryngeal epidermoid carcinomas treated in four institutions, in three different countries, according with demographic, clinical, therapeutic and pathologic variables. With regard to clinical variables, worse rates of overall survival were observed in patients older than 65 years, classified as ASA III or IV, and tumors with tongue base extension. Among the patients who underwent surgical treatment, the worse rates of overall survival were in the cases of involved surgical margins, with vascular invasion present, and in cases with pathological metastatic lymph nodes pN2 or pN3 staging. The rates of overall survival and cancer specific survival did not differ among patients who underwent surgical treatment or chemoradiotherapy. Conversely, improved local recurrence-free survival rates were registred in patients who underwent surgical treatment. In multivariate analysis, the independent prognostic factors for the risk of death, were ASA III or IV classification, and pathological lymph nodes pN2 or pN3 staging among the patients who underwent surgery; age above 65 years, tumors with tongue base extension, extension into the perilaryngeal soft tissues and the presence of more than one structure invaded by tumor in patients who underwent chemoradiotherapy. In conclusion, several variables are potential prognostic factors related too verall survival, however only ASA III or IV and stage pN2 or pN3 were in dependent prognostic factors for the risk of death in patients undergoing surgical treatment. In patients undergoing chemoradiotherapy: age above 65 years, tumors extension to the base of tongue, perilaryngeal soft tissue invasion and presence of two or more structure sinvaded were independent prognostic factors for the risk of death

O câncer de laringe é uma das neoplasias mais comuns das vias aéreas superiores. No Brasil a maioria dos casos são diagnosticados nos estádios clínicos III ou IV. Nos tumores T4a os resultados terapêuticos são discordantes na literatura. Alguns estudos referem melhores taxas de sobrevida global com o tratamento cirúrgico e outros demonstraram taxas de sobrevida global semelhantes entre pacientes submetidos a laringectomia total e os submetidos a quimioradioterapia. Concomitante ao surgimento destas novas modalidades terapêuticas, descritas nas últimas décadas, observou-se nos Estados Unidos queda nas taxas de sobrevida dos pacientes com tumores de laringe. O objetivo deste estudo foi avaliar fatores prognósticos em pacientes portadores de carcinoma epidermóide de laringe cT4aN0-3M0 tratados em quatro instituições - em três diferentes países - de acordo com variáveis demográficas, clínicas, terapêuticas e anatomopatológicas. Em relação as variáveis clínicas, foram observadas piores taxas de sobrevida global nos pacientes com mais de 65 anos, nos classificados como ASA III ou IV, nos casos com tumores com extensão para base de língua. Dentre os pacientes submetidos ao tratamento cirúrgico as piores taxas de sobrevida global foram observados nos casos com margens de ressecção comprometidas, nos com invasão vascular presente e os com estádio linfonodal pN2 ou pN3. As taxas de sobrevida global e câncer específica não diferiu entre os pacientes submetidos ao tratamento cirúrgico ou quimioradioterápico. Por outro lado as melhores taxas de sobrevida livre de recorrência local foi evidenciada nos pacientes submetidos ao tratamento cirúrgico. Na análise multivariada, foram fatores prognósticos independentes para o risco de óbito, a classificação pelo ASA (III ou IV) e o estadiamento linfonodal patológico (pN2 ou pN3) entre os pacientes submetidos à cirurgia, a faixa etária superior a 65 anos, a extensão do tumor para base de língua, para partes moles perilaríngeas e a presença de mais de uma estrutura invadida pelo tumor nos pacientes submetidos a quimioradioterapia. Em conclusão, diversas variáveis são potenciais fatores prognósticos relacionados a sobrevida global, no entando somente a classificação como ASA III ou IV e o estádio pN2 ou pN3 foram identificados como fatores prognósticos independente para o risco de óbito nos pacientes submetidos ao tratamento cirúrgico. Nos pacientes submetidos ao tratamento com quimioradioterapia a faixa etária superior a 65 anos, extensão para base de língua, para partes moles perilaríngeas e presença de duas ou mais estruturas invadidas foram os fatores prognósticos independentes para o risco de óbito / Laryngeal cancer is one of the most common neoplasms of the upper airways. In Brazil most cases are diagnosed at stages III or IV. In T4a tumors, there are discordant treatment results in the literature. Some studies refer to improved rates of overall survival with surgical treatment while other studies show similar rates of overall survival between patients submitted to total laryngectomy or chemoradiotherapy. Concomitant to the emergence of these new therapeutic modalities described in recent decades, a decline in survival rates of patients with laryngeal tumors has been registred in the United States. The aim of this study was to assess prognostic factors in patients with clinical stage cT4aN0-3M0 laryngeal epidermoid carcinomas treated in four institutions, in three different countries, according with demographic, clinical, therapeutic and pathologic variables. With regard to clinical variables, worse rates of overall survival were observed in patients older than 65 years, classified as ASA III or IV, and tumors with tongue base extension. Among the patients who underwent surgical treatment, the worse rates of overall survival were in the cases of involved surgical margins, with vascular invasion present, and in cases with pathological metastatic lymph nodes pN2 or pN3 staging. The rates of overall survival and cancer specific survival did not differ among patients who underwent surgical treatment or chemoradiotherapy. Conversely, improved local recurrence-free survival rates were registred in patients who underwent surgical treatment. In multivariate analysis, the independent prognostic factors for the risk of death, were ASA III or IV classification, and pathological lymph nodes pN2 or pN3 staging among the patients who underwent surgery; age above 65 years, tumors with tongue base extension, extension into the perilaryngeal soft tissues and the presence of more than one structure invaded by tumor in patients who underwent chemoradiotherapy. In conclusion, several variables are potential prognostic factors related too verall survival, however only ASA III or IV and stage pN2 or pN3 were in dependent prognostic factors for the risk of death in patients undergoing surgical treatment. In patients undergoing chemoradiotherapy: age above 65 years, tumors extension to the base of tongue, perilaryngeal soft tissue invasion and presence of two or more structure sinvaded were independent prognostic factors for the risk of death

Abstract Incidence trends of laryngeal cancer in Finland were analyzed, especially in relation to survival, in a patient series of 5766 patients diagnosed in 1956–1995 and identified from the Finnish Cancer Registry. The age-adjusted incidence rate decreased from 6.5 to 3.5 per 100 000 person-years in males and remained unchanged among females. Only minor improvement occurred in survival. In a hospital-based material from Northern Finland (353 patients with laryngeal squamocellular carcinoma, LSCC, diagnosed in 1976–1995), the incidence among males decreased only for supraglottic cancer, diminishing the supraglottic to glottic incidence ratio from 1.4:1 to 0.5:1. Evaluation of diagnostic delays and their impact on survival and risk of recurrence was undertaken in a sample of 66 LSCC patients. In only 38% of the patients was malignancy suspected at the initial visit to a physician; infection was the most common misdiagnosis (41%). Half of the first consultations resulted in referral, whereas 17% of the patients were neither referred nor controlled. The median patient delay was 2 months and median professional delay 3 months. The latter exceeded 12 months in 17% of the patients. The delays were not significantly related to any other clinical parameter, nor were they interrelated. Professional delay of 12 months or more resulted in increased relative hazard of death (HR = 4.74, p = 0.05), equalling the effect of advanced stage (stage IV). One-third of the patients developed a recurrence. In univariate analysis, professional delay of 12 months or more increased the risk of local (p = 0.019) and neck (p = 0.019) recurrence. In a multivariate model, professional delay of 12 months or more indicated an adjusted relative hazard ratio (HR) of 4.6 for local recurrence (p = 0.02) and 9.5 for neck recurrence (p = 0.015). Immunohistochemical factors p53, apoptosis, angiogenesis and proliferation were included in a multivariate model evaluating prognostic factors of LSCC in addition to clinical and sociodemographic factors. Advanced stage (stages III–IV) (relative hazard ratio of death (HR) 8.9, p = 0.01), supraglottic site (HR 5.6, p = 0.02) and high apoptotic index (≥ 0.3) (HR 11.1, p = 0.05) were the best indicators of impaired prognosis. Professional delay and enhanced apoptotic rate could be helpful in selecting LSCC patients for more aggressive primary treatment.

No município de São Paulo, em 1970 foram relatados 169 casos novos de câncer de laringe, com uma taxa de incidência ajustada por idade pela população mundial de 25,5/100.000 (30-74 anos) e 299 casos em 1985, elevando a taxa para 37,6 (ambos nosexo masculino). O Tabagismo e o consumo de álcool são os fatores de risco mais bem estabelecidos para o câncer de laringe. Com relação aos fatores ocupacionais, o único carcinógeno estabelecido é a exposição à névoa de ácidos inorgânicosfortes. É proposto um estudo caso-controle de base hospitalar, para investigar e quantificar o papel da exposição ocupacional no desenvolvimento do câncer de laringe e é discutida uma nova proposta de avaliação de exposição que visa melhorar avalidade interna desse tipo de estudo / Laryngeal cancer comprises 3,1% of the new cases of cancer in men in the world, representing the tenth most common malign neoplasm for males. In São Paulo City, 169 new cases of laryngeal cancer were reported in 1970, representing 25,5/100.000 and 299 cases in 1985, increasing the rate to 37,6/100.000 (both for males, age standardized truncated rate, 30-74 years). The best established risk factors for laryngeal cancer are tobacco and alcohol. In respect to occupational factors, the only established carcinogen is exposure to strong inorganic acid mists. However, asbestos, pesticides, paints, gasoline and diesel engine emissions, dusts, among other factors are reported in the literature as occupational agents that increase the risk of laryngeal cancer. A hospital-based case-control study was conducted, in seven hospitals in São Paulo, to investigate occupational risk factors for laryngeal cancer. The study included 122 laryngeal cancers and 187 controls, selected by frequency matching on sex and age. Detailed information on smoking, alcohol consumption, and occupational history was collected. Occupational hygienists assessed the exposure to 49 agents. Odds Ratios (OR) and 95% confidence intervals (95% CI) were estimated by unconditional logistic regression, and were adjusted for sex, age, smoking and alcohol. Laryngeal cancer was associated with exposure to respirable free crystalline silica (OR 1,83, 95% CI 1,00 - 3,36), soot (from coal, coke, fuel oil, wood) (OR 1,78, 95% CI 1,03 - 3,03), fumes (OR 2,55, CI 95% 1,14 - 5,67) and to live animals (OR 1,80, 95% CI 1,02 - 3,19). This study showed that occupational exposures to: respirable free crystalline silica, soot (from coal, coke, fuel oil, wood), fumes and live animals are risk factors for laryngeal cancer.

INTRODUÇÃO: A presença de metástase cervical é um dos principais fatores prognósticos em pacientes com carcinoma epidermoide de laringe. A análise rotineira dos linfonodos não inclui a avaliação da reatividade linfonodal, nem pesquisa de micrometástases, podendo ser desprezada informações prognóstica relevantes neste grupo de pacientes. OBJETIVOS: Relacionar a reatividade linfonodal com a recidiva e mortalidade de pacientes com carcinoma epidermoide de laringe estadiados como pT3 e pT4, pN0, identificar através de marcadores imuno-histoquímicos (AE1/AE3, citoqueratina 7 e citoqueratina 5/6) a incidência de micrometástases e relacionar com o prognóstico dos pacientes. MÉTODO: Entre 2002 e 2005, foram selecionados 105 pacientes estadiados como pN0 com carcinoma epidermoide de laringe submetidos à laringectomia total e esvaziamento cervical seletivo dos níveis II, III e IV bilateralmente, com a maioria dos pacientes (69) submetidos à radioterapia adjuvante. Todos os linfonodos foram analisados conforme os padrões de reatividade: hiperplasia folicular (associada à resposta imune humoral), hiperplasia paracortical (associada à resposta imune celular), histiocitose sinusal, linfonodo normal e depleção linfonodal. Apenas os dois primeiros padrões são considerados como linfonodos estimulados e os demais como não estimulados. O padrão linfonodal mais frequente de cada caso foi considerado na análise da recidiva da doença e mortalidade. Todos os linfonodos foram submetidos à análise imuno-histoquímica com o clone de citoqueratinas AE1/AE3, e nos casos de positividade utilizou-se as citoqueratinas 5/6 e 7 para a diferenciação entre micrometástases e inclusões epiteliais benignas. RESULTADOS: Foram analisados um total de 3.648 linfonodos, com uma média de 34,7 linfonodos por paciente. O padrão de reatividade mais frequente foi a histiocitose sinusal (50 casos) seguido da hiperplasia paracortical (35 casos) e hiperplasia folicular (20 casos), não havendo relação entre esta análise e a recidiva (p = 0,98) ou mortalidade (p = 0,49). A resposta imune humoral esteve relacionada com maior mortalidade (p = 0,05), com sobrevida global em 5 anos de 76%, comparado a 60% de sobrevida no grupo com imunidade celular estimulada (log-rank = 0,05). A pesquisa imuno-histoquímica utilizando o clone de citoqueratinas AE1/AE3 foi positiva em 17 pacientes (16,2%) tendo esta análise relação com a recidiva da doença (p = 0,03) e com a mortalidade (p = 0,04). No grupo com AE1/AE3 positivo, a radioterapia associou-se a uma tendência de proteção à recidiva regional (p = 0,06). A citoqueratina 5/6 confirmou a presença de micrometástases em 4 (3,81%) casos e estas não apresentaram relação prognóstica com a recidiva (p = 0,66) e mortalidade (p = 0,66). A citoqueratina 7 diagnosticou inclusões epiteliais benignas em 11 (10,47%) casos. CONCLUSÕES: Pacientes com imunidade celular estimulada (hiperplasia paracortical) apresentaram menor mortalidade comparados aos pacientes com resposta imune humoral (hiperplasia folicular) (p = 0,05). A positividade na pesquisa com o clone de citoqueratinas AE1/AE3 apresentou relação com significância com a recidiva (p = 0,03) e com a mortalidade (p = 0,04). A incidência de micrometástases na pesquisa com citoqueratina 5/6 foi de 3,86% (4 casos de 105) e de inclusões epiteliais benignas foi de 10,47% (11 casos de 105). / INTRODUCTION: The presence of neck metastasis is one of the most important prognostic factors in patients with squamous cell carcinoma of the larynx. Immunohistochemical analysis for lymph node reactivity, micrometastasis and presence of benign epithelial inclusions are not included in routine anatomopathological exam, leading to potential missing of important prognostic information in this group of patients. OBJECTIVES: 1- To analyze if lymph node reactivity is associated with recurrence and mortality in patients with pT3/pT4, pNo SCCa of the larynx. 2- To identify, with the aid of hystochemical markers AE1/AE3, cytokeratin 7 and cytokeratin 5/6), the incidence of micrometastasis and benign intraepithelial inclusions and to evaluate the relationship of these findings with the patient`s prognosis. METHODS: One hundred and five patients with SCCa of the larynx underwent total laryngectomy and bilateral selective neck dissection of the levels II, III, and IV between 2002 and 2005. Sixty-nine patients also received postoperative radiotherapy. All 105 patients were N0 at the time of diagnosis. The lymph nodes were analyzed according to their reactivity pattern: Follicular hyperplasia (associated to humoral immune response), paracortical hyperplasia (associated to cellular immune response), sinusoidal histiocytosis, normal lymph node, lymphoid depletion. Only the he first two patterns are associated with stimulated lymph nodes. The predominant pattern on each case was recorded and the relationship between these patterns and tumor recurrence and mortality was assessed. All the lymph nodes were tested for cytokeratin AE1/AE3, and the positive cases were than tested for cytokeratin 5/6 and 7 to further differentiate micrometastasis from benign epithelial inclusions. RESULTS: A total of 3648 lymph nodes were analyzed (average of 34.7 lymph nodes/patient). The most common reactivity pattern was paracortical hyperplasia (49 cases), followed by sinusoidal histiocytosis (36 cases) and follicular hyperplasia (20 cases). There was no association between these different patterns and recurrence (p=0.98) or mortality (p=0.49). The humoral immune response was associated with increased mortality (p=0.005). The 5-year overall survival was 76%, compared to 60% on the stimulated cellular response group (LogRank=0.05). Seventeen patients (16.2%) were positive for cytokeratin AE1/AE3, and this was associated with tumor recurrence (p=0.03%) and mortality (p=0.004). In the positive cytokeratin group, the use of radiation therapy showed a tendency to protect against regional recurrence (p=0.06). The use of cytokeratin 5/6 confirmed the presence of micrometastasis in 4 cases (3.81%), but this finding was not associated with local recurrence (p=0.66) or mortality (p=0.66). Cytokeratin 7 has diagnosed benign epithelial inclusions in 11 cases (10.47%). CONCLUSION: Patients with stimulated cellular immunity experienced decreased mortality when compared to humoral immune response (p=0.05). AE1/AE3 positivity was significantly related to recurrence (p=0.03) and mortality (p=0.04). The incidence of micrometastasis was 3.86% (4 cases in 105 patients), and of benign ephitelia inclusions was 10.47% (11 cases in 105 patients).

In addition, we investigated the cytotoxic effect of a widely used chemotherapeutic agent 5Fu on laryngeal squamous cell cancer cell lines and evaluated the role of p53 in 5Fu treatment. We found that the apoptosis and G1/S cell arrest mediated by 5Fu in laryngeal cancers is p53-independent but p21 WAF1/CIP1-dependent. We further demonstrated the effect of 5Fu on HPV16-associated laryngeal cancer cells. Using cytotoxicity assay and Annexin V staining, we proved that 5Fu induces apoptosis in all of the transfected cells in a dose- and time-dependent manner, suggesting that the process was not prevented by HPV16 E6 or E7. 5Fu induced the accumulation of active pRb and cyclin dependent kinase inhibitor p21WAF1/CIP1 together with an increase in Bak and Bax expression and a decrease in Bcl-2 levels in all the transfected cells. In addition, G1/S phase cell cycle arrest was associated with the antiproliferation activity of 5Fu in all cell lines. Through RT-PCR, 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in transfected UMSCC 12 cells. / Our results suggest that HPV16 E6 and E7 oncoproteins do not prevent 5Fu medicated apoptosis and G1/S cell arrest in laryngeal cancers. The anti-cancer effect of 5Fu is probably decided by the level of p21 WAF1/CIP1 while the sensitivity of laryngeal cancer cells responded to 5Fu treatment is associated with the increase of Bak or/and the decrease in Bcl-2, not with the HPV16 viral proteins and p53 status. 5Fu also presented some effects on the E6 and E7 oncoproteins of HPV16 in laryngeal cancer. However, the anti-viral effect of 5Fu still needs further investigation. / Our study indicated that (1) the evasion of apoptosis mediated by HPV16 E6 and E7 plays a critical role in laryngeal carcinogenesis; (2) HPV16 E6 or E7 plays an important role in regulating the expression of Bak, Bax and Bcl-2; (3) The degradation of Bak by HPV16 E6 is not caused by interacting with the promoter of Bak; (4) The induction of Bcl-2 is mediated through HPV16 E7; (5) HPV16 transfection does not interfere with the apoptosis and cell cycle arrest mediated by 5Fu in human laryngeal squamous cancer cells. / There is a growing body of evidence that human papillomavirus type 16 (HPV16) is involved in the development of human laryngeal cancer, especially in Chinese population. The two oncoproteins, HPV16 E6 and E7 that target host cell tumor suppressor proteins p53 and Rb respectively, may generate antiapoptotic effects and induce cell immortalization. However, the effect of both oncoproteins on apoptosis in laryngeal cancers is not completely clear. In this study, we demonstrated the possible mechanism of high risk HPV16 in laryngeal carcinogenesis and evaluated the effect of 5Fu on HPV16-positive laryngeal cancer cells. / We employed two human laryngeal cancer cell lines---UMSCC12 (with truncated p53) and UMSCC11A (with mutant but functional p53) in this study. These two cell lines were stably transfected with HPV16 E6, E7 or empty vector, pcDNA3.1, which provided a good foundation for further study on the carcinogenic mechanism of HPV16 E6 or E7 in human laryngeal cancers. Through Annexin V staining and protein stability assay, we found that the transfection of HPV16 E6 and E7 induced fewer spontaneous apoptosis in both UMSCC11A and UMSCC12 cells accompanied with enhanced protein stability of Bcl-2 and increased protein degradation of Bak. Similar results were obtained when E6- and E7-transfected cells exposed to apoptosis stimuli---TNF-alpha/CHX. These results indicate that stable transfection of E6 and E7 in human laryngeal cancer cells on one hand shortened the half-life of Bak protein, and on the other hand, enhanced the steady-state levels of Bcl-2 protein. In order to gain insight into the role of Bak and Bcl-2 in regulating apoptosis in HPV-associated laryngeal cancer cells, we performed transient transfection of Bcl-2 into E6- and E7-transfected cells. It is found that HPV16 E7 statistically enhanced the expression of Bcl-2 in laryngeal cancer, indicating that the induction of Bcl-2 require the transfection of HPV16 E7. Furthermore, Luciferase assay was performed to investigate whether the viral proteins E6 and E7 altered the stability of Bak through interaction with the promoter of Bak. Negative results were obtained, suggesting that E6 or E7 do not alter the transcription activity of Bak, indicating the degradation of Bak by E6 or E7 may be mediated through other mechanisms. / Liu Han-ching. / "August 2006." / Advisers: C. A. van Hasselt; George G. Chen. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1569. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 245-274). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Du Jing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 70-89). / Abstracts in English and Chinese. / ABSTRACT --- p.I / ACKNOWLEDGMENTS --- p.IV / PUBLICATIONS --- p.V / LIST OF FIGURES --- p.VI / LIST OF TABLES --- p.VII / ABBREVIATIONS --- p.VIII / CONTENTS --- p.X / Chapter CHAPTER ONE: --- INTRODUCTION AND LITERATURE / Chapter 1.1 --- Laryngeal carcinoma and HPV --- p.1 / Chapter 1.2 --- HPV --- p.2 / Chapter 1.3 --- Human papillomavirus E6 protein --- p.6 / Chapter 1.3.1 --- Transformation by HPV E6 --- p.7 / Chapter 1.3.2 --- Inhibition of apoptosis by E6 --- p.8 / Chapter 1.3.3 --- Alteration of gene transcription --- p.11 / Chapter 1.3.4 --- E6 interation with other proteins --- p.12 / Chapter 1.3.5 --- E6 as a therapeutic target --- p.14 / Chapter 1.4 --- HPV E7 protein --- p.15 / Chapter 1.4.1 --- Regulation of viral life cycle by HPV E7 --- p.16 / Chapter 1.4.2 --- Degradation of retinoblastoma tumor suppressor by HPV E7 --- p.18 / Chapter 1.4.3 --- Inhibition of p53 by HPV E7 --- p.22 / Chapter 1.4.4 --- Interaction with other proteins by HPV E7 --- p.24 / Chapter 1.5 --- Objective --- p.26 / Chapter CHAPTER TWO: --- GENERAL MATERIALS AND METHODS --- p.28 / Chapter 2.1 --- Materials --- p.28 / Chapter 2.1.1 --- Materials for cDNA and RNA manipulation --- p.28 / Chapter 2.1.2 --- Culture media and transfection reagents --- p.28 / Chapter 2.1.3 --- Antibodies --- p.29 / Chapter 2.1.4 --- Materials for protein manipulation --- p.29 / Chapter 2.1.5 --- Kits --- p.30 / Chapter 2.1.6 --- Instrumentation --- p.31 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Plasmid construction --- p.32 / Chapter 2.2.1.1 --- DNA preparation --- p.34 / Chapter 2.2.1.2 --- DNA ligation --- p.34 / Chapter 2.2.1.3 --- Transformation of competent E. coli --- p.35 / Chapter 2.2.2 --- Mini preparation --- p.35 / Chapter 2.2.3 --- Clone selection and confirmation --- p.37 / Chapter 2.2.4 --- Sequencing gel electrophoresis --- p.37 / Chapter 2.2.5 --- Cell culture and cytokine treatment --- p.39 / Chapter 2.2.6 --- Plasmid transfection --- p.39 / Chapter 2.2.7 --- Confirming construction of stable cell lines by RT-PCR --- p.40 / Chapter 2.2.7.1 --- Total cellular RNA extraction --- p.40 / Chapter 2.2.7.2 --- First strand cDNA synthesis --- p.41 / Chapter 2.2.7.3 --- Polymerase chain reaction (PCR) --- p.41 / Chapter 2.2.8 --- Fluorescence microscopy and imaging --- p.43 / Chapter 2.2.9 --- DNA fragmentation assay --- p.44 / Chapter 2.2.10 --- Protein detection --- p.46 / Chapter 2.2.10.1 --- Preparation of protein extract --- p.46 / Chapter 2.2.10.2 --- SDS-PAGE electrophoresis and protein transfer --- p.47 / Chapter 2.2.10.3 --- Immunoblotting analysis --- p.47 / Chapter 2.2.11 --- Statistical analysis --- p.48 / Chapter CHAPTER THREE: --- RESULTS --- p.49 / Chapter 3.1 --- Plasmid construction --- p.49 / Chapter 3.2 --- Expression of HPV16 viral oncogenes in transfected UMSCC12 --- p.51 / Chapter 3.3 --- HPV16 E6 and E7 protect apoptosis induced by TNF-alpha and CHX --- p.53 / Chapter 3.4 --- Detection of apoptosis with fluorescence staining --- p.55 / Chapter 3.5 --- Regulation of the expression of apoptosis-associated proteins by E6 and E7 oncoproteins --- p.57 / Chapter CHAPTER FOUR: --- DISCUSSION --- p.59 / Chapter CHAPTER FIVE: --- CONCLUSION AND FUTURE PERSPECTIVE --- p.68 / REFERENCES --- p.70 / APPENDIX DNA SEQUENCING RESULTS --- p.90

INTRODUÇÃO: A presença de metástase cervical é um dos principais fatores prognósticos em pacientes com carcinoma epidermoide de laringe. A análise rotineira dos linfonodos não inclui a avaliação da reatividade linfonodal, nem pesquisa de micrometástases, podendo ser desprezada informações prognóstica relevantes neste grupo de pacientes. OBJETIVOS: Relacionar a reatividade linfonodal com a recidiva e mortalidade de pacientes com carcinoma epidermoide de laringe estadiados como pT3 e pT4, pN0, identificar através de marcadores imuno-histoquímicos (AE1/AE3, citoqueratina 7 e citoqueratina 5/6) a incidência de micrometástases e relacionar com o prognóstico dos pacientes. MÉTODO: Entre 2002 e 2005, foram selecionados 105 pacientes estadiados como pN0 com carcinoma epidermoide de laringe submetidos à laringectomia total e esvaziamento cervical seletivo dos níveis II, III e IV bilateralmente, com a maioria dos pacientes (69) submetidos à radioterapia adjuvante. Todos os linfonodos foram analisados conforme os padrões de reatividade: hiperplasia folicular (associada à resposta imune humoral), hiperplasia paracortical (associada à resposta imune celular), histiocitose sinusal, linfonodo normal e depleção linfonodal. Apenas os dois primeiros padrões são considerados como linfonodos estimulados e os demais como não estimulados. O padrão linfonodal mais frequente de cada caso foi considerado na análise da recidiva da doença e mortalidade. Todos os linfonodos foram submetidos à análise imuno-histoquímica com o clone de citoqueratinas AE1/AE3, e nos casos de positividade utilizou-se as citoqueratinas 5/6 e 7 para a diferenciação entre micrometástases e inclusões epiteliais benignas. RESULTADOS: Foram analisados um total de 3.648 linfonodos, com uma média de 34,7 linfonodos por paciente. O padrão de reatividade mais frequente foi a histiocitose sinusal (50 casos) seguido da hiperplasia paracortical (35 casos) e hiperplasia folicular (20 casos), não havendo relação entre esta análise e a recidiva (p = 0,98) ou mortalidade (p = 0,49). A resposta imune humoral esteve relacionada com maior mortalidade (p = 0,05), com sobrevida global em 5 anos de 76%, comparado a 60% de sobrevida no grupo com imunidade celular estimulada (log-rank = 0,05). A pesquisa imuno-histoquímica utilizando o clone de citoqueratinas AE1/AE3 foi positiva em 17 pacientes (16,2%) tendo esta análise relação com a recidiva da doença (p = 0,03) e com a mortalidade (p = 0,04). No grupo com AE1/AE3 positivo, a radioterapia associou-se a uma tendência de proteção à recidiva regional (p = 0,06). A citoqueratina 5/6 confirmou a presença de micrometástases em 4 (3,81%) casos e estas não apresentaram relação prognóstica com a recidiva (p = 0,66) e mortalidade (p = 0,66). A citoqueratina 7 diagnosticou inclusões epiteliais benignas em 11 (10,47%) casos. CONCLUSÕES: Pacientes com imunidade celular estimulada (hiperplasia paracortical) apresentaram menor mortalidade comparados aos pacientes com resposta imune humoral (hiperplasia folicular) (p = 0,05). A positividade na pesquisa com o clone de citoqueratinas AE1/AE3 apresentou relação com significância com a recidiva (p = 0,03) e com a mortalidade (p = 0,04). A incidência de micrometástases na pesquisa com citoqueratina 5/6 foi de 3,86% (4 casos de 105) e de inclusões epiteliais benignas foi de 10,47% (11 casos de 105). / INTRODUCTION: The presence of neck metastasis is one of the most important prognostic factors in patients with squamous cell carcinoma of the larynx. Immunohistochemical analysis for lymph node reactivity, micrometastasis and presence of benign epithelial inclusions are not included in routine anatomopathological exam, leading to potential missing of important prognostic information in this group of patients. OBJECTIVES: 1- To analyze if lymph node reactivity is associated with recurrence and mortality in patients with pT3/pT4, pNo SCCa of the larynx. 2- To identify, with the aid of hystochemical markers AE1/AE3, cytokeratin 7 and cytokeratin 5/6), the incidence of micrometastasis and benign intraepithelial inclusions and to evaluate the relationship of these findings with the patient`s prognosis. METHODS: One hundred and five patients with SCCa of the larynx underwent total laryngectomy and bilateral selective neck dissection of the levels II, III, and IV between 2002 and 2005. Sixty-nine patients also received postoperative radiotherapy. All 105 patients were N0 at the time of diagnosis. The lymph nodes were analyzed according to their reactivity pattern: Follicular hyperplasia (associated to humoral immune response), paracortical hyperplasia (associated to cellular immune response), sinusoidal histiocytosis, normal lymph node, lymphoid depletion. Only the he first two patterns are associated with stimulated lymph nodes. The predominant pattern on each case was recorded and the relationship between these patterns and tumor recurrence and mortality was assessed. All the lymph nodes were tested for cytokeratin AE1/AE3, and the positive cases were than tested for cytokeratin 5/6 and 7 to further differentiate micrometastasis from benign epithelial inclusions. RESULTS: A total of 3648 lymph nodes were analyzed (average of 34.7 lymph nodes/patient). The most common reactivity pattern was paracortical hyperplasia (49 cases), followed by sinusoidal histiocytosis (36 cases) and follicular hyperplasia (20 cases). There was no association between these different patterns and recurrence (p=0.98) or mortality (p=0.49). The humoral immune response was associated with increased mortality (p=0.005). The 5-year overall survival was 76%, compared to 60% on the stimulated cellular response group (LogRank=0.05). Seventeen patients (16.2%) were positive for cytokeratin AE1/AE3, and this was associated with tumor recurrence (p=0.03%) and mortality (p=0.004). In the positive cytokeratin group, the use of radiation therapy showed a tendency to protect against regional recurrence (p=0.06). The use of cytokeratin 5/6 confirmed the presence of micrometastasis in 4 cases (3.81%), but this finding was not associated with local recurrence (p=0.66) or mortality (p=0.66). Cytokeratin 7 has diagnosed benign epithelial inclusions in 11 cases (10.47%). CONCLUSION: Patients with stimulated cellular immunity experienced decreased mortality when compared to humoral immune response (p=0.05). AE1/AE3 positivity was significantly related to recurrence (p=0.03) and mortality (p=0.04). The incidence of micrometastasis was 3.86% (4 cases in 105 patients), and of benign ephitelia inclusions was 10.47% (11 cases in 105 patients).