Dissertations / Theses on the topic 'Treatment of squamous cell carcinoma of the oral cavity'

Feline oral squamous cell carcinoma (SCC) is a devastating disease that responds poorly to traditional treatment modalities. The tumor location directly impacts the patient's ability to eat and drink, and immediate intervention to alleviate clinical signs is important. To design better treatment strategies it is paramount to understand the underlying biological behavior of this poorly defined tumor. This research takes a comprehensive approach in attempt to understand this disease. A number of assays have been developed and applied to elucidate underlying biology. New imaging modalities have been used to better stage the disease and define tumor location. Finally, patients were treated with a new radiation therapy modality, stereotactic radiation therapy (SRT), and outcome was correlated with the biological assays for potential predictive value.

The goal of the prospective study described in Chapter 2 was to compare gross tumor volume measurements using ¹⁸F-FDG PET vs. those using computed tomography (CT) for SRT planning in cats with oral SCC. Twelve cats with confirmed oral SCC underwent pretreatment ¹⁸F-FDG PET/CT. Gross tumor volumes based on contrast-enhanced CT and ¹⁸F-FDG PET were measured and compared between cats. Mean PET gross tumor volume was significantly smaller than mean CT gross tumor volume in the mandibular/maxillary SCC group (n=8, P=0.002) and for all cats (n=12, P=0.006), but not for cats with lingual/laryngeal SCC (n=4, P=0.57). Mismatch fraction analysis revealed that most of the lingual/laryngeal patients had a large region of high-¹⁸F-FDG activity outside of the CT gross tumor volume. This mismatch fraction was significantly greater in the lingual/laryngeal group than the mandibular/maxillary group ( P=0.028). The effect of poor spatial resolution of PET imaging was greater when the absolute tumor volume was small. Findings from this study indicated that ¹⁸F-FDG PET warrants further investigation as a supplemental imaging modality in cats with oral SCC because it detected presumed regions of primary tumor that were not detected on CT images.

For canine and feline patients with tumors in the head region, simultaneous irradiation of the primary tumor and mandibular and retropharyngeal lymph nodes (LNs) is often indicated. The purpose of this study described in Chapter 3 was to assess the reliability of a planning target volume (PTV) expansion protocol for secondary targets (LNs).

Information about the molecular biology of feline oral SCC is still limited. In Chapter 4, 22 archived tumor samples of feline oral SCC were evaluated to develop immunohistochemical assays and to determine if there was correlation to clinical parameters. Immunohistochemistry for Ki67, MVD, and EGFR was performed and scored. Patient survival information was obtained from the medical records. These molecular markers as well as MI were correlated with tumor locations and patient survival time. The 22 tumors showed wide variation in Ki67, MI, MVD, and EGFR. Tongue SCC expressed higher MVD than mandibular/maxillary SCC (P=0.088).

Cancer stem cell or tumor initiating cell (TIC) theory and telomere biology are actively studied fields in human head and neck (H&N) cancer. In feline oral SCC, which has been advocated as a feline model for human H&N cancer, our knowledge about the TIC and telomere/telomerase biology is limited. Protein expression levels of putative TIC markers of human H&N cancer, CD44 and Bmi-1, were immunohistochemically evaluated for their possible role as prognostic markers in 20 patients with feline oral SCC who underwent SRT. This patient population was part of a clinical trial and information relevant to PFI and ST was available. A combined technique of fluorescent in-situ hybridization and immunofluorescent staining was used to determine telomere length ratio (fractions of very short telomere/average length telomere in the putative cancer stem cells) in the putative TICs that were positive for CD44 and Bmi-1. This was also correlated with treatment outcome. (Abstract shortened by UMI.)

The Tumour-Node-Metastasis (TNM) classification system of tumour stage does not always reflect the actual tumour mass present at diagnosis. Recent reports propose that volumetric analysis may allow improved stratification of disease recurrence and survival in head and neck squamous cell cancer (SCC). This study aims to assess the prognostic value of tumour volume on the outcome of patients with oral cavity and oropharyngeal SCC.
A retrospective review of 73 patients was completed. Tumours were outlined semi-automatically in digitized computed tomography scans, and volumes computed based on surface triangulations of three-dimensional reconstructions with novel software developed at McGill.
Results illustrate significant interstage variability within the current TNM model. Moreover, in oral cavity and oropharyngeal SCC, tumour volume as well as T-stage are significant and independent predictors of disease free survival and overall survival.

Radiotherapy is an effective therapeutic modality employed in the management of malignant tumors in the oral cavity. Its use in head and neck results in adverse effects. The measurement of quality of life allows to gather the most common problems in oral cancer patients in a structured way, ranking its intensity and enabling a more accurate clinical control. This study aimed to evaluate the quality of life of post- irradiated diagnosed with squamous cell carcinoma of the oral cavity. Eleven patients over six months of completion of therapy treated at the Hospital Haroldo JuaÃaba â Instituto do CÃncer do Cearà were included in the study. University of Washington quality of life questionnaire, specific to head and neck cancer, was applied. Data related to socio-demographic, clinical, pathological and therapeutic profile were collected. Unstimulated salivary flow was measured by spitting method. Categorical data were exposed in the form of absolute and percentage frequency, and quantitative data as mean  standard deviation, followed by their minimum and maximum. Statistical analysis of quantitative data was performed by nonlinear Spearman correlation, considering a 95% confidence. Most patients were elderly (72.72%), male (81.81%), with lower education (90.9%), away from their work activities since the completion of cancer treatment (90.9%), with a history of smoking (90.9%), alcohol abuse (90.9%) and exposure to sunlight before the onset of the disease (90.9%), with lesions in the mouth floor (45.45%) moderately differentiated type (100%), stage IV (54,54%), subjected to other treatment modalities in addition to radiation therapy (100%). Among the domains assessed, chewing, saliva and speech presented the lowest mean scores (31.8, 42.3 and 60.6, respectively). All participants presented moderate to severe hyposalivation. Statistically significant correlation was found between age and speech (p = 0.043); time of completion of radiation therapy and recreation (p = 0.027); swallowing and pain (p = 0.039); activity and recreation (p = 0.030); swallowing and chewing (p = 0.007); chewing and speaking (p = 0.048); and shoulder and mood (p = 0.004). We conclude that the post- irradiated patients diagnosed with squamous cell carcinoma of the oral cavity tend to consider their quality of life ranging from fair to outstanding and that chewing, saliva and speech domains are those with greater commitment.
A radioterapia à uma modalidade terapÃutica eficaz utilizada no tratamento de tumores malignos da cavidade oral. Sua utilizaÃÃo em regiÃo de cabeÃa e pescoÃo leva ao surgimento de efeitos adversos. A mensuraÃÃo da qualidade de vida permite reunir os problemas mais comuns aos pacientes com cÃncer oral de forma estruturada, classificando sua intensidade, tornando-se possÃvel um controle clÃnico mais apurado. O presente estudo teve como objetivo avaliar a qualidade de vida de pÃs-irradiados com diagnÃstico de carcinoma espinocelular de cavidade oral. Onze pacientes com mais de seis meses de conclusÃo do tratamento, atendidos no Hospital Haroldo JuaÃaba â Instituto do CÃncer do CearÃ, foram incluÃdos no estudo. Foi aplicado o questionÃrio de qualidade de vida da Universidade de Washington, especÃfico para o cÃncer de cabeÃa e pescoÃo. Foram coletados dados relacionados ao perfil sociodemogrÃfico, clÃnico-patolÃgico e terapÃutico. Realizou-se a mensuraÃÃo do fluxo salivar nÃo estimulado atravÃs do mÃtodo de spitting. Dados categÃricos foram expostos em forma de frequÃncia absoluta e percentual e os dados quantitativos em forma de mÃdia  desvio-padrÃo, seguido de sua mÃnima e mÃxima. A anÃlise estatÃstica dos dados quantitativos foi realizada atravÃs da correlaÃÃo nÃo linear de Spearman, considerando uma confianÃa de 95%. Predominaram pacientes idosos (72,72%), do sexo masculino (81,81%), de baixa escolaridade (90,9%), afastados de suas atividades laborais desde a conclusÃo do tratamento oncolÃgico (90,9%), com histÃrico de tabagismo (90,9%), abuso de Ãlcool (90,9%) e exposiÃÃo à luz solar anterior ao surgimento da doenÃa (90,9%), com lesÃes localizadas em soalho de boca (45,45%) do tipo moderadamente diferenciado (100%), estadiamento IV (54,54%), submetidas a outras modalidades de tratamento alÃm da radioterapia (100%). Dentre os domÃnios avaliados, mastigaÃÃo, saliva e fala apresentaram as menores mÃdias de escores (31,8; 42,3 e 60,6, respectivamente). Todos os participantes apresentaram hipossalivaÃÃo de moderada à severa. Constatou-se correlaÃÃo estatisticamente significante entre: idade e fala (p=0,043); tempo de conclusÃo da radioterapia e recreaÃÃo (p=0,027); dor e deglutiÃÃo (p=0,039); atividade e recreaÃÃo (p=0,030); deglutiÃÃo e mastigaÃÃo (p=0,007); mastigaÃÃo e fala (p=0,048); e ombro e humor (p=0,004). Conclui-se que os pacientes pÃs-irradiados com diagnÃstico de carcinoma espinocelular de cavidade oral tendem a considerar de mÃdia a excelente sua qualidade de vida e que os domÃnios de mastigaÃÃo, saliva e fala sÃo os que apresentam maior comprometimento.

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HPV e Expressão de p16 como Biomarcadores de Prognósticos em Carcinoma de Células Escamosas da Cavidade Bucal

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CAPES
Biomarcadores de progressão tumoral são importantes na determinação do prognóstico e resposta ao tratamento em carcinoma de células escamosas da cavidade bucal. Este estudo teve como objetivo avaliar a frequência de infecção pelo HPV e a expressão de p16 como biomarcador de prognóstico em carcinoma de células escamosas da cavidade bucal. Dados clínico-patológicos e tecido tumoral de 90 indivíduos com carcinoma de células escamosas da cavidade bucal foram obtidos por entrevista e análise de prontuários. Detecção de HPV foi realizada à partir de amostras de tecido tumoral por PCR utilizando o conjunto de primers PGMY09/11. A seguir, as mesmas amostras foram submetidas à PCR com os primers MY09/11 e nested PCR com os primers GP5+/6+. Expressão de p16 foi detectada por imunohistoquímica. Análise estatística foi feita através de associação de variáveis, utilizando o teste de qui-quadrado e exato de Fisher. Sobrevida doença-específica (SDE) e sobrevida livre de doença (SLD) foram estimadas usando método de Kaplan-Meier. Comparação entre as curvas de sobrevida foram realizadas com o teste Log-Rank. Comparação entre status da infecção pelo HPV e expressão de p16 foram analisadas pelo teste Cox. Todos os testes com P ≤ 0.05 foram considerados significantes. A frequência de infecção pelo HPV na população estudada foi de 3,70% detectadas apenas com a nested PCR. Expressão de p16 foi observada em 21,87% das amostras analisadas. A SDE foi de 25,5 meses (95% IC = 20,03 - 31,00), enquanto a SLD foi de 30 meses (95% IC = 25,63 – 34,48). Maior SDE foi observada em indivíduos com tumores em estádio inicial (I-II) (P=0,001), tumores < 2cm (P=0,001), sem metástase em linfonodos regionais (P=0,006) e aqueles submetidos a tratamento cirúrgico (P<0,0001). Em conclusão, o status da infecção pelo HPV não mostrou-se um bom marcador de prognóstico e parece não ser determinante na tumorigênese em CCE da cavidade bucal, uma vez que apresentou baixa frequência de infecção. A expressão de p16 não foi um indicador determinante da presença de HPV nas amostras estudadas e um maior número de casos é necessário para avaliar sua aplicabilidade como marcador de prognóstico na população estudada.
Tumor progression biomarkers are important in determining prognosis and treatment response in squamous cell carcinoma of the oral cavity. This study aimed to evaluate the frequency of HPV infection and p16 expression as a biomarker of prognosis in squamous cell carcinoma of the oral cavity. Clinicopathological data and tumor tissue of 90 patients with squamous cell carcinoma of the oral cavity were obtained by interview and review of medical records. HPV detection was performed starting from tumor tissue samples by PCR using the primer set PGMY09/11. Next, the same samples were subjected to PCR with primers MY09 / 11 primers and nested PCR with GP5+/6+ primers. P16 expression was detected by immunohistochemistry. Statistical analysis was performed using variable association, using the chi-square and Fisher exact test. Disease-specific survival (DSS) and disease-free survival (DFS) were estimated using Kaplan-Meier. Comparison between the survival curves were performed using the log-rank test. Comparison status of HPV infection and p16 expression were analyzed by Cox test. All tests with P≤0,05 were considered significant. The frequency of HPV infection in this population was 3,70% detected only with nested PCR. p16 expression was observed in 21.87% of the analyzed samples. The DSS was 25.5 months (95% CI = 20.03 to 31.00), while the DFS was 30 months (95% CI = 25.63 to 34.48). Most DSS was observed in subjects with initial stage tumors (I-II) (P=0,001), tumor <2 cm (P=0,001), without regional lymph node metastasis (P=0,006) and those undergoing surgical treatment (P<0,0001). In conclusion, the status of HPV infection not proved to be a good prognostic marker and does not seem to be decisive in tumorigenesis in SCC of the oral cavity, since it showed low frequency of infection. The expression of p16 was not a key indicator of the presence of HPV in all samples and a greater number of cases it is necessary to evaluate its applicability as a prognostic marker in this population.

Orientadores: Luiz Paulo Kowalski, Cristina Kurachi
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O tratamento mais empregado para os carcinomas de células escamosas da boca é a ressecção cirúrgica, sendo ou não acompanhada de radio e/ou quimioterapia. O tratamento é simplificado, diminuindo número de recidivas e aumentando a sobrevida, quando as lesões encontram-se em estágios iniciais, conforme localização anatômica e diagnóstico de margens cirúrgicas livres. Frente a isto, o desenvolvimento e aperfeiçoamento de técnicas para um diagnóstico precoce, assim como de uma acurada definição das margens cirúrgicas livres e correta delimitação da extensão do câncer boca, com o intuito de melhorar a qualidade de vida e a taxa de sobrevida desses pacientes, tornam-se de grande importância. A espectroscopia de fluorescência é uma ferramenta diagnóstica não invasiva que pode auxiliar na detecção do câncer em tempo real, com o potencial de fornecer sensibilidade e especificidade semelhantes ao diagnóstico clínico de profissionais experientes. É uma técnica relativamente simples, rápida e acurada que consiste em avaliar a composição bioquímica e a estrutura do tecido pelo espectro de fluorescência emitido por ele, após aplicação de um feixe de luz. Quando há progressão de um estado normal para um estado alterado, isso é refletido nas características espectrais da fluorescência dos tecidos, podendo ser correlacionada com o exame histopatológico destes tecidos. O objetivo do presente estudo consistiu em discriminar, na mucosa bucal, tecido sadio de neoplásico, por meio de espectroscopia de fluorescência avaliando as margens ressecadas cirurgicamente. As avaliações ocorreram nos pacientes do Departamento de Cirurgia de Cabeça e Pescoço e Otorrinolaringologia do AC Camargo Câncer Center. O estudo obteve aprovação dos Comitês de Ética em Pesquisa das instituições participantes. A amostra consistiu de 75 indivíduos nos quais se realizou a espectroscopia de fluorescência dos quais 45 pacientes eram portadores de carcinoma oral e 30 voluntários com mucosa oral clinicamente normal. 29 casos (64.4%) do sexo masculino e a média de idade foi de 61.3 anos. Foram realizadas biópsias e os resultados destas duas metodologias foram comparados, usando o diagnóstico histopatológico como padrão ouro, para identificar características espectrais de entre tecidos clinicamente não alterados das margens cirúrgicas da mucosa de voluntários. Os espectros foram classificados e comparados com a histopatologia para determinação da eficiência na discriminação diagnóstica empregando-se a fluorescência. A análise inicial foi qualitativa e após consistiu de processamentos matemáticos dos espectros com excitação nos comprimentos de 532 e 406nm. Observou-se a variabilidade entre os indivíduos, entre os sítios anatômicos, entre regiões da mesma lesão e entre tecido clinicamente normal de voluntários e tecido das margens cirúrgicas em momentos in situ. Foram observadas também grandes diferenças entre espectros in situ e ex vivo, em concordância com resultados de outros estudos. A acurácia da técnica variou em função do tipo de análise empregada, mas pode-se constatar o seu potencial de uso como instrumento auxiliar para avaliar margens cirúrgicas no câncer de boca
Abstract: The most widely used treatment for squamous cell carcinoma of the mouth is surgical resection, whether or not accompanied by radiation and/or chemotherapy. The treatment is simplified, reducing the number of recurrences and increasing survival when the lesions are in the early stages, according to anatomical site and diagnosis of disease-free surgical margins. The development and improvement of techniques for early diagnosis, as well as an accurate definition of disease-free surgical margins and correct delineation of the extent of the mouth cancer, is an important part of improving the quality of life and survival rate for these patients. Fluorescence spectroscopy is a noninvasive diagnostic tool that can aid in real-time cancer detection, with the potential to provide similar sensitivity and specificity to that of the clinical diagnoses of experienced professionals. It is a relatively simple, fast and accurate technique that assesses the biochemical composition and structure of the tissue by the fluorescence spectrum emitted after the application of a beam of light. When there is progression from a normal state to an altered state, this is reflected in the spectral characteristics of the fluorescence of the tissues, which may be correlated with the histopathological examination of these tissues. The aim of this study was to discriminate, in oral mucosa, healthy tissue through fluorescence spectroscopy by evaluating surgically resected margins. Assessments occurred in patients of the Head and Neck Surgery and Otorhinolaryngology Department of A.C. Camargo Cancer Center. The study was approved by the Research Ethics Committee of the institution. The sample consisted of 75 individuals who underwent fluorescence spectroscopy, of which there were 45 individuals with oral carcinoma and 30 healthy volunteers with normal oral mucosa. Twenty-nine cases (64.4%) were male and the mean age was 61.3 years. Biopsies were performed and the results of these two methods were compared using histopathology as the gold standard to identify spectral characteristics from clinically unchanged tissues to surgical margins of the mucosa of volunteers. The spectra were classified and compared with histopathology for determining the efficiency of diagnostic discrimination of employing fluorescence. The initial analysis was qualitative and consisted of following mathematical processing of the spectra with excitation in lengths of 532 and 406 nm. Variability was observed among individuals, anatomical sites, regions of the same lesion and clinically normal tissue of volunteers and the tissue of surgical margins in situ. Also observed were large differences between spectra in situ and ex vivo, in agreement with results of other studies. The accuracy of the technique varied according to the type of analysis used, but its potential use is noted as an aid to evaluated surgical margins in oral cancer
Doutorado
Estomatologia
Doutora em Estomatopatologia

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JUSTIFICATION: Oral cancer is considered worldwide as a major public health problem, more common in developing countries. The evolution can be insidious, and often diagnosed only in advanced stages with mutilating surgeries, associated or not with adjuvant treatment. As in other cancers, early diagnosis is a priority activity to perform less aggressive treatments and improving survival. An activity that can contribute to the early diagnosis of lesions with or without evident clinical repercussion is exfoliative cytology; technique is not yet implemented for the diagnosis of oral lesions in the Amazonas´s state. OBJECTIVES: The aim of this study is to evaluate the efficacy of the use of exfoliative cytology for the diagnosis of oral cavity and oropharyngeal lesions, prior to its surgical removal. MATERIAL AND METHODS: Patients with an incisional or excisional biopsy of oral cavity lesions attended at the Dental North Specialty Center and the Oncology Control Center Foundation of the State of Amazonas, both in Manaus-AM, were selected. Before completing the biopsy, and after patient's consent, a form was filled out to obtain clinical data, followed by photodocumentation of the lesion and collection of cytological material for the preparation of conventional smears and in a liquid medium. These were fixed and then stained by the Papanicolaou technique. The results of the cytology techniques were compared with each other and with those obtained in the histopathological evaluation. RESULTS: The study sample was composed of 50 patients, 30 females and 20 males. In 70% of the cases, the lesions were histological interpreted as benign and 30% as malignant. Squamous cell carcinoma was the most prevalent lesions, with 15 cases, the benign lesions preferential site was lip and the malignant lesions were tongue, the profile´s patients diagnosed with malignancy was of male patients, smoker and alcohol use. When comparing the two methods of cytology we obtained 100% sensitivity, specificity 97%, accuracy 97%, Kappa 0.91. Regarding the accuracy of the cytological and histopathological diagnosis, sensitivity was 86.6%, specificity 100%, PPV 100%, NPV 94.5%, Kappa 0.958 and accuracy 96%. CONCLUSIONS Traditional and liquid based cytology methods were able to identify and classify cell changes with characteristics of malignancy and have high sensitivity and specificity, without significant differences between the two techniques tested. Cytology techniques have proven reproducible and, if well indicated, can be routinely used for the early detection of malignant lesions.
JUSTIFICATIVA: O câncer de boca é considerado em todo o mundo como um grave problema de saúde pública, sendo mais incidente em países em desenvolvimento. Sua evolução pode ser insidiosa, sendo na maioria das vezes diagnosticado apenas em estágios avançados, em que cirurgias mutiladoras são realizadas, associadas ou não a tratamento adjuvante. Como em outras neoplasias, a precocidade do diagnóstico é atividade prioritária para a realização de tratamentos menos agressivos e na melhoria da sobrevida. Uma atividade que pode contribuir para o diagnóstico precoce de lesões orais e de orofaringe, com ou sem repercussão clínica mais evidente, é a citologia esfoliativa, técnica ainda não implementada para avaliação das referidas lesões no Estado do Amazonas. OBJETIVOS: Avaliar a eficácia do uso da citopatologia esfoliativa para diagnóstico de lesões de cavidade oral antes de sua remoção cirúrgica. MÉTODOS: Foram selecionados pacientes com indicação de biópsia incisional ou excisional de lesões de cavidade oral e orofaringe, atendidos no Centro de Especialidade Odontológica Norte e na Fundação Centro de Controle de Oncologia do Estado do Amazonas, ambos em Manaus-AM. Antes da realização da biópsia, e após anuência do paciente, foi realizado preenchimento de formulário para obtenção de dados clínicos, seguido de fotodocumentação da lesão e de coleta de material citológico para confecção de esfregaços convencional e em meio líquido. Estes foram fixados e então corados pela técnica de Papanicolau. Os resultados das técnicas de citologia foram comparados entre si e com aqueles obtidos na avaliação histopatológica. RESULTADOS: A amostra foi constituída de 50 pacientes, na qual 30 eram do sexo feminino e 20 do sexo masculino. E em 70% dos casos a lesão foi interpretada à histopatologia como de natureza benigna e em 30%, como maligna. A localização preferencial das lesões benignas foi em lábio e das lesões malignas, em língua, sendo que destas, a mais prevalente foi o carcinoma escamocelular, com 15 casos. O perfil dos pacientes com diagnóstico de malignidade foi de pacientes do sexo masculino, tabagistas e etilistas. Quando os dois métodos de citologia foram comparados entre si foram obtidos os seguintes resultados: sensibilidade 100%, especificidade 97%, acurácia 97%, Kappa 0,91. Com relação à acuidade do diagnóstico citológico com o histopatológico, a sensibilidade foi de 86,6%, a especificidade de 100%, o VPP 100%, o VPN 94,5%, Kappa 0,958 e acurácia 96%. CONCLUSÕES: Os métodos de citologia tradicional e em meio líquido foi capaz de identificar e classificar as alterações celulares características de malignidade e possuem alta sensibilidade e especificidade, sem que houvesse diferenças significativas entre as duas técnicas testadas. As técnicas de citologia se mostraram reprodutíveis e se bem indicadas podem ser utilizadas rotineiramente para detecção precoce de lesões malignas.

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
Therapeutic modalities for the treatment of oral cancer result in local adverse effects that can cause interruptions of the radiotherapy and consequently to influence in the patient survival. For the reduction and control of the adverse effects, Dental Care Protocol is applied to patients with oral cancer by dental surgeon team before, during and after radiotherapy and chemotherapy. Objective: The aim of this study was to investigate the adhesion of patients with oral cancer to a Dental Care Protocol and its Impact in the interruption and survival. Patients and methods: In this study 133 cases of oral cancer undergoing radiotherapy were selected. The patients were classified according to the period of dental adhesion: no adhesion (group I), adhesion less than or equal to 6 months (group II) and adhesion higher than 6 months (group III). Clinic and pathological aspects, occurrence of interruption of radiotherapy, disease-free survival and overall survival were investigated. Results: The incidence of radiotherapy interruption due to symptoms was statistically significant in group III compared to group I (p = 0.01). The frequency and duration of interruption due to symptoms were not statistically significant between groups. The conclusion of radiotherapy rate was statistically significant in the group that exceeded 6 months of dental adhesion (p = 0.02). Patient’s survival was higher in group III (p = 0.01) when compared to the other groups. Conclusion: The adhesion to a dental care protocol did not have any impact on the radiotherapy interruption due to the occurrence of symptoms, however, patients who had higher adhesion to the Dental Care Protocol showed a higher rate of disease-free survival and overall survival.
Modalidades de tratamentos para o câncer de cavidade oral resultam em efeitos adversos locais que podem gerar interrupções da radioterapia e consequentemente influenciar na sobrevida do paciente. Para a redução e controle dos efeitos adversos, protocolos de preparo do paciente oncológico são estabelecidos por equipes odontológicas antes, durante e pós-tratamento radioterápico e quimioterápico. Objetivo: Verificar a adesão dos pacientes portadores de câncer de cavidade oral ao protocolo preventivo odontológico e seu impacto na interrupção da radioterapia e sobrevida do paciente. Pacientes e método: Neste estudo foram selecionados 133 casos de câncer de cavidade oral submetidos à radioterapia. Os pacientes foram classificados de acordo com o tempo de adesão odontológica: sem adesão (grupo I), adesão inferior ou igual a 6 meses (grupo II) e adesão superior a 6 meses (grupo III). Foram investigadas as características clínico-patológicas, ocorrência de interrupção da radioterapia, sobrevida livre de doença e sobrevida global. Resultados: A ocorrência de interrupção por sintomas foi estatisticamente significante no grupo III quando comparado ao grupo I (p=0,01). A frequência e a duração de interrupção por sintomas não foram estatisticamente significante entre os grupos. A conclusão da radioterapia foi estatisticamente significante no grupo com adesão superior a 6 meses (p=0,02). A sobrevida dos pacientes foi maior no grupo III (p=0,01) quando comparado aos demais grupos. Conclusão: A adesão ao protocolo não teve impacto sobre interrupção da radioterapia devido ocorrência da interrupção por sintomas, no entanto, pacientes que tiveram adesão ao protocolo preventivo odontológico apresentaram um melhor índice de sobrevida livre de doença e de sobrevida global.

La morbi-mortalité élevée des carcinomes épidermoïdes de la cavité orale (CECO), qui peuvent se développer à partir de lésions orales à potentiel malin (LOPM), rend indispensable le développement de nouvelles stratégies thérapeutiques. Le décryptage de l’hétérogénéité moléculaire aux différentes étapes de la carcinogénèse orale pourrait permettre de personnaliser les stratégies thérapeutiques de prévention et de traitement de ces cancers. Notre objectif était de caractériser l’hétérogénéité transcriptomique des LOPM et des CECO.Nous avons d’abord défini des signatures transcriptomiques associées aux changements histologiques de la carcinogénèse orale observés dans le modèle murin induit par le 4-NQO, montrant la pertinence de l’analyse de la dynamique temporelle du transcriptome pour améliorer la prévention des CECO. Cependant, ce modèle ne représentant qu’un sous-groupe particulier des CECO, nous avons ensuite étudié l’hétérogénéité inter-lésionnelle des LOPM en identifiant deux sous-types transcriptomiques principaux nommés « classical » et « immunological », qui sont caractérisés par différents biomarqueurs de risque de CECO.Au stade invasif, nous avons également étudié l’hétérogénéité transcriptomique des CECO HPV-négatifs entre les patients non-fumeurs non-buveurs (NFNB) et les patients fumeurs buveurs (FB). Le microenvironnement immunitaire était la principale différence biologique entre NFNB et FB, suggérant un bénéfice accru des immunothérapies chez les NFNB. Le profil transcriptomique de réponse antivirale observé dans les CECO des NFNB pourrait être en faveur de leur origine virale. En conclusion, l’hétérogénéité transcriptomique des LOPM et CECO suggère de personnaliser les stratégies thérapeutiques des patients porteurs de ces lésions
Oral squamous cell carcinomas (OSCC), which may develop from oral premalignant lesions (OPL), are associated with a substantial morbidity and mortality. A better understanding of the molecular heterogeneity at different steps of oral carcinogenesis may help to refine prevention and treatment strategies of patients suffering from OPL and OSCC. Our goal was to decipher transcriptomic hetereogeneity of OPL as well as OSCC. Using the 4-NQO murine model of oral carcinogenesis, we first identified transcriptomic signatures that characterized the dynamics of gene expression changes through different stages of disease progression, and that could be relevant for refining prevention strategies. Because this model represents only a subgroup of patients suffering from OSCC, we then investigated inter-OPL molecular heterogeneity. We identified two distinct gene expression subtypes, which were named classical and immunological and were characterized by different biomarkers of cancer risk. At invasive steps, we investigated transcriptomic heterogeneity between HPV-negative OSCC from never-smoker never-drinker (NSND) and smoker drinker (SD) patients. The immune microenvironment was the main biological difference between OSCC from NSND and SD, suggesting higher clinical benefit of immunotherapies in OSCC from NSND. The antiviral gene expression profile of OSCC from NSND could suggest a viral origin.In conclusion, we investigated transcriptomic heterogeneity of OPL as well as OSCC, that could help to refine their prevention and treatment strategies

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Medicina Dentária
A terapia fotodinâmica (PDT, do inglês, photodynamic therapy) é uma opção terapêutica recente e valiosa, dirigida para a destruição das células tumorais, com potencial para ser incluída no tratamento principal de combate ao cancro, bem como adjuvante de outras terapêuticas. Atualmente é considerada uma estratégia terapêutica de sucesso, clinicamente, aprovada para o tratamento de lesões potencialmente malignas (LPM) e malignas da cavidade oral. A PDT envolve a administração de um fotossensibilizador (PS, do inglês, photosensitizer) que, por si só, não possui efeito farmacológico. Contudo, este é, posteriormente ativado por irradiação, com luz visível de comprimento de onda (λ) adequado ao local a tratar. Com a foto-ativação do PS ocorre a produção de espécies reativas de oxigénio (ROS, do inglês, reactive oxygen species) que dão inicio à morte celular. O processo está associado com a indução de uma forte reação inflamatória local, potenciando a resposta imunitária e conduzindo à destruição efetiva das células tumorais. O efeito citotóxico ocorre, apenas, no local de ativação da luz e o PS acumula-se preferencialmente nas células malignas, pelo que esta terapêutica apresenta elevada seletividade e um baixo número de efeitos secundários. No contexto clínico, a PDT é utilizada em diversas áreas da medicina como a oftalmologia, a dermatologia e a oncologia, entre outras. No entanto, a sua utilização no tratamento do cancro ainda é limitada.
Photodynamic therapy (PDT) is a new and valuable therapeutic option, directed to the destruction of tumor cells, with the potential to be included in the primary treatment to combat cancer, as well as an adjunct to other therapies. A successful therapeutic strategy, clinically approved for the treatment of premalignant lesions (LPM) and malignant oral cavity is considered currently. PDT involves the administration of a photosensitizer (PS) which, by itself, does not possess pharmacological effect. However, this is subsequently activated by irradiation with visible light of wavelength (λ) appropriate to the site to be treated. With the photo-activation of the PS occurs the production of reactive oxygen species (ROS, English, reactive oxygen species) that give beginning to cell death. The process is associated with the induction of a strong local inflammatory response, enhancing the immune response and leading to the effective destruction of tumor cells. The cytotoxic effect occurs only in the light activation site and the PS accumulates preferentially in malignant cells, whereby this treatment has high selectivity and a low number of side effects. In the clinical context, PDT is used in many areas of medicine such as ophthalmology, dermatology and oncology, among others. However, their use in cancer treatment is still limited.

碩士
國立陽明大學
衛生福利研究所
98
Background: Oral cavity Squamous Cell Carcinoma was the important issue in Taiwan. The prevalence and mortality increase rapidly. Several studies have discussed the prognosis of the patients under different treatments in western countries. However, there are few studies concerning the territory in Taiwan. This study is to explore the treatment outcome of oral cavity Squamous Cell Carcinoma by using the secondary databases of government. Objective: To investigate the effect of first treatment modality on survival in patients with oral cavity squamous cell carcinoma. Methods: The study was retrospective cohort study. Patients who diagnosed with oral cavity squamous cell carcinoma in 2004-2005 were collected from Taiwan Cancer Data Base (TCDB) and Cancer Registry System (CRS), and using encrypted identification number for linking the related database. Stage was used as a stratification variable in the analysis. Using stepwise Cox' s regression analysis, to examined the effect of first treatment modality in four months after first diagnosis on survival in patients with oral cavity squamous cell carcinoma. Results: There were 7,064 patients included. The treatment rate in four months was 88.82％; the three common treatment modalities were surgery(39.32%)、postoperative radiotherapy(17.90%)、postoperative radiotherapy then chemotherapy(8.05%). surgery alone had the best prognosis in each stage. The stageⅠ postoperative radiotherapy and postoperative radiotherapy then chemotherapy had a significantly higher adjusted hazard ratio than surgery alone(1.89 and 1.86 respectively). The stageⅡ was 1.80 and 2.37 respectively; the stageⅢ was 1.66 and 1.37 respectively; the stageⅣA was 1.09 and 1.00 respectively. Conclusions: Treatment modality is a crucial attribute to prognosis in patients with oral cavity squamous cell carcinoma. Careful considering the patients’ characteristics and using the most adequate treatment are importment.

O cancro oral é a 6º neoplasia mais comum em todo mundo e o carcinoma espino- celular representa 90% dos casos. A taxa de sobrevivência aos 5 anos é cerca de 50% devido ao estádio avançado do tumor no momento do diagnóstico. Esta realidade motiva uma intensa investigação no sentido de aprofundar o conhecimento do processo de carcinogénese bem como a procura de novas estratégias, alvo e esquemas terapêuticos. A medicina dentária tem um papel fundamental, nomeadamente na prevenção, realização do diagnóstico precoce e, no encaminhamento dos pacientes para os centros de referência, bem como no acompanhamento dos doentes quer na fase prévia, durante e após os tratamentos. O objetivo deste estudo foi realizar uma revisão narrativa da neoplasia oral avaliando os dados epidemiológicos do carcinoma da cavidade oral, factores de risco envolvidos, principais lesões potencialmente malignas, diagnóstico, tratamentos e demonstrar o importante papel do médico dentista neste processo.
Oral cancer is the 6th most common neoplasm in the world and Squamous cell carcinoma represents 90% of the cases. The survival rate at 5 years old is about 50% due to the advanced stage of the tumor at the time of diagnosis. This reality motivates an intense investigation in the sense of improving the search of knowledge in the process of carcinogenesis as well as the search of new strategies, targets and therapeutic schemes. Dental medicine plays a key role, particularly in the prevention, doing the early diagnosis and referring the patients to specific centers as well as in their follow-up, both in the pre-treatment phase, during and after treatment. The objective of this study was to perform a narrative review of oral neoplasm by evaluating the epidemiological data of oral cavity carcinoma, risk factors involved, main potentially malignant lesions, diagnosis and treatments, as well as to demonstrate the important role of the dentist in this process.

O cancro é um dos principais causadores de milhões de mortes em todo o mundo e sendo o cancro oral, especificamente, a sexta neoplasia mais frequente a nível mundial. Todos os anos são diagnosticados mais de 500 mil novos casos, sendo que as altas taxas de mortalidade e mortalidade não se têm alterado ao longo dos anos. A maior incidência de cancro oral encontra-se na Ásia e na Europa do Sul. Em Portugal, mais precisamente em 2012, foram diagnosticados cerca de 1924 novos casos de cancro oral, dos quais 967 ocorreram em homens. O carcinoma espinocelular é o tipo histológico mais comum, sendo que 90% dos casos de cancro oral são deste tipo. Sabe-se também que esta variante é mais frequente no sexo masculino entre a 5ª e 6 ª década de vida apesar de, a incidência no sexo feminino, ter vindo a aumentar, devido à contínua exposição ao tabaco, álcool e a outros factores de risco. Como foi dito anteriormente, o cancro oral tem uma alta taxa de mortalidade e de morbilidade e, apesar dos avanços no diagnóstico, no tratamento e no conhecimento de quais os factores de risco desta patologia, a taxa de sobrevivência ainda é inferior a 50% o que revela que, o grande problema, passa pelo diagnóstico do cancro em estádios avançados. Assume-se então que, grande parte dos casos de cancro oral, poderiam ter sido evitados se houvesse maior conhecimento e grau de alerta sobre a doença o que tendencialmente, levaria a diagnósticos mais precoces. Neste sentido, este estudo tem como propósito a avaliação do nível de conhecimento geral e do grau de alerta de uma população do interior do país, mais precisamente do Nordeste Transmontano, bem como, efectuar o registo da percepção dos inquiridos relativamente a esta patologia, passando pelo reconhecimento da doença, pelo conhecimento epidemiológico e etiológico, e pela melhor percepção a nível de sinais e sintomas clínicos próprios desta patologia.
Cancer ir a major cause of millions of deaths around the world, and oral cancer, specifically, is the sixth most common cancer worldwide. Each year 500,00 new cases are diagnosed, being a cancer associated with high rates of mortality and morbility. The highest incidence of oral cancer is in Asia and in Southern Europe. In Portugal, more precisely in 2012 were diagnosed about 1924 new cases of oral cancer, 967 occurred in men. As mentioned, oral cancer has a high mortality rate and poor prognosis, and despite advances in detection, treatment and knowledge of what are the risk factors of this disease, the survival rate is still below 50% which reveals that the major problem,remains in the diagnosis of cancer in advanced stages. It is assumed, that most cases of oral cancer could be prevented if there was greater awareness and knowledge about the disease which in turn would lead to earlier diagnosis. This study aims to evaluate the general level of knowledge and degree of alert of a specific population, more precisely from the northeast of Portugal and register the perception of this population regarding this pathology, through recognition of the disease, the epidemiological and etiological knowledge, and for better understanding the signs and symptoms of this condition.

Os carcinomas de células escamosas da cabeça e pescoço foram os sextos cancros mais comuns em Portugal em 2010. Uma detecção precoce, uma melhor estratégia terapêutica levaram a um aumento da sobrevivência e da qualidade de vida dos doentes. No entanto, o risco de desenvolver um segundo cancro primário permanece elevado nesta população, constituindo um importante factor de morbilidade. Aproximadamente um terço das mortes dos carcinomas da cabeça e pescoço são imputáveis aos segundos primários cancros. Um segundo cancro primário apesar de poder ser considerado uma recaída, é diferente de uma recorrência tumoral, pelo facto de não se originar a partir do tumor primário, apresentando um processo carcinogénico distinto que origina um novo tumor. A sua detecção precoce revela-se um factor fundamental para aumentar as hipóteses de um tratamento com intuito curativo. Para tal, é fundamental um melhor conhecimento dos mecanismos que levam ao aparecimento destes segundos tumores, quais as localizações mais frequentes e qual a probabilidade e tempo necessário para ocorrerem. O objectivo deste estudo foi comparar os diversos relatos na literatura científica, sobre a realidade da distribuição dos segundos cancros primários e a sua frequência após um tratamento inicial para um carcinoma da cabeça e pescoço. O conhecimento desta realidade será de grande importância para o médico dentista, permitindo uma adequada gestão dos doentes com cancro oral. A revisão bibliográfica foi realizada a partir das bases PubMed, B-on, Cancer Network, Science Direct e repositório institucional da Universidade Fernando Pessoa.
Head and Neck squamous cell carcinoma (HNSCC) was the sixth most common cancer in Portugal in 2010. Earlier detection, better therapeutic management has led to an increase survival in patients' lifespan and quality of life. However, the risk of developing second primary malignancy remains elevated in this population and is still an important morbidity factor. Approximately one-third of HNSCC deaths are attributable to second primary cancers. A second primary cancer may be considered a relapse, but differs from tumour recurrence, by the fact that it doesn’t originate from the primary tumor and presents its own carcinogenic process that will generate a new tumor. Early detection is a fundamental factor to increase the hypotheses of a treatment with a curative objective. For these reasons it is fundamental to have a better knowledge of the appearance mechanisms of secondary tumours, their most frequent locations and how likely and how long they can occur. The purpose of this study is to compare the different relations in the scientific literature and articles, to analyse the reality of the distribution of second primary cancers and their frequency after initial treatment for a head and neck carcinoma. Knowledge of this reality will be important for the dentist, allowing adequate management of patients with oral cancer. A bibliographic review was carried from PubMed, B-on, Cancer Network, Science Direct and the Fernando Pessoa University institutional repository.

Oral squamous cell carcinoma (OSCC) is the most common head and neck cancer, with a worldwide incidence of 275,000 new cases annually (Warnakulasuriya, 2009). Globally, the head and neck carcinoma represents a major cause of morbidity and mortality and is the sixth most commonly occurring cancer (Warnakulasuriya, 2009). A majority (>90%) of the head and neck cancers are squamous in origin and thus are linguistically referred to as head and neck squamous cell carcinoma (HNSCC) (Warnakulasuriya, 2009). HNSCC includes cancers of the oral cavity, larynx and pharynx; oral cancer being the most common (Warnakulasuriya, 2009). Although, HNSCC is the sixth most common cancer globally (Warnakulasuriya, 2009), the Indian scenario is graver. According to GLOBOCAN 2008 (http://globocan.iarc.fr), the worldwide age standardized incidence rate (ASR) for HNSCC (and thus OSCC) is 5.3 and 2.5 per 100,000 males and females respectively (Ferlay et al., 2010). In India, the ASR is 9.8 and 5.2 per 100,000 males and females respectively, clearly demonstrating a remarkably high incidence rate of OSCC (Ferlay et al., 2010; http://globocan.iarc.fr). OSCC is a peculiar cancer which is largely preventable and rarely presents as a familial disorder. The most common etiological factors associated with OSCC include tobacco and alcohol consumption (Johnson, 2001). Additionally, high risk human papillomaviruses (HPV strains 16 and 18) as well as genetic predispositions have been implicated. The treatment of OSCC mainly relies on surgical resection of the tumor. The site, size, depth of infiltration and proximity to the bone of the tumor determine whether a combination of surgery with radiation therapy or chemotherapy would be advised (Scully and Bagan, 2009). The concomitant chemo-radiation therapy is the most commonly used strategy in locally advanced cancer. Taxanes (e.g., paclitaxel and docetaxel) and platinum-based induction chemotherapy (e.g., cisplatin) are the options in the treatment of locally advanced cancer. Epidermal growth factor receptor (EGFR) targeted with cetuximab in combination with radiotherapy has been successfully tested in a large randomized trial and thus is currently a new option (Scully and Bagan, 2009). The success of cetuximab has paved the path for the development and implementation of molecules targeting various signaling pathways. Despite extensive research on oral squamous cell carcinoma (OSCC), the five-year survival rate has not changed in several decades with the exception of the targeted treatment strategies involving cetuximab as discussed above. The current chemotherapeutic approaches lack selectivity and are flagitious. Thus, effective treatment of OSCC requires the identification of molecular targets to design appropriate therapeutic strategies. To this end, the present study took three distinct approaches in order to validate the use of existing targets and to reveal novel prognostic biomarkers and therapeutic targets. 1) Targeting the PI3K-AKT-MTOR pathway in OSCC and identification of determinants of its sensitivity. 2) Gene expression analysis of ectopically overexpressed TSC2 to identify new therapeutic targets and prognostic biomarkers as well as to elucidate the genes regulated by it. 3) Expression profiling of CYP1B1 in order to validate the use of CYP1B1 based prodrug therapy in OSCC. Investigations pertaining to the changes in gene and protein expression profiles in malignant as well as pre-malignant lesions have documented the deregulation of the PI3K-AKT-MTOR (phosphoinositide 3-kinase-AKT-mechanistic target of rapamycin) and EGFR (epidermal growth factor receptor) pathways in OSCC which are being widely targeted in many therapeutic strategies (Molinolo et al., 2007; Chakraborty et al., 2008; Matta and Ralhan, 2009; Molinolo et al., 2009; Stransky et al., 2011). The PI3K-AKT-MTOR pathway is a central hub for controlling cellular proliferation and growth in response to various intracellular as well as extracellular stimuli. Crucial signaling cascades including WNT, RAS, HIF-1α and AMPK cross-talk with the PI3K-AKT-MTOR pathway at a variety of molecular junctions. Thus, making this pathway sensitive to perceiving various growth modulatory conditions, ranging from the presence of growth factors to hypoxia and nutrient deprivation (Sengupta et al., 2010; Yang and Guan, 2007). The aberrant expression of the PI3K-AKT-MTOR pathway in OSCC advocated the targeting of this coveted pathway (Chakraborty et al., 2008). In various cancers, the monotherapeutic treatments with inhibitors like LY294002 (PI3K inhibitor) and rapamycin (MTOR inhibitor) demonstrated reduced efficacies. Such reduced efficacies were attributed to the drug toxicity and non-specific action of LY294002 (Davies et al., 2000; Sun et al., 2005; Ikezoe et al., 2007; Wang et al., 2008; Liu et al., 2009), or the ablation of a feedback inhibition loop leading to the reactivation of the PI3K-AKT-MTOR pathway by rapamycin (O'Reilly et al., 2006; Carracedo et al., 2008). Thus, rapamycin or its analogues demonstrated mediocre efficacy due to cytostatic effects in clinical trials, primarily due to the paradoxical activation of major survival kinases namely MAPK and AKT (O'Reilly et al., 2006; Carracedo et al., 2008). The present study aimed at increasing the efficacy of these drugs by incorporating a combinatorial approach. The MTT assay demonstrated that prolonged monotherapeutic treatments with rapamycin led to a modest growth inhibition in three OSCC (KB, SCC131 and SCC084) and HeLa cell lines. Western blot analysis of the phosphorylation status of AKT and RPS6KB1 revealed that monotherapeutic treatments with rapamycin for 96 hr led to the reactivation of the PI3K-AKT-MTOR pathway. Thus, the modest growth inhibitory effect of rapamycin was attributed to the reactivation of the PI3K-AKT-MTOR pathway. A combinatorial treatment approach was hence believed to circumvent this problem in order to increase the efficacy of targeting the PI3K-AKT-MTOR pathway. The PI3K inhibitor LY294002 was used combinatorially with rapamycin. This prolonged dual combinatorial treatment regime was distinctly more efficacious than either of the drugs alone and led to a reduction in cellular viability accompanied by increased sub-G1 population, indicating marked cell death that was characterized as caspase-3 dependent apoptosis. The differential sensitivity of the cell lines towards this combinatorial treatment revealed a novel determinant of the sensitivity, the transactivation of EGFR. The cell lines (SCC131 and SCC084) that were capable of transactivating EGFR were relatively resistant to the dual targeting of PI3K and MTOR in comparison to cell lines that did not transactivate EGFR (HeLa and KB). Further, targeting PI3K, MTOR and EGFR simultaneously was more efficacious in the presence of EGFR transactivation than dually targeting PI3K and MTOR. The results conclusively proved that the combinatorial therapeutic approach dually targeting PI3K and MTOR is a promising treatment strategy as compared to a monotherapeutic treatment and a major factor determining the sensitivity towards this treatment is the status of autophosphorylation of EGFR (Tyr1173) which governs the potential for EGFR transactivation by the combinatorial treatment. Thus, this study demonstrated that the status of EGFR autophosphorylation (Tyr1173) can be used as a biomarker to predict the sensitivity towards the combinatorial targeting of PI3K and MTOR in OSCC. The PI3K-AKT-MTOR pathway is negatively regulated by TSC2 (tuberous sclerosis complex 2; tuberin) (Tee et al., 2002). The importance of the TSC2 gene in the regulation of cell growth and proliferation is irrefutable. TSC2 facilitates the crosstalk between a variety of cellular signals, making it a crucial hub where many cellular networks integrate like AKT, MAPK and AMPK (Clements et al., 2007; Rosner et al., 2007; Rosner et al., 2008). It is a tumor suppressor gene and is downregulated in many cancers including OSCC (Chakraborty et al., 2008). In order to identify the genes regulated by TSC2 in OSCC, we stably overexpressed TSC2 in KB cells and the changes in the gene expression profiles caused by this ectopic overexpression were observed using a whole genome expression microarray. The results showed differential regulation of 268 genes (107 genes were upregulated and 161 genes were downregulated, p<0.05, fold change ≥ 1.5). A majority of these genes were functionally associated with transcription, cell growth and proliferation, apoptosis, cell cycle and neurogenesis. Functional annotation and network analysis was performed by using the DAVID v6.7 and IPA version 8.7 softwares. The microarray data revealed a novel aspect in the crosstalk between WNT signaling and TSC2, namely the transcriptional regulation of WNT signaling by TSC2. Further, in the context of therapeutic applications, the microarray analysis revealed multiple genes that were functionally categorized to be involved in response to radiation, UV and drugs (e.g., SERPINB13 and IL1B). Future studies on the regulation of such genes that are involved in responses to drugs and radiation may give insights into the role of TSC2 in resistance or sensitivity towards chemotherapy and radiation therapy. Moreover, EREG, a member of the epidermal growth factor family, was found to be the most downregulated gene in the microarray analysis. Previous reports have documented elevated levels of EREG in tuberous sclerosis lesions and its association with poor clinical prognosis in OSCC patients (Li et al., 2008; Shigeishi et al., 2008), making its regulatory aspects intriguing. Additionally, published data on the transcriptional functions of TSC2 instigated us to analyze the role of TSC2 in the regulation of EREG. TSC2 has been shown to modulate the transcription mediated by members of the steroid receptor superfamily of genes (Henry et al., 1998) and was shown to bind specifically to ERα and inhibit estrogen induced proliferation (Finlay et al., 2004). Also, TSC2 has been shown to possess C-terminal transcriptional activation domains (Tsuchiya et al., 1996). We have therefore attempted to investigate the transcription related functional aspects of TSC2 by exploiting the observed transcriptional repression of EREG. The physiological roles of TSC1 and TSC2 that are independent of the PI3K-AKT-MTOR pathway have been termed as ‘non-canonical’ (Neuman and Henske, 2011). The repression of EREG by TSC2 was observed to be insensitive to rapamycin, suggesting that it was independent of MTORC1 and thus a non-canonical function of TSC2. To determine whether the repression in EREG was at the level of the promoter, we performed a dual luciferase reporter assay. The results showed that the EREG promoter was repressed by stable as well as transient overexpression of TSC2. In order to elucidate the mechanism of transcriptional regulation by TSC2, we performed the ChIP analysis to observe the in vivo binding of TSC2 to the EREG promoter. In the ChIP analysis with the anti-TSC2 antibody, we observed that TSC2 did not bind to the EREG promoter between the regions -857 bp to -302 bp or -325 bp to +165 bp. Further, in silico analysis revealed an interesting trend among the transcription factors that were differentially regulated by TSC2 and had putative binding sites on the EREG promoter. A majority of these transcription factors (17/21) were downregulated by the overexpression of TSC2. This observation suggested that the repression of EREG could be an indirect effect due to repression of transcription factors caused by overexpression of TSC2. On the whole, this study revealed novel functions of TSC2 in OSCC with implications in determining novel biomarkers and therapeutic targets. As discussed previously, OSCC has a very flagitious treatment regime. A prodrug approach is thought to aid in targeting chemotherapy (Rooseboom et al., 2004). CYP1B1, a member of the cytochrome P450 family, has been implicated in chemical carcinogenesis (Bandiera et al., 2005; Sliwinski et al., 2010). There exists a general accordance that this protein is overexpressed in a variety of cancers (e.g., colon, lung, renal, bladder, prostate, breast, endometrial and esophageal cancers), making it an ideal candidate for a prodrug therapy (McFadyen et al., 1999; Murray et al., 2001; McFadyen et al., 2004; Sissung et al., 2006; Wen and Walle, 2007; Sliwinski et al., 2010). The activation of the prodrug facilitated by CYP1B1 would enable the targeting of chemotherapy to tumor tissues in which CYP1B1 is specifically overexpressed as a result reducing the non-specific side effects that the current chemotherapy elicits (Rooseboom et al., 2004). This study was aimed at validating the use of CYP1B1 as a target for the prodrug therapy in OSCC. The expression profile of CYP1B1 was analysed in a panel of 51 OSCC tumors, their corresponding normal tissues, an epithelial dysplasia lesion and its matched normal tissue by qRT-PCR, Western blotting and Immunohistochemistry. Counterintuitively, CYP1B1 was found to be downregulated in 77.78% (28/36) tumor tissues in comparison to their corresponding normal tissues as well as in the epithelial dysplasia lesion compared to its matched normal tissue at the transcriptional level, and in 92.86% (26/28) of tumor tissues at the protein level. This clearly demonstrated the downregulation of CYP1B1 at the transcriptional and translational levels in tumor tissues in comparison to their corresponding normal tissues. These observations indicate that caution should be observed as this therapy may not be applicable universally to all cancers. Since CYP1B1 has been shown to be involved in the activation of pro-carcinogens (Murray et al., 2001; Bandiera et al., 2005; Sissung et al., 2006), its inhibition could facilitate the development of a prophylactic therapy for oral cancer. Overall, this study has identified the transactivation of EGFR as a determinant of sensitivity towards combinatorial targeting of PI3K and MTOR in OSCC and has demonstrated that the autophosphorylation of EGFR (Tyr1173) can be used as a marker to judge the sensitivity towards this treatment. In the clinical perspective, the identification of such markers would aid in predicting the efficacy of targeted therapies. Such investigations would enable the strategic treatment of OSCC patients, thus decreasing the time lost in trial and errors for determining the appropriate treatment. Additionally, this study elucidated a novel role of TSC2 in the transcriptional repression of EREG, a prognostic biomarker for OSCC. Further, the study revealed potential prognostic biomarkers as well as therapeutic targets that are regulated by TSC2 by using a whole genome expression microarray. Moreover, the counterintuitive downregulation of CYP1B1 in OSCC tumors suggested the possibility of a prophylactic therapy for oral cancer but also advised a precautionary note for the application of prodrug treatments based on CYP1B1 overexpression in OSCC.

碩士
國立陽明大學
口腔生物研究所
101
Abstract Hepatocyte growth factor activator inhibitor type I (HAI-1) is a transmembrane protein which is expressed in epithelial cell. It can inhibit the activity of the serine protease and further downregulate the HGF/c-Met pathway. The elimination of HAI-1 can significantly increase cancer cell proliferation, migration and anti-apototic effects in various tumors. However, the roles of HAI-1 in the development of oral squamous cell carcinoma (OSCC) and apoptotic pathway under anti-cancer drug cisplatin treatment are still unknown. In this project, we found that HAI-1 expression was significantly associated with clinical stage and nodal stage in OSCC patients. Furthermore, the low expression of HAI-1 is correlated with poor survival rate in OSCC patients. We also found that the cell proliferation and migration were induced and the cisplatin cytotoxicity was inhibited in HAI-1 knockdown cells. The percentage of apoptotic cell was decreased in HAI-1 knockdown cells after cisplatin treatment. Western blot analysis revealed that the level of phosphorylated Met and Akt were up-regulated and cleaved caspase 9 and caspase 3 were down-regulated in HAI-1 knockdown cells. We also demonstrated that the HAI-1 knockdown tumor size in nude mice was large than control group after five weeks cisplatin treatment. Furthermore, the cisplatin cytotoxicity and apoptotic effect were induced under the cisplatin and EGCG combined treatment in oral cancer lines. In conclusion, we suggest that low expression of HAI-1 might promote tumorigenicity and cisplatin resistance in oral squamous cell carcinoma.

碩士
高雄醫學大學
醫學檢驗生物技術學研究所
102
According to cancer epidemiology report from Ministry of Health and Welfare, oral squamous cell carcinoma (OSCC) is the sixth of cancer incidence and the fifth of cancer mortality in Taiwan. Clioquinol (CQ) is the metal chelator which binds to divalent ion, such as zinc, iron, copper and so on. CQ was used as an antiseptic and oral intestinal amebicide in the past. However, CQ has been notified by its anticancer function nowadays. By acting as chelator and/or ionophore, CQ regulates the copper ion, which is specifically accumulated around the tumor site, to induce cell apoptosis. It has been reported that the concentration of copper in saliva and serum is relatively higher in OSCC patients than in healthy people. To decipher the underlying mechanisms of how CQ-copper exerts the anti-cancer function in oral cancer cells, liquid chromatography-tandem mass spectrometry was used for analyzing the differences on proteins between the DMSO-treated oral cancer cells and the CQ-copper-treated group. The present data demonstrated that CQ-copper combined treatment could cause Apoptosis Inducing Factor translocate from mitochondria inner-membrane to nuclear, then further damage DNA in a Caspase-3-independent action in HSC-3 cells. Meanwhile, CQ-copper combined treatment could also induce G2 arrest through regulating several G2/M phase transition regulators. The decrease of Cyclin A2, Cyclin B1, Cyclin B2, CDC25B, and the induction of p21 led to the consequent suppression of CDK1 activity. Taken together, CQ exerts the anti-cancer effects via induction of apoptosis and cell cycle arrest simultaneously in oral cancer cells.

Cisplatin is an effective chemotheraputic agent for head and neck squamous cell carcinoma particularly in conjunction with radiation therapy. Unfortunately, its cytotoxic profile and associated systemic side effects limit its clinical efficacy. A localized delivery system was developed for cisplatin by processing calcium polyphosphate (CPP) in a multistep gelling protocol, with the goal of limiting its systemic toxicity and enhancing its overall clinical applicability. In addition, a novel method for processing the material was examined utilizing cold isostatic pressure (CIP) to allow for miniaturization of the system into an implantable device. The integration of cisplatin into the matrix was examined for efficient and dose dependent loading via dissolution of the final product and measurement of platinum concentrations by inductively coupled plasma optical emission spectrometry (ICP-OES). Drug release was measured in vitro by placing the CPP-cisplatin matrix into TRIS buffer solution while measuring the platinum concentration at given intervals from 0.5 hours to 14 days. The cytotoxicity of the cisplatin against L1210 cells was examined using an MTT assay following a 12-hour elution. The material demonstrated a predictable and dose dependent loading of cisplatin, although the release of the drug showed variability exemplified by a more pronounced burst release with aging of the stock CPP glass particulate. The CPP/cisplatin matrix exhibited cytotoxic effects after processing. This work suggests that further evaluation of this material as a matrix for cisplatin delivery should be undertaken in an attempt to normalize release, maximize the concentration within the system and further optimize the bead format in order to improve the potential for clinical usage.

博士
國立清華大學
核子工程與科學研究所
104
According to the Ministry of Health and Welfare, oral cancer was the fifth most common throughout the population in 2013. Oral cancer has a high recurrence rate and frequently metastasizes to cervical lymph nodes. Lymph node metastasis has a poor prognosis and is a prognostic index for oral cancer. Boron neutron capture therapy (BNCT) is a promising new modality for cancer treatment. In BNCT, boronophenylalanine (BPA) accumulates in a tumor, while being present at a much lower level in a normal tissue. After boron drug treatment, the tumor is irradiated with thermal neutron inducing α-particles and 7Li. These two particles have a high linear energy transfer. All of their energy is deposited in cells, effectively killing tumor cells while doing little damage to normal tissue. The therapeutic success of BNCT depends on the high boron concentration in the tumor, the high tumor-to-the normal tissue boron ratio, the homogenous distribution of boron in the tumor and the high-quality thermal neutron beam. Therefore, this involves a series of investigations to evaluate the therapeutic effect of BPA-mediated BNCT against oral cancer. The program has four parts. In Part I.a, low dose of gamma irradiation enhances boronophenylalanine uptake in head and neck carcinoma cells for boron neutron capture therapy. In Part I.b, the boron concentration is increased in the tumor using low-dose gamma radiation to improve the therapeutic efficiency of BPA-mediated BNCT in an orthotopic oral cancer animal model. In Part II, the pharmacokinetics and tumor to normal tissue boron ratios are obtained by 18F-BPA-PET scan before BNCT and that obtained by ICP-AES analysis following real-time BNCT treatment in an orthotopic oral cancer model. In Part III, macro- and micro-distributions of BPA are analyzed in a subcutaneous xenograft model for BNCT. In Part IV, the administration of BPA is optimized to maintain a high boron concentration in tumor and narrow down the range of normal tissue to blood boron ratios in BNCT in a mouse model.

CDC73, also known as HRPT2, is a tumour suppressor gene whose expression is lost or downregulated in parathyroid, renal, breast, uterine and gastric cancers. However, the reports regarding the role of CDC73 in oral squamous cell carcinoma (OSCC) are lacking. As part of the Paf1 complex, it remains associated with ribonucleic acid (RNA) polymerase II and is involved in transcript site selection, transcriptional elongation, histone H2B ubiquitination, histone H3 methylation, poly(A) length control and, coupling of transcriptional and posttranscriptional events. It has been reported to negatively regulate cellularproliferation by targeting oncogenes CCND1 (cyclin D1) and MYC (c-Myc). Moreover, it has also been indicated to inhibitβ-catenin-mediated transcription. Taken together, these findings strongly suggest that it contributes to the expression of genes whose products have an important role in the suppression of tumor development and cell death. In this study, we have attempted to study the transcriptional and posttranscriptional regulation of CDC73 and its role in OSCC. The main findings of the present study are listed below. 1. To begin with, the expression analysis of CDC73 was performed both at the RNA and the protein levels by qRT-PCR and IHC, respectively. As expected, a majority of the OSCC samples showed downregulation of CDC73 both at the RNA and the protein levels compared to their normal oral tissues. 2. Loss-of-heterozygosity (LOH), mutation and promoter methylation are the hallmarks of a tumor suppressor gene (TSG). Therefore, to characterize CDC73 as a TSG in OSCC and to look into the mechanisms that could be the cause of CDC73 downregulation in OSCC, LOH, mutation and promoter methylation of CDC73 were studied. The results showed that LOH, mutation and promoter methylation are not the major causes of CDC73 downregulation in OSCC. 3. To identify the alternate mechanisms as the cause of CDC73 downregulation in OSCC, a combination of bioinformatics and molecular approaches were used. The results showed that the upregulation of an inhibitory transcription factor WT1 (Wilms tumor protein 1) and an oncogenic microRNA-155 are the major causes of its downregulation in OSCC. 4. The luciferase reporter assay of SCC131 cells co-transfected with a WT1 construct and a CDC73 promoter construct showed that WT1 over expression represses CDC73 expression in a dose-dependent manner. 5. Due to the presence of zinc fingers in its C-terminal half, WT1 has been found to be a potent transcriptional regulator of genes. Therefore, to determine if WT1 down regulates CDC73 via binding its promoter, the chromatin immunoprecipitation (ChIP) assay was performed. The results showed the binding of WT1 to the CDC73 promoter in vivo. Binding of WT1 to the CDC73 promoter was further confirmed in vitro by the electrophoretic mobility shift assay (EMSA). 6. The 5-aza-2’-deoxycytidine (AZA) treatment of SCC131 cells led to upregulation of WT1 with a concomitant downregulation of CDC73. The COBRA technique demonstrated that the upregulation of WT1 upon the 5-AZA treatment was due to its promoter methylation. 7. To determine if the WT1-mediated reduction of CDC73 expression has a functional relevance in cell growth and proliferation, we knocked down CDC73 expression by transient over expression of WT1 in SCC131 cells and quantitated cell proliferation by the MTT assay. As expected, the results demonstrated that the reduced CDC73 level was associated with an increased cell proliferation. Cotransfection of CDC73 with WT1 in SCC131 cells attenuated the pro-oncogenic effect of WT1 by apoptosis induction. 8. After validating CDC73 as the target of WT1 by bioinformatics and in vitro assays, we quantitated the expression levels of WT1 and CDC73 by qRT-PCR in OSCC samples and their matched normal oral tissue samples. The results showed an inverse correlation between the expression levels of WT1 and CDC73 in a majority of the samples. To exclude the possibility of alternate mechanisms as the cause of CDC73 downregulation in OSCC, we selected a subset of OSCC samples with downregulated level of CDC73 and analysed them for LOH at the CDC73 locus and promoter methylation. Further, some of these OSCC samples were also analyzed for mutations in CDC73. The results showed that these OSCC samples did not have LOH, promoter methylation or any mutation, again validating the fact that CDC73 is a biological target of oncogenic WT1, and the transcriptional repression of CDC73 by WT1 could be a major mechanism for CDC73 downregulation in OSCC. 9. Recent studies have shown that a growing class of noncoding RNAs called microRNAs (miRNAs) is involved in posttranscriptional regulation of genes. There is a growing body of literature supporting the potential role of miRNAs in tumorigenesis. The importance of CDC73 in orchestration of several cellular functions and its role in tumorigenesis make it an attractive candidate for miRNA-mediated regulation of cell growth and proliferation. Using bioinformatics approaches, we identified an oncogenic microRNA-155 (miR-155) that could posttranscriptionally regulate CDC73 expression. 10. Consistent with its oncogenic role, miR-155 was found dramatically upregulated in OSCC samples and was found to be another mechanism for downregulation of CDC73 in a panel of human cell lines and a subset of OSCC samples in the absence of LOH, mutations and promoter methylation. 11. miRNAs regulate posttranscriptional gene expression generally via binding to their cognate sites in the 3’UTR. Therefore, a luciferase reporter construct was made by cloning the 3’UTR of CDC73 downstream to the luciferase reporter gene and the reporter assay was performed. Our experiments clearly indicated that the mature miR-155 regulates CDC73 expression by interacting with its 3’UTR in a site specific manner. 12 Ectopic expression of miR-155 in HEK293 cells dramatically reduced CDC73 levels, enhanced cell viability and decreased apoptosis. Conversely, the delivery of a miR-155 antagonist (antagomir-155) to KB cells over expression miR-155 resulted in increased CDC73 level, decreased cell viability, increased apoptosis and marked regression of engrafts in nude mice. Cotransfection of miR-155 with CDC73 in HEK293 cells abrogated its pro-oncogenic effect. Reduced cell proliferation and increased apoptosis of KB cells were dependent on the presence or absence of the 3’UTR in CDC73. In nutshell, the knockdown of CDC73 expression due to over expression of WT1 and miR-155 not only adds a novelty to the list of mechanisms responsible for its downregulation in different tumors, but the restoration of CDC73 levels by the use of inhibitors to WT1 and antagomir-155 may also have an important role in therapeutic intervention of cancers, including OSCC.

碩士
國立陽明大學
口腔生物研究所
102
Hepatocyte growth factor activator inhibitor type 2 (HAI-2) is one of the Kunitz-type serine protease inhibitor. The function of HAI-2 is to inactivate the serine protease and the downstream signaling transduction. The previous study found that the HAI-2 expression was usually down-regulated in many cancers and the downstream receptor tyrosine kinase, such as c-Met. Cisplatin is one of the commonly used anti-cancer drugs. It could form DNA adduct and induce cancer cells apoptosis. But the role of HAI-2 expression in OSCC under cisplatin treatment is unknown. We found that HAI-2 expression was significantly associated with the primary tumor stage in OSCC patients. To figure out the role of HAI-2 in oral squamous cell carcinoma (OSCC), we established HAI-2 knock-down and HAI-2 overexpressed cancer cell lines. The data showed that the cell growth and migration abilities were up-regulated in HAI-2 knockdown cell lines and were down-regulated in HAI-2 overexpression cell line. On the other hand, the cytotoxicity was promoted in HAI-2 knockdown cell lines under cisplatin treatment. The data revealed that the apoptosis trigger by cisplatin in HAI-2 knockdown cells was lower than scramble cell lines. Western blot analysis showed that the level of matriptase-2, phosphorylated c-Met and phosphorylated Akt were increased and cleaved caspase 3 and 9 were decreased in HAI-2 knockdown cell lines under cisplatin treatment. In conclusion, we suggest that HAI-2 might suppress the tumorigenesis and low expression of HAI-2 could inhibit the apoptosis triggered by cisplatin in OSCC.

碩士
嘉南藥理科技大學
生物科技系暨研究所
100
Areca nut (AN) is a human carcinogen responsible for high oral cancer incidence in Taiwan. More than 70% patients keep on AN chewing when they took medical treatment in Chi Mei Medical Center. It is thus reasonable to speculate that their oral tumor cells, including lymphocytic and epithelial origins, have received a constant and long-term stimulation from AN. To simulate this in vivo stimulation and observe its on these cells, we subjected Jurkat leukemia T and oral epidermoid carcinoma Meng-1 (OECM-1) cells to long-term treatment with noncytotoxic concentrations of AN extract (ANE) or partially purified 30-100 kDa fraction of ANE (ANE 30-100K) and analyze the effects of these treatment on these cells. The results have demonstrated that both ANE- and ANE 30-100K-selected Jurkat T cells exhibited stronger resistance to low oxygen as well as to cisplatin and 5-fluorouracil (5-FU) as compared to non-selected parental cells. To access the possible mechanism, we next searched for upregulated proteins presumed to responsible for such phenotypic changes in these selected cells. By using immunoblotting or two-dimensional gel electrophoresis and liquid chromatography-mass spectrophotometer (LC-MASS), expression level of DRAM/LC3-II and secretion peroxiredoxin-1 (PRDX-1) were identified to be elevated in both selected cells. Immunohistochemistry revealed that PRDX-1 protein is mainly localized at epithelial layer of normal tissues but is expressed in most tumor cells. Moreover, after comparison of 4 pairs of OSCC tissues from AN and non-AN users, our preliminary data indicate on elevated expression level of PRDX1 in the specimens from AN users. In addition, ANE 30-100K also stimulated PRDX-1 expression in non-tumor oral fibroblasts (CMT-415) in a concentration- dependent manner. We are currently try to inhibit PRDX-1 by siRNA to assess whether this enzyme contribute to increase resistance of low oxygen and drugs in both selected cells.

Tese de doutoramento do Programa Interuniversitário de Doutoramento em Envelhecimento e Doenças Crónicas, apresentada à Faculdade de Medicina da Universidade de Coimbra
Head and neck squamous cell carcinoma (HNSCC) is an emergent health problem worldwide. These tumors present heterogeneity at phenotypic, aetiological, biological and clinical level. In developed countries, smoking and alcohol are implicated in the increase of HNSCC cases, and human papillomavirus is an important risk factor, especially in the rise of oropharyngeal tumors without smoke and alcohol habits. A significant percentage of HNSCC patients develop loco-regional and distant recurrences. Even with progresses in surgery, radiation and chemotherapy, approximately half of all patients die of the disease. Risk stratification for HNSCC is essencial in order to decrease mortality and improve quality of life of the patients. The great HNSCC heterogeneity makes difficult to understand the molecular carcinogenesis process as well as to develop early detection and therapeutic strategies for these tumors. Nowadays, the majority of genome-wide molecular profiling studies of HNSCC are limited to single approaches, which hampers the identification of accurate and robust biomarkers of early diagnosis and prognosis. Indeed, there is a lack of proven biomarkers for predicting clinical outcomes and response to treatment. The present work aimed to perform a molecular characterization of HNSCC in order to predict recurrence/metastasis development and signaling pathways associated to targeted therapy and resistance to conventional drugs through the identification of different molecular groups with apparently different survival profiles using genomic, epigenetic and transcriptomic approaches. We analyzed the same HNSCC patients through different molecular technologies, being the identified biomarkers and molecular signatures validated with TCGA (The Cancer Genome Atlas) data. First, we performed a direct genetic and epigenetic characterization of HNSCC patients, using specific Multiplex Ligation-dependent Probe Amplification (MLPA) and Methylation-Specific MLPA (MS-MLPA) probe panels. We reported different genetic signatures related to tumor stage and anatomic site as well as tobacco use. Additionally, specific genomic and epigenetic signatures associated to patients' risk of recurrence/metastasis development after treatment of primary tumor and also to survival were identified. The genetic analysis of non-tumor samples (from surgical margins) revealed some imbalances similar to those identified in the tumor samples, which reinforce the importance of molecularly analyze the high-risk patients even before the visible morphological changes and also the suspicious lesions in order to early diagnose these tumors and their recurrences. Secondly, we moved forward to a high-throughput genomic and transcriptomic approaches and we identified molecular signatures with capability to predict the recurrent/metastatic disease development and clinical outcome. In these studies using either direct probe panels or genome-wide approaches we identified the most common chromosomal regions with imbalances and altered genes. As expected, whole-genome techniques revealed new chromosomal regions and genes that seem to have a role in HNSCC development and behavior. Overall, through these comprehensive genomic, epigenetic and transcriptomic characterization we identified biomarkers and molecular signatures of prognosis and survival, which open the door for personalized medicine in HNSCC patients. Finally, we applied these genomic and epigenetic technologies to perform a molecular characterization of four paradigmatic HNSCC patients in order to prove the benefit of these molecular knowledgement to the clinical management of the HNSCC patients. Several chromosomal regions and genes related to radiation and/or chemotherapy resistance and to patients' prognosis and survival were identified, which could help and guide the type or intensify of treatment modalities. Moreover, molecular characterization of commercial HNSCC cell lines and primary cell cultures established from these patients was conducted, which revealed the ploidy and the complex structural chromosomal rearrangements of HNSCC tumors. This comprehensive characterization enables cell models for further studies both in radiation and pharmacogenomics fields, as well as to understand the molecular mechanisms of HNSCC development and progression. With this work we performed a robust molecular characterization of HNSCC, using different omic approaches in the same tumor samples, which allowed the identification of new prognostic biomarkers and molecular signatures with potential to be translated to clinical practice.
O carcinoma epidermóide da cabeça e pescoço (CECP) é um problema emergente de saúde em todo o mundo. Estes tumores são heterogéneos a nível fenótipico, etiológico, biológico e clínico. Nos países desenvolvidos, o tabaco e o álcool estão implicados no aumento do número de casos de CECP e o papiloma vírus humano é um fator de risco importante para o aumento dos tumores da orofaringe não relacionados com hábitos tabágicos e de álcool. Uma percentagem significativa de doentes com CECP desenvolve recidivas loco-regionais e à distância. Mesmo com os progressos na cirurgia, radioterapia e quimioterapia, cerca de metade de todos os doentes morre devido ao CECP. A estratificação do risco de CECP é essencial de forma a contribuir para a diminuição da mortalidade e melhoria da qualidade de vida destes doentes. A heterogeneidade do CECP dificulta por um lado a compreensão dos processos moleculares da carcinogénese e por outro lado o desenvolvimento de estratégias de deteção precoce e de terapêutica. Atualmente, a maioria dos estudos moleculares de grande escala são restritos, o que dificulta a identificação robusta e precisa de biomarcadores de diagnóstico e prognóstico. De facto, há falta de biomarcadores para predizer o desenlace clínico e resposta ao tratamento. O presente trabalho teve como objetivo caraterizar molecularmente o CECP de forma a prever o desenvolvimento de recidivas/metástases e a identificação de vias de sinalização associadas a terapias alvo e resistência às terapias convencionais, através da identificação de diferentes grupos moleculares com diferentes sobrevivências, usando abordagens de genómica, epigenética e transcriptómica. Neste estudo, analisámos os mesmos doentes com CECP usando diferentes tecnologias moleculares, tendo validado os biomarcadores e assinaturas moleculares identificados usando dados do portal TCGA (The Cancer Genome Atlas). Em primeiro lugar, realizámos uma caraterização genética e epigenética do CECP direcionada, utilizando painéis de sondas específicos de Multiplex Ligation-dependent Probe Amplification (MLPA) e Methylation-Specific MLPA (MS-MLPA). Identificaram-se diferentes assinaturas genéticas relacionadas com o estadio do tumor e as localizações anatómicas, bem como com o consumo de tabaco. Adicionalmente, uma assinatura genética e epigenética associada ao risco dos doentes desenvolverem recidivas/metástases após o tratamento do tumor primário e também associada à sobrevivência, foi identificada. A análise genética das amostras não tumorais (provenientes das margens cirúrgicas) revelou alguns desequilíbrios similares aos identificados nas amostras tumorais, o que reforça a importância de analisar molecularmente os doentes de elevado risco mesmo antes de qualquer alteração morfológica visível e também das lesões suspeitas, de forma a diagnosticar precocemente estes tumores e as suas recidivas. Na segunda parte do estudo utilizámos abordagens genómicas e transcriptómicas de larga escala e, identificámos assinaturas moleculares capazes de prever o desenvolvimento de recidivas/metástases e evolução clínica dos doentes. Estes estudos, usando quer painéis de sondas direcionados quer abordagens de todo o genoma, permitiram identificar as regiões cromossómicas e genes mais comummente alterados. As técnicas de análise de todo o genoma revelaram novas regiões cromossómicas e genes que parecem desempenhar um papel no desenvolvimento e evolução clínica do CECP. No geral, através desta caraterização genómica, epigenética e trasncriptómica, identificámos biomarcadores e assinaturas moleculares de prognóstico e sobrevivência, o que abre novas portas para a medicina personalizada no CECP. Finalmente, utilizámos estas tecnologias de genómica e epigenética para caraterizar quatro doentes paradigmáticos com CECP de forma a provar o benefício deste conhecimento molecular na conduta clínica. Várias regiões cromossómicas e genes relacionados com a resistência à radiação, quimioterapia, prognóstico e sobrevivência foram identificados, o que poderia ajudar na escolha do tipo e intensidade das modalidades de tratamento. Adicionalmente, foi realizada a caraterização molecular de linhas comerciais de CECP e de culturas primárias estabelecidas a partir destes doentes de CECP, o que revelou a ploidia e rearranjos estruturais complexos destes tumores, garantindo modelos celulares para futuros estudos no campo da radiação e farmacogenómica e ainda para uma melhor compreensão dos mecanismos moleculares de desenvolvimento e progressão do CECP. Este trabalho permitiu, de uma forma robusta, caracterizar molecularmente o CECP, usando diferentes abordagens ómicas nas mesmas amostras tumorais, ajudando assim a identificar novos biomarcadores de prognóstico e assinaturas moleculares com potencial translação à clínica.

博士
國立中正大學
分子生物研究所
104
Background: Radiation therapy (RT) is the current standard adjuvant approach for oral squamous cell carcinoma (OSCC) patients. Activation of the PI3K/AKT/mTOR signaling pathway has been linked to the decreased radiation responsiveness in human oral cancer. Thus, it limits efficacy of radiotherapy. In addition, this pathway has been considered to be a major contributor in radiation resistance in various tumors. Although a number of studies suggest that blockage of this pathway can in vitro and in vivo enhance the radiation response, its role in radioresistance of OSCC is not fully explored and little is known about the radiosensitization capacity of patient-derived OSCCs. In this study, we used patient-derived OSCCs and a radiation-resistant cell line for two purposes: to in vitro and in vivo evaluate the anti-tumor effects and to understand the mechanisms for the targeting of PI3K/AKT/mTOR signaling pathway in regulation of radiosensitization. Methods: We have established primary cells isolated from patient-derived OSCC tumor tissue for further molecular analyses. In this study, we used patient-derived OSCCs, OSCC-derived cell lines, and a radiation-resistant cell line in vitro and in vivo to examine the radiosensitization effects of single or dual PI3K/AKT/mTOR inhibitors and to explore the possible mechanism for the PI3K/AKT/mTOR inhibitor to serve as a potential candidate drug for radiosensitization. We carried out clonogenic survival assays, cell cycle analysis, apoptotic and autophagic assays and Western blotting to evaluate the possible mechanism. Results: The effect of the allosteric mTORC1-specific inhibitor, RAD001, and the dual mTORC1/mTORC2 inhibitor, AZD2014, on the sensitivity of OSCC cells toward radiation was investigated. We found that the mTORC1/mTORC2 inhibitor combined with radiation resulted in a significant reduction in the colony formation of OSCC cell lines and patient-derived OSCCs. By contrast, treatment with RAD001 plus radiation only slightly enhanced radiosensitization in SCC4. However, treatment with RAD001 increased AKT phosphorylation, which may limit effectiveness of mTORC1-targeting therapies for suppressing tumor growth. Pre-treatment with AZD2014 in irradiated oral cancer cells induced cell cycle arrest at the G1 and G2/M phases, which led to disruption of cyclin D1-CDK4 and cyclin B1-CDC2 complexes. Moreover, AZD2014 synergized with radiation to promote both apoptosis and autophagy by increasing the levels of caspase-3 and LC3 in patient-derived OSCCs. Therefore, inhibition of mTORC1/mTORC2 can sensitize OSCC cells to radiation, leading to cell-growth inhibition. Although the dual mTORC1/ mTORC2 inhibitor significantly enhanced the radiosensitivity of OSCC cells, no additional effects of this inhibitor were observed in radiation-resistant OSCC cells. Next, we further examined the effect of PI3K and mTOR inhibition on the irradiated oral cancer cells. Compared to a combination of PI3K or mTOR inhibitors with radiation, a dual blockade of the PI3K and mTOR kinases significantly improved radiation efficacy in OSCC-derived cell lines, patient-derived OSCC cells and radiation-resistant OML1-R cells. The dual PI3K/mTOR inhibition enhanced the effect of radiation by inhibiting AKT/mTOR signaling pathways and caused cell cycle arrest at the G1 phase, which is associated with downregulation of cyclin D1/CDK4 activity, leading to growth inhibition. Nevertheless, inhibition of these signaling pathways slightly increased the autophagy modulator, LC3. In nude mice xenografted with radiation-resistant OML1-R cells, the combined treatment was more effective than RT alone in reducing tumor growth. Immunohistochemistry showed that the combination therapy can suppress the S6 kinase activity and down-regulate the expression of eIF4E. These findings indicate that activation of the PI3K/AKT/mTOR signaling pathway has a role in radioresistance of OSCC. Conclusion: We determined that either mTORC1/mTORC2 or PI3K/mTOR inhibitor in combination with radiation exhibits a synergistic inhibition of the AKT/mTOR axis and induces cell cycle arrest. This treatment also triggers autophagy, resulting in sensitization of OSCC cells to radiation and cell-growth inhibition. More importantly, BEZ235 is a strong radiosensitizer to overcome radioresistance in treatment of OSCC with RT. Our results show the therapeutic potential of drugs targeting the PI3K/AKT/mTOR signaling pathway, which should be new candidate drugs for radiosensitization in radiotherapy for OSCC patient.

O cancro oral é um dos 10 cancros mais comuns no mundo, apresentando taxas de mortalidade elevadas. É na maioria das vezes diagnosticado tardiamente. Contudo sabe-se que existe um grupo de lesões, designadas, de potencialmente malignas, que têm capacidade de evoluir e progredir até cancro oral. Entre este encontra-se a leucoplasia oral, caracterizada como uma mancha ou placa branca que não pode ser identificada clínica ou patologicamente como nenhuma outra lesão branca e sem etiologia conhecida para além do tabaco (OMS, 1978). Existem vários tipos de leucoplasias, contudo há uma rara, sem etiologia conhecida e com caracter bastante agressivo, designada, leucoplasia verrucosa proliferativa. É caracterizada como uma leucoplasia de progressão lenta, com características multifocais, que se apresenta clinicamente com aspeto verrucoso e com prolongamentos exofíticos e proliferativos. Apresenta elevadas taxas de transformação maligna e de recidiva após tratamento, não havendo ainda um tratamento totalmente eficaz. Devido a sua elevada taxa de transformação maligna e a evolução para carcinoma verrucoso e carcinoma de células escamosas, é importante que seja feito um diagnóstico precoce, para que se tente travar a sua evolução, o que melhora o prognóstico e a morbilidade associada à lesão. Diminuindo, assim, também, a incidência de cancro oral, ou pelo menos a incidência de cancro oral extensivo.
Oral Cancer is one of the 10 most common cancers in the world, with high mortality rates. Is most often diagnosed late. However, it is known that there is a group of potentially malignant lesions that have the ability to evolve into oral cancer. Spot or white plaque that can not be identified clinically or pathologically as no other white lesion and no known etiology other than tobacco (WHO, 1978). There are several types of leukoplakias, however there is a leukoplakia without kown etiology, rare and with a very aggressive character, called proliferative verrucous leukoplakia. Is characterized as a slow progression leukoplakia, with multifocal features, that presents clinically verrucous appearance and with exophytic and proliferative projections. Presents with high rates of malignant transformation and relapse after treatment, with no totally effective treatment. Because of its high rate of malignant transformation and evolution in to verrucous carcinoma and squamous cell carcinoma, it is important that exists early diagnosis to try to halt its evolution and reducing the incidence of oral cancer or extensive oral cancer.