As células T regulatórias (Tregs) suprimem a população de células T efetoras e podem capacitar células tumorais a evadir a resposta imune do hospedeiro. Além disso, estas células têm sido correlacionadas com angiogênese em alguns tipos de câncer. O objetivo deste estudo foi avaliar a frequência de células Tregs e sua correlação com angiogênese em carcinomas espinocelulares (CEC) de boca. Amostras de um total de 61 pacientes com CEC de boca, localizados no lábio e na língua e assoalho bucal obtidos dos Departamentos de Patologia dos Hospitais de Base e Estadual da cidade de Bauru, São Paulo, Brasil, no período de 2005 a 2012 foram analisados quanto às características clínicas e demográficas. Avaliou-se também, o índice histopatológico de malignidade e a expressão imunoistoquímica de células Tregs (FOXP-3), densidade microvascular intratumoral (MVD) e VEGF-A. A associação da marcação do anticorpo anti-FOXP-3 e as variáveis clínicas e microscópicas foram avaliadas através do teste qui quadrado. A análise estatística da expressão dos marcadores entre CECs de lábio e CECs de Língua e assoalho bucal foi realizada através do teste t. As correlações entre a frequência de células Treg e MVD, células Tregs e VEGF-A, foram obtidas através do coeficiente de correlação de Pearson, bem como a correlação entre MVD e VEGF-A. Não houve associação da expressão de FOXP-3 com as características clínicas, demográficas e índice de malignidade tumoral. Foram observados valores similares da frequência de células Tregs, índices de MVD e expressão de VEGF-A em CECs de lábio e CECs de língua e assoalho bucal. Uma correlação positiva entre a frequência de células Tregs e a MVD foi observada, embora não estatisticamente significativa (P-valor=0,682). Além disso, uma correlação positiva estatisticamente significativa foi detectada entre a frequência de células Tregs e a expressão de VEGF-A (P-valor=0,029). Não foi observada correlação entre a MVD e a expressão de VEGF-A. Estes resultados sugerem que embora exista uma associação entre a frequência de células Tregs e a angiogênese em CECs de boca, a expressão de FOXP-3 parece não estar associada ao desenvolvimento tumoral. / Regulatory T (Treg) cells suppress effector T-cell populations and can enable tumor cells to evade the host immune response. Moreover, Tregs cells have been correlated with angiogenesis in certain types of cancers. The aim of this study was to evaluate the frequency of Treg cells and its correlation with angiogenesis in oral squamous cell carcinomas (OSCC), as well as its implication on growth and tumor aggressiveness. Samples from a total of 61 patients with OSCC, located on the lip and tongue and floor of the mouth were analyzed for clinical and demographic characteristics. The histopathological malignancy index and immunoexpression of Treg cells (FOXP-3), IMD (CD105) and VEGF-A were also evaluated. The association of FOXP-3 expression and the clinical and microscopic changes were evaluated using the chi square test. Statistical analysis of the expression of markers between SCCs lip and SCCs of the tongue and floor of the mouth was performed using the t test. Pearsons rank correlation was performed to evaluate the correlation between the frequencies of both, Tregs cells and VEGF-A expression and Tregs cells and IMD determinations, as well as between VEGF-A expression and IMD values. There was no association of FOXP-3 expression with clinical and demographic characteristics, and tumor malignancy index. Similar values were observed for the frequency of Treg cells, IMD values and VEGF-A expression in OSCCs of the lip and tongue and floor of mouth. A positive correlation between FOXP-3 expression with IMD values was also detected, however, statistically that was non-significant (P-value=0.682). Furthermore, a significant positive correlation was observed between the frequency of tumor-infiltrating Tregs with VEGF-A expression (P-value=0.029). No correlation was observed between IMD values and with VEGF-A expression. These results suggest that although there is an association between the frequency of Treg cells and angiogenesis in OSCC, FOXP-3 expression was not associated with increased tumor aggressiveness.

A proteína moesina, uma das proteínas do complexo ERM (ezrina, radixina e moesina), participa do processo de migração de células tumorais controlando a ligação entre o citoesqueleto de actina e os receptores transmembrana. As proteínas ERM vêm sendo investigadas como ligantes de outras glicoproteínas, como a podoplanina, cuja expressão é encontrada em células malignas de diversas neoplasias, incluindo o carcinoma espinocelular (CEC) de boca. O objetivo desse estudo foi avaliar as expressões imuno-histoquímicas da moesina e da podoplanina pelas células malignas no front de invasão de 84 pacientes com CEC de boca e suas associações com a evolução clínica e com o prognóstico dos pacientes. A associação entre a expressão imuno-histoquímica da moesina e da podoplanina pelas células malignas e as variáveis demográficas, clínicas e microscópicas foi avaliada pelo teste qui-quadrado ou teste exato de Fisher. As análises de sobrevida global e livre de doença em 5 e 10 anos foram calculadas pelo estimulador produto-limite de Kaplan-Meier e a comparação das curvas de sobrevida realizada pelo teste de log-rank. Os resultados mostraram que houve expressão da moesina pelas células malignas na região do front de invasão tumoral, entretanto, nenhuma associação estatisticamente significativa foi encontrada entre esta proteína e as características clínicas, demográficas e microscópicas. A expressão da podoplanina, pelas células malignas, foi significativamente associada à radioterapia (p=0,004), à invasão muscular (p=0,006) e ao comprometimento linfonodal (p=0,013). Não houve associação significativa entre a expressão das duas proteínas nos CECs de boca (p=0,460). A forte expressão da moesina pelas células malignas constituiu um fator de prognóstico desfavorável para os pacientes com CEC de boca e estadiamento clínico II e III. O comprometimento linfonodal histopatológico também se mostrou fator de prognóstico significativo para a recidiva da doença (p=0,018). Estes resultados sugerem que a expressão de moesina, pelas células malignas juntamente com o comprometimento linfonodal pode contribuir para determinar os pacientes com CEC de boca que apresentam um pior prognóstico. Além disso, verificamos que as proteínas moesina e podoplanina se expressam pelas células neoplásicas nos CEC de boca mas não parecem estar associadas no processo de invasão tumoral. / The moesin protein, one of the proteins of the ERM complex (ezrin, radixin and moesin) takes part in the migration of tumor cells process by controlling the relation between actin cytoskeleton and transmembrane receptors. The ERM proteins have been investigated as ligants of other glycoproteins, such as podoplanin, which are found in malignant cells of malignant, including oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the immunohistochemical expressions of moesin and podoplanin by malignant cells in the invasive front of 84 patients with oral squamous carcinoma and its association with clinical outcome and patients\' prognosis. Chi- square or Fisher\'s exact test was used to analyze the association between the moesin and podoplanin expressions by malignant cells and demographic, clinical and microscopic variables in oral squamous cell carcinoma patients. The 5 and 10 years survival rates were calculated by Kaplan-Meier method and the comparison of survival curves were performed using log-rank test. The results showed that there was moesin expression by malignant cells in the invasive front, however, no statistically significant association was found between this protein and demographic, clinical and microscopic features. The expression of podoplanin by malignant cells was significantly associated with radiotherapy (p=0.004), with muscular invasion (p=0.006) and lymph node involvement (p=0.013). There was no significant association between the expression of two proteins in OSCC (p=0.460). The strong expression of moesin by malignant cells was a factor of unfavorable prognosis for patients with OSCC and clinical stage II and III. The histopathological lymph node involvement was also significant prognostic factor for disease recurrence (p=0.018). These results suggest that the expression of moesin by malignant cells and lymph node involvement may help to determine patients with squamous cell carcinoma who have a poor prognosis. Furthermore, we found that moesin and podoplanin proteins are expressed by neoplastic cells in oral squamous cell carcinoma but not appear to be associated in the process of tumor invasion.

Um dos objetivos da pesquisa científica, atualmente, é encontrar biomarcadores que possam auxiliar na definição da probabilidade de progressão das lesões orais displásicas, e ainda sejam capazes de identificar os principais agentes moleculares envolvidos na carcinogênese de um determinado tipo de tumor. Assim, o objetivo deste estudo foi investigar a expressão de β-catenina, ciclina D1 e Ki-67 em 15 espécimes de epitélio oral normal, 45 queilites actínicas displásicas e em 30 carcinomas espinocelulares de lábio. Essa amostra foi constituída por pacientes tratados na Faculdade de Medicina de Botucatu (Brasil) e no Hospital Clínico San Cecílio de Granada (Espanha). O grau de displasia epitelial e de diferenciação tumoral foi classificado com base nos critérios definidos pela Organização Mundial da Saúde. A avaliação dos biomarcadores foi realizada por meio da técnica imunohistoquímica, dividindo a espessura do epitélio em quatro compartimentos (basal, suprabasal, terço médio e terço superior) para o grupo controle e para as queilites actínicas e em três compartimentos (basal, suprabasal e região interna) para o grupo dos carcinomas espinocelulares de lábio. Para a comparação da média de expressão de cada marcador, nas diferentes localizações do epitélio foi utilizado o teste estatístico de Kruskal-Wallis. Para a correlação da expressão dos três marcadores entre os grupos foi utilizada a correlação de Spearman, com nível de significância de 5%. Os resultados demonstraram uma perda discreta da expressão membranosa de β-catenina na camada basal das queilites actínicas com displasia epitelial intensa (Cis) e nos carcinomas espinocelulares de lábio, assim como uma expressão citoplasmática e nuclear, discreta e diretamente proporcional à desorganização epitelial nas camadas basal e suprabasal das queilites actínicas e carcinomas espinocelulares de lábio. Notou-se também um aumento da expressão de ciclina D1 e Ki-67 na camada basal à medida que aumentava a desorganização epitelial. Houve uma associação estatisticamente significativa da expressão de ciclina D1 e Ki-67 na camada suprabasal do grupo controle (p=0,030) e das queilites actínicas (p=0,001) e ainda na região interna dos carcinomas espinocelulares de lábio (p=0,000). Não houve correlação significativa entre as expressões nucleares de ß-catenina e de ciclina D1. Nossos resultados reforçam que a β-catenina, a ciclina D1 e o Ki-67, podem ser utilizados como biomarcadores preditivos para o câncer de lábio. Além disso, sugerem que a β-catenina e a ciclina D1 participam da carcinogênese labial, em eventos independentes da via de sinalização/Wnt. / One of the goals of scientific research today is to find predictive biomarkers that can help define the probability of progression of dysplastic oral lesions, and are still able to identify key molecular agents involved in the carcinogenesis of a particular type of tumor. The objective of this study was to investigate β-catenin, cyclin D1 and Ki-67 expression in 15 specimens of normal oral epithelium, 45 dysplastic actinic cheilitis and 30 squamous cell carcinoma of the lip. This sample consisted of patients treated at the Botucatu Medicine School (Brazil) and the Clinical Hospital San Cecilio of Granada (Spain). The degree of epithelial dysplasia and tumor differentiation was classified based on the criteria defined by the World Health Organization. The evaluation of biomarkers was performed by immunohistochemical technique, dividing the thickness of the epithelium into four compartments (basal, suprabasal, middle third and upper third) for the control group and actinic cheilitis and three compartments (basal, suprabasal and inner region) to the group of squamous cell carcinoma of the lip. For comparing the average expression of each marker in different locations of the epithelium we used the statistical test of Kruskal-Wallis. For the correlation of the three markers expression between the groups was used Spearman, with 5% significance level. The results showed a slight loss of membranous expression of β-catenin in the basal layer of actinic cheilitis with severe epithelial dysplasia (Cis) and squamous cell carcinoma of the lip, and a cytoplasmic and nuclear expression, slight and directly proportional to the epithelial disorganization in layers basal and suprabasal of actinic cheilitis and squamous cell carcinoma of the lip. It was also noted an increase in expression of cyclin D1 and Ki-67 in the basal layer as increased epithelial disorganization. There was a statistically significant association of cyclin expression D1 and Ki-67 in the suprabasal layer of the control group (p=0.030) and actinic cheilitis (p=0.001) and also in the inner region of squamous cell carcinoma of the lip (p=0.000). There was no significant correlation between the nuclear expression of ß-catenin and cyclin D1. Our results emphasize that β-catenin, cyclin D1 and Ki-67 can be used as predictive biomarkers for lip cancer. Moreover, they suggest that β-catenin and cyclin D1 acts on the lip carcinogenesis, in independent events signaling pathway/Wnt.

Diversas alterações genéticas estão associadas à patogênese do carcinoma epidermoide (CE), neoplasia maligna mais comum de cabeça e pescoço. Algumas dessas alterações comprometem proteínas pertencentes à via de sinalização do Akt, envolvida em diferentes fenômenos celulares. Este trabalho teve como objetivo estudar a expressão da proteína pAkt em linhagens celulares de carcinomas epidermoides de cabeça e pescoço, de forma a verificar possíveis alterações na transcrição dessa molécula em células de CE tratadas com Curcumina. Foram utilizadas duas linhagens celulares de CE de cabeça e pescoço (FaDu e SCC9) e uma linhagem de queratinócitos normais (HaCat) divididas em dois grupos: a. Grupo controle não tratado; b. Células tratadas com Curcumina. A proliferação celular foi monitorada através do teste de viabilidade celular e a análise da expressão de proteína foi realizada através da técnica do Western Blotting que revelou supressão do pAkt na linhagem celular SCC9 nos tempos de 24 e 48 horas. Desta forma, conclui-se que a Curcumina na via do Akt em carcinomas epidermoides de cabeça e pescoço tem importante ação supressora do gene pAkt. / Several genetic alterations are associated with the pathogenesis of squamous cell carcinoma (SCC), the most common malignant neoplasm of the head and neck. Some of these changes compromise the proteins belonging to the Akt signaling pathway, involved in various cellular phenomena. The objective of this study to explores the expression of the pAkt protein in cell lines of the squamous cell carcinomas of the head and neck to verify possible changes in the transcription of this molecule in EC cells treated with curcumin. The study used two cell lines of EC head and neck (FaDu and SCC9) and a normal line of keratinocytes (HaCat), split into two groups: A. the controlled group, untreated; B. Cells treated with curcumin. The cell proliferation it was observed by cell viability test and analysis of protein expression performed through Western blotting technique revealed suppression of pAkt in SCC9 cell line at 24 and 48 hours. Thus, it is concluded that the Curcumin on the path of Akt in squamous cell carcinoma of the head and neck has a significant suppressive effect of gene Akt.

O câncer de cavidade oral é uma das neoplasias mais comuns no Brasil e no mundo, porém seu prognóstico ainda é incerto principalmente devido ao diagnóstico tardio e presença de metástases. A análise de fatores relacionados ao prognóstico dessa doença é de suma importância e, o receptor de quimiocina denominado CXCR4, está sendo relacionado a um pior prognóstico devido a maior capacidade de invasão das células que o expressam, em diversas neoplasias. Apesar dessa relação estar demonstrada em vários tipos de cânceres, com relação ao de cavidade oral pouco se sabe até o momento. Assim, objetivo desse trabalho foi analisar a expressão imuno-histoquímica do receptor de quimiocina CXCR4 em carcinomas epidermóides de cavidade oral, e relacioná-la com variáveis clínicas e histológicas. Foram obtidos 94 blocos de carcinomas epidermóides oriundos de instituições parceiras para obtenção de cortes histológicos convencionais, corados com hematoxilina e eosina (HE), e cortes de TMA (tissue microarray). Foi realizado imuno-histoquímica para anticorpo anti-CXCR4 (ab124824, ABCAM, EUA) e análise da marcação em lâminas de TMA utilizando o software Image J (versão 1.49u). A intensidade de marcação imuno-histoquímica foi correlacionada com dados clínicos (TNM, tabagismo, etilismo e sobrevida) e histopatológicos (diferenciação histológica, infiltrado inflamatório e infiltração vascular, linfática e perineural) dos pacientes. Dos casos analisados 74,4% exibiram uma marcação fortemente positiva para o CXCR4, enquanto que o epitélio não tumoral mostrou uma marcação negativa ou fracamente positiva (71,1%; p=0,011). Tumores classificados como \"bem diferenciados\" apresentaram marcação fortemente positiva para a proteína estudada (53,3%; p=0,049). Não houve associação entre a marcação imuno-histoquímica do CXCR4 com sobrevida global em 5 anos (?2= 0.3, p=0.565). Os resultados sugerem que a alta expressão dessa proteína não influencia no prognóstico e na sobrevida desses pacientes. / Oral cancer is one of the most common neoplasia in Brazil and the world. Mainly due to a late diagnosis and presence of metastases its prognosis is still uncertain. Finding biological markers related to the prognosis of this disease is of paramount importance. The chemokine receptor CXCR4 is being related to a worse prognosis in several neoplasms because cells expressing it acquire a greater capacity of invasion. Although this relationship is demonstrated in several types of cancers, in the oral cavity it is uncertain. The aim of this study was to analyze by immunohistochemistry the CXCR4 chemokine receptor expression in oral squamous cell carcinomas, and related to clinical and histological variables. Conventional histological sections stained with hematoxylin and eosin (HE) were acquired from 94 blocks of oral squamous cell carcinoma for histological analysis. TMA (tissue microarray) was assembled from these blocks for anti-CXCR4 immunohistochemistry (ab124824, ABCAM, USA). Staining analysis was performed using Image J software (version 1.49u). The immunohistochemical signal intensity was correlated with clinical (TNM, smoking, alcoholism and survival) and histopathological parameters (histological differentiation, inflammatory infiltration, vascular, lymphatic and perineural infiltration). From the cases studied 74.4% showed a strong positivity for CXCR4, and the non-tumoral epithelium was negative or weakly positive (71.1%; p = 0.011). Tumors histologically well differentiated were strongly positive for the protein studied (53.3%; p = 0.049). There was no association between CXCR4 signal and global survival in 5 years (?2= 0.3, p=0.565). These results suggest that a high expression of CXCR4 it is not related to prognosis and survival of patients of patients with oral squamous cell carcinoma.

Submitted by Maria Aparecida (cidazen@gmail.com) on 2017-11-27T17:43:41Z No. of bitstreams: 1 Marta Belfort.pdf: 1659970 bytes, checksum: 7aca8a1f671f32a3e25959a2840b065f (MD5) / Made available in DSpace on 2017-11-27T17:43:41Z (GMT). No. of bitstreams: 1 Marta Belfort.pdf: 1659970 bytes, checksum: 7aca8a1f671f32a3e25959a2840b065f (MD5) Previous issue date: 2017-08-28 / FAPEMA,CNPQ. / Penile cancer (PeCa) is a rare neoplasm in developed countries; however, its incidence is high in underdeveloped countries. In Brazil, regions North and Northeast are those with the higher number of cases for the disease. Among the factors associated with this neoplasm we highlight: poor hygiene, phimosis, chronic inflammation and infection by human papilloma virus (HPV). Changes in the PI3K/AKT/PTEN cell signaling pathway have been reported for several malign neoplasms, but little is known about the involvement of this pathway in penile tumors. Thus, the aim of this study was to verify the role of HPV infection and the occurrence of copy number alterations (CNA) in genes from the signaling pathway mediated by receptors of growth factors and PI3K in a population characterized by advanced tumors and high frequency of high risk HPV. To achieve that, we collected tumor tissue samples (both fresh and in formalin-fixed paraffin-embedded tissue-FFPE) from 34 patients from two reference hospitals: Instituto Maranhense de Oncologia Aldenora Belo (IMOAB) and the Hospital Universitário Presidente Dutra from the Universidade Federal do Maranhão (HU-UFMA). Fresh tumors were submitted to detection and genotyping of HPV by Nested PCR (Polymerase Chain Reaction) and direct sequencing. CNA analyzes were carried out in FFPE tissue from a subgroup HPV positive (91.2%), of which 88.2% were at high oncogenic risk. TaqMan® Copy Number Assays (Life TechnologiesTM) and CopyCaller software version v2.0 were performed to determine copy number for EGFR, HER3, HER4, AKT1, AKT2, PI3KCA and PTEN. Increase of 3 and 4 copies was considered gains, while increase of 5 or more copies was considered amplifications. The presence of a single gene copy was referred to as loss, while the absence of two copies was named deletion. Clinical and histopathological parameters were analyzed as to the presence of HPV and to CNAs. Our data showed that EGFR/PI3K/AKT/PTEN signaling pathway is highly altered in PeCa. The results showed that 100% of the tumors presented an increase of the number of copies for HER3; out of those, 93.9% were amplified, with 84.4% having 10 or more copies. EGFR also showed an increase of copies in 87.8% of tumor samples, out of which 65.6% were amplifications, with 48.2% having more than 10 copies. Furthermore, HER4 and AKT1 also presented an increase in the number of copies of 20.6% and 15%, respectively. AKT1 had a higher frequency of tumors with a regular number of copies (78.8%). On the other hand, PI3KCA, HER4, PTEN and AKT2 presented a higher frequency of samples with deletions, presenting 56%, 52.9%, 39% and 36%, respectively. Loss of copies was also frequent on tumors, so that genes AKT2, PTEN and PI3KCA appeared in heterozygosis in 60.6%, 54.5% and 37.5%, respectively. Our data show the occurrence of genetic alterations that may justify the differential expression of growth factor receptors and the downstream genes of the PI3K/ AKT pathway in penile carcinoma. However, in this study there was no association between CNAs and clinical and histopathological variables. On the other hand, taking into consideration the high frequency of HPV in the evaluated tumors, we suggest that CNAs are related to HPV integration into genome host. Finally, we highlight the high frequency of tumors with amplifications in HER3 and EGFR, reinforcing these markers as targets for specific therapies in CaPe. / Câncer de pênis (CaPe) é uma neoplasia rara em países desenvolvidos, entretanto, sua incidência é elevada em países subdesenvolvidos. No Brasil, as regiões Norte e Nordeste são as regiões com os maiores números de casos da doença. Dentre os fatores de risco, destaca-se a má higiene do órgão genital, fimose, inflamação crônica e infecção pelo papilomavírus-humano (HPV). Tem sido reportada alterações na via de sinalização celular PI3K/AKT/PTEN em diversas neoplasias malignas, entretanto, pouco se sabe sobre o envolvimento dessa via nos tumores de pênis. Assim, buscou-se neste trabalho verificar o papel da infecção por HPV e a ocorrência de alteração no número de cópias (CNAs) em genes da via de sinalização controlada por receptores dos fatores de crescimento e PI3K, em uma amostra caracterizada por tumor avançado e alta frequência de HPV de alto risco. Para isso, foram coletadas amostras de tecido tumoral (fresco e de tecido fixado com formalina e embebido em parafina - FFPE) de 34 pacientes provenientes de dois hospitais de referência, Instituto Maranhense de Oncologia Aldenora Belo (IMOAB) e o Hospital Universitário Presidente Dutra da Universidade Federal do Maranhão (HU-UFMA). As amostras de tumor fresco foram submetidas a detecção e genotipagem de HPV por Nested PCR (Polymerase Chain Reaction) e sequenciamento direto. As análises de CNAs foram realizadas em amostras de FFPE, HPV positivas (91,2%), das quais 88,2% eram de alto risco oncogênico. Utilizou-se o ensaio TaqMan® Copy Number Assays (Life TechnologiesTM) e o software CopyCaller versão 2.0 para determinação do número de cópias dos genes EGFR, HER3, HER4, AKT1, AKT2, PIK3CA e PTEN. Aumento de 3 e 4 cópias foi considerado ganho, enquanto aumento de 5 ou mais cópias foi considerado amplificação. A presença de uma única cópia gênica foi nomeada perda, enquanto a ausência de duas cópias foi nomeada deleção. Os parâmetros clínicos e histopatológicos foram analisados quanto a presença de HPV e também quanto a CNAs. A via de sinalização celular EGFR/PI3K/AKT/PTEN revelou-se altamente alterada nos tumores de pênis. Observou-se que 100% dos tumores apresentaram aumento de número de cópias do gene HER3. Destes, 93,9% apresentaram-se amplificados, sendo que 84,4% apresentavam 10 ou mais cópias. O gene EGFR apresentou aumento de cópias em 87,8% das amostras, das quais 65,6% eram amplificações, sendo que 48,2% apresentavam mais de 10 cópias. Além desses, os genes HER4 e AKT1 também apresentaram aumento de cópias gênicas, em 20,6% e 15%, respectivamente. AKT1 apresentou maior porcentagem de tumores com número normal de cópias (78,8%). Por outro lado, os genes PI3KCA, HER4, PTEN e AKT2 apresentaram maiores frequências de amostras com deleções, com 56%, 52,9%, 39% e 36%, respectivamente. As perdas também foram frequentes nos tumores, de modo que os genes AKT2, PTEN e PI3KCA apresentaram-se em heterozigose em 60,6%, 54,5% e 37,5%, respectivamente. Nossos dados mostram a ocorrência de alterações genéticas que podem justificar a expressão diferencial dos receptores de fatores de crescimento e de genes downstream da via PI3K/AKT em carcinoma peniano. No entanto, não foi encontrada associação entre CNAs e as variáveis clínicas e histopatológicas. Por outro lado, considerando-se a alta frequência de HPV nos tumores avaliados, levanta-se a possibilidade de haver uma relação entre a integração do HPV no genoma do hospedeiro e a ocorrência de CNAs em CaPe. Finalmente, destacamos a alta frequência de tumores com amplificações em HER3 e EGFR, abrindo a possibilidade desses marcadores serem utilizados como alvos para terapias específicas em CaPe.

There is some evidence to suggest that human papilloma virus (HPV) may play a causal role in head and neck carcinoma (HNSCC). The aim of this study was to investigate the prevalence of HPV DNA in HNSCC and to determine whether any correlation exists with p16 or survival. An initial pilot study of sixty formalin-fixed HNSCC was carried out in order to optimise the methodology for the PCR and immunohistochemistry. A further 84 benign lesions, 12 dysplasias and additional 80 HNSCC were also included. In the pilot study the prevalence of all HPV types was 67% of which 18% were high risk-HPV (HR-HPV) and for the combined carcinoma sample it was 59% of which 25% were HR-HPV. The overall HPV prevalence was 51% and 42% for benign lesions and dysplasias with HR-HPV accounting for 14% and 8% respectively. A total of four alpha HPV types were identified and eleven beta HPV types. Multiple HPV types co-existed in the same tissue and in some cases both alpha and beta HPV. The results may suggest that HR-HPV may play a role in a small subset of HNSCC. An association was found between HPV status and gender, age group, survival, nodal metastasis and T3 tumour size and smoking. HPV16 was predominantly present in female patients and was associated with an improved overall survival and recurrence free survival. p16 positivity varied from 76-78% in carcinomas, 51% in benign lesions and 66% in dysplasias. p16 status was not associated with disease recurrence or nodal metastasis. Positive p16 staining and high staining intensity was associated with a poorer overall survival and the male gender, an older age group, anatomic site, and T2 tumour size. Overall HPV status was not correlated with p16 expression but a correlation found between p16 and HPV16 may suggest that p16 could potentially act as a surrogate marker of HPV16. However, the lack of concordance would suggest that in isolation p16 may not be a reliable marker for HR-HPV and should not be relied upon in isolation. Our findings could suggest that HPV16 and p16 status may be independent predictors for prognosis and disease recurrence.

Choi, Siu Cheong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 132-148). / Abstracts in English and Chinese. / Acknowledgement --- p.i / Abstract --- p.ii / Abbreviation --- p.v / List of Figures --- p.viii / List of Tables --- p.xii / Table of Content --- p.xiii / Chapter Chapter 1: --- General Introduction / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.1.1 --- Treatment resistance in cancer --- p.1 / Chapter 1.1.1.1 --- Surgery --- p.2 / Chapter 1.1.1.2 --- Chemotherapy --- p.3 / Chapter 1.1.1.3 --- Radiotherapy --- p.3 / Chapter 1.1.1.4 --- Hormonal therapy --- p.4 / Chapter 1.1.2 --- Hypoxia/reoxygenation and its correlation with treatment resistance --- p.5 / Chapter 1.1.3 --- Aim of the study --- p.6 / Chapter Chapter 2: --- The drug sensitivity in HepG2 cells and A431 cells / Chapter 2.1 --- Introduction --- p.8 / Chapter 2.1.1 --- Treatment of cancer --- p.8 / Chapter 2.1.2 --- Drug resistance --- p.9 / Chapter 2.2 --- Materials and Methods --- p.10 / Chapter 2.2.1 --- Cell culture --- p.10 / Chapter 2.2.2 --- Drugs --- p.10 / Chapter 2.2.3 --- MTT assay --- p.11 / Chapter 2.3 --- Results --- p.12 / Chapter 2.3.1 --- The drugs to which G10HR and G20HR cells were more resistant --- p.12 / Chapter 2.3.2 --- "The drugs of which GP, G10HR and G20HR cells have similar response" --- p.12 / Chapter 2.3.3 --- The drugs to which A10HR and A20HR cells were more resistant --- p.17 / Chapter 2.3.4 --- The drugs to which A10HR and/or A20HR cells were more sensitive --- p.17 / Chapter 2.3.5 --- "The drugs which AP, A10HR and A20HR cells have similar response" --- p.18 / Chapter 2.4 --- Discussion --- p.24 / Chapter 2.4.1 --- Camptothecin and 10-hydroxy camptothecin --- p.27 / Chapter 2.4.2 --- Etoposide --- p.30 / Chapter 2.4.3 --- Hydrogen peroxide --- p.32 / Chapter 2.4.4 --- Interferons --- p.32 / Chapter 2.4.4.1 --- Interferon alpha --- p.33 / Chapter 2.4.4.2 --- Interferon gamma --- p.34 / Chapter 2.4.5 --- Methotrexate --- p.35 / Chapter 2.4.6 --- Vincristine --- p.36 / Chapter Chapter 3: --- The resistance mechanism of doxorubicin in A431 cells / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.1.1 --- Chemotherapeutic resistance --- p.38 / Chapter 3.1.2 --- Tumor hypoxia --- p.39 / Chapter 3.1.3 --- Structure and function of doxorubicin --- p.39 / Chapter 3.1.4 --- Clinical use of doxorubicin --- p.40 / Chapter 3.1.5 --- Mechanisms of doxorubicin resistance --- p.41 / Chapter 3.1.6 --- Structure and function of P-glycoprotein --- p.42 / Chapter 3.1.7 --- Drug resistance contributed by P-glycoprotein and the solution --- p.43 / Chapter 3.1.8 --- Epigenetic modulation of mdr1 --- p.45 / Chapter 3.2 --- Materials and Methods --- p.47 / Chapter 3.2.1 --- Cell culture --- p.47 / Chapter 3.2.2 --- MTT assay --- p.47 / Chapter 3.2.3 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.47 / Chapter 3.2.4 --- Western blot analysis --- p.48 / Chapter 3.2.5 --- Doxorubicin efflux assay --- p.50 / Chapter 3.2.6 --- Drug sensitivity of A431 cells treated with verapamil --- p.50 / Chapter 3.2.7 --- Treatment with DNA methyltransferase inhibitor --- p.51 / Chapter 3.2.8 --- Drug sensitivity of A431 cells treated with 5-Aza-dC --- p.51 / Chapter 3.2.9 --- Methylation-specific PCR (MSP) --- p.51 / Chapter 3.2.10 --- Bisulfite genomic DNA sequencing --- p.52 / Chapter 3.3 --- Results --- p.54 / Chapter 3.3.1 --- Drug sensitivity of A431 cells to doxorubicin --- p.54 / Chapter 3.3.2 --- Expression profile of mdrl and P-glycoprotein in A431 cells --- p.54 / Chapter 3.3.3 --- Dox efflux-pump activity in A431 cells --- p.57 / Chapter 3.3.4 --- Drug sensitivity of A431 cells in the presence of verapamil --- p.59 / Chapter 3.3.5 --- Expression profile of mdrl in A431 cells in the presence of 5- Aza-dC --- p.59 / Chapter 3.3.6 --- Drug sensitivity of A431 cells in the presence of 5-Aza-dC --- p.62 / Chapter 3.3.7 --- Methylation status of mdrl promoter region --- p.64 / Chapter 3.3.8 --- Bisulfite genomic DNA sequencing of the mdrl promoter --- p.64 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4: --- The resistance mechanism of cisplatin in HepG2 cells / Chapter 4.1 --- Introduction --- p.70 / Chapter 4.1.1 --- Tumor hypoxia and chemotherapeutic resistance --- p.70 / Chapter 4.1.2 --- Cisplatin and its action mechanism --- p.71 / Chapter 4.1.3 --- Mechanisms of cisplatin resistance --- p.74 / Chapter 4.1.4 --- Mismatch repair genes --- p.79 / Chapter 4.1.5 --- Epigenome and drug resistance in cancer --- p.80 / Chapter 4.2 --- Materials and Methods --- p.84 / Chapter 4.2.1 --- Cell culture --- p.84 / Chapter 4.2.2 --- MTT assay --- p.84 / Chapter 4.2.3 --- Reverse transcription polymerase chain reaction (RT-PCR) --- p.84 / Chapter 4.2.4 --- Oligonucleotide transfection --- p.85 / Chapter 4.2.5 --- Treatment with DNA methyltransferase inhibitor --- p.86 / Chapter 4.2.6 --- Drug sensitivity of HepG2 cells treated with 5-Aza-dC --- p.87 / Chapter 4.2.7 --- Treatment with histone deacetylase inhibitor --- p.87 / Chapter 4.2.8 --- Drug sensitivity of HepG2 cells treated with TSA --- p.87 / Chapter 4.3 --- Results --- p.89 / Chapter 4.3.1 --- Drug sensitivity of HepG2 cells to cisplatin --- p.89 / Chapter 4.3.2 --- Expression profile of the MMR genes in HepG2 cells --- p.89 / Chapter 4.3.3 --- Drug sensitivity of HepG2 cells to cisplatin after the knock- down of PMS2 --- p.91 / Chapter 4.3.4 --- Expression profile of MMR genes in the presence of 5-Aza-dC --- p.95 / Chapter 4.3.5 --- Drug sensitivity of HepG2 cells to cisplatin after the addition of 5-Aza-dC --- p.95 / Chapter 4.3.6 --- Expression profile of MMR genes in the presence of trichostatin A --- p.98 / Chapter 4.3.7 --- Sensitivity of HepG2 cells to cisplatin after the addition of trichostatin A --- p.98 / Chapter 4.4 --- Discussion --- p.101 / Chapter Chapter 5: --- The role of PMS2 in cisplatin-induced apoptosis / Chapter 5.1 --- Introduction --- p.105 / Chapter 5.1.1 --- Apoptosis --- p.105 / Chapter 5.1.2 --- Extrinsic pathway of apoptosis --- p.106 / Chapter 5.1.3 --- Intrinsic pathway of apoptosis --- p.106 / Chapter 5.1.4 --- Cisplatin-induced apoptosis --- p.107 / Chapter 5.1.5 --- MMR and apoptosis --- p.109 / Chapter 5.2 --- Materials and Methods --- p.111 / Chapter 5.2.1 --- Cell culture --- p.111 / Chapter 5.2.2 --- Flow cytometric analysis of apoptosis --- p.111 / Chapter 5.2.3 --- Oligonucleotide transfection --- p.111 / Chapter 5.2.4 --- Western blot analysis --- p.111 / Chapter 5.2.5 --- Drug and antibodies --- p.112 / Chapter 5.3 --- Results --- p.113 / Chapter 5.3.1 --- Cisplatin induced apoptosis --- p.113 / Chapter 5.3.2 --- Knockdown of PMS2 by siRNA --- p.113 / Chapter 5.3.3 --- Cisplatin-induced apoptosis involved caspases --- p.115 / Chapter 5.3.4 --- Protein expressions of anti-apoptotic genes --- p.119 / Chapter 5.3.5 --- Protein expressions of pro-apoptotic genes --- p.119 / Chapter 5.3.6 --- Protein expressions of apoptotic proteins after knockdown of PMS2 --- p.122 / Chapter 5.4 --- Discussion --- p.124 / Chapter Chapter 6: --- General discussion and conclusion / Chapter 6.1 --- Diverse sensitivity for hypoxia/reoxygenation treated cells to anticancer drugs --- p.128 / Chapter 6.2 --- Resistance mechanism of doxorubicin in A10HR and A20HR cells --- p.129 / Chapter 6.3 --- Resistance mechanism of cisplatin in G10HR and G20HR cells --- p.129 / Chapter 6.4 --- The role of PMS2 as a direct signaling molecule and the alteration of apoptotic proteins in cisplatin-induced apoptosis --- p.130 / Chapter 6.5 --- Future work --- p.131 / References --- p.132

A neoplasia escamosa da superfície ocular constitui é uma das lesões mais frequentes que envolve a conjuntiva ou a córnea e tem como principais fatores de risco a exposição solar (radiação ultravioleta), a infecção pelo vírus do papiloma humano (HPV) e os estados de imunodeficiência, especialmente a infecção pelo vírus da imunodeficiência humana (HIV). Em suas formas mais avançadas, a neoplasia pode cursar com infiltração do globo ocular, da órbita e, mais raramente, com ocorrência de metástases para linfonodos ou a distância. O presente estudo teve como objetivo investigar os dados clínicos, histopatológicos, a presença de HPV e a expressão das proteínas p16 e p53 em 45 casos de neoplasia escamosa da superfície ocular diagnosticados no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, no período de 2005-2015. Avaliação histopatológica das lesões, estudo imuno-histoquímico para a detecção das proteínas e hibridização in situ cromogênica (CISH) para a detecção do DNA de HPV de alto e baixo grau foram realizados em todas as amostras. As lesões foram mais frequentes em homens de cor branca, com idade superior a 40 anos. A avaliação histopatológica revelou 31 casos (69%) de carcinoma invasivo e 14 casos (31%) de carcinoma in situ. Em 6 casos (13%) foi detectado DNA de HPV de alto grau por CISH. A frequência de expressão de proteínas p53 e p16 foi alta nas lesões, 89% e 53%, respectivamente. Em nosso estudo, a neoplasia escamosa da superfície ocular predomina em homens de cor branca, com idade acima dos 40 anos, com presença de HPV de alto grau em 13% dos casos. A expressão de p16 não apresentou um valor preditivo positivo alto quanto a possibilidade de associação com o vírus. / Ocular surface squamous neoplasia (OSSN) is one the most frequent lesions involving the conjunctiva or cornea and its main risk factors are solar exposure (ultraviolet radiation), human papilloma virus (HPV) infection and immunodeficiency states, especially human immunodeficiency virus (HIV) infection. In advanced cases one can observe eyeball or orbital infiltration and, rarely, lymph node or distant metastases. The present study aimed to investigate the clinical and histopathological data, as well as the presence of HPV and the expression of proteins p16 e p53 in 45 cases of OSSN diagnosed at the Clinics Hospital of Ribeirão Preto Medical School, University of São Paulo, from 2005 through 2015. Histopathological examination, immunohistochemical study for proteins p16 and p53, and chromogenic in situ hybridization (CISH) for low and high grade HPV were performed in all samples. Lesions were more frequent in white males, above 40 years old. Histopathological examination revealed 31 cases (69%) of invasive carcinoma and 14 cases (31%) of carcinoma in situ. In 6 cases (13%) high grade HPV was detected by CISH. The expressions of p53 and p16 were high, 89% and 53%, respectively. In our study, increased incidence of OSSN was observed in white males, above 40 years old, with high grade HPV in 13% of the cases. The expression of p16 did not show a high positive predictive value for HPV positivity in OSSN.

O carcinoma espinocelular (CEC) é a segunda forma de neoplasia cutânea mais prevalente. Os mecanismos exatos envolvidos na progressão desse tipo de tumor ainda não estão elucidados. Estudos recentes têm mostrado que a citocina IL33 é uma citocina reguladora da resposta imune adaptativa, principalmente como potente indutor do perfil Th2. Juntamente com seu receptor ST2, apresenta-se com os níveis elevados em alguns tipos de câncer, corroborando para a evidência de que essa citocina contribui para a carcinogênese. Baseado nessas informações, testamos a hipótese de que a presença de IL33 em carcinoma espinocelular, poderia estar relacionada a um melhor prognóstico. Neste estudo foram utilizadas amostras de carcinoma espinocelular, em diferentes gradações de malignidade tumoral (Grau I, Grau II e Grau III). Os resultados mostraram um infiltrado inflamatório mais intenso em tumores com Grau I e II. Imunorreatividade para IL33 foi observada em tumores de Grau I e II tanto por células epiteliais como por células do infiltrado inflamatório. A análise por microscopia confocal evidenciou que um grande número de células TCD4+ e TCD8+ que expressavam IL33 foi observado em tumores de Grau II. Esses resultados indicam a presença de um intenso infiltrado inflamatório e expressão de IL33 em amostras de carcinoma espinocelular com níveis menores de malignidade tumoral. / Squamous cell carcinoma (SCC) is the second most common form of cutaneous neoplasm. The exact mechanisms involved in the progression of this type of tumor have not yet been elucidated. Recent studies have shown that the cytokine IL33 is a cytokine regulating the adaptive immune response, mainly as a potent inducer of Th2 profile. Together with its ST2 receptor, its presents with elevated levels in some types of cancer, corroborating to evidence that this cytokine contributes to carcinogenesis. Based on this information, we tested the hypothesis that the presence of IL33 in squamous cell carcinoma could be related to a better prognosis. In this study, squamous cell carcinoma samples were used in three different gradations of tumor malignancy (Grade I, Grade II and Grade III). The results showed that a more intense inflammatory infiltrate in Grade I and II tumors. Immunoreactivity for IL33 was observed in Grade I and Grade II tumor, by epithelial cells and by inflammatory infiltrate cells. The analysis by confocal microscopy evidenced that a great number of TCD8+ and TCD4+ cells expressing IL33 was observed in grade II tumors. These results indicate the presence of an intense inflammatory infiltrate and expression of IL33 in samples of squamous cell carcinoma with lower levels of tumor malignancy.