Analisamos uma coorte de mulheres no sul do Brasil, com objetivo de identificar associações epidemiológicas para persistência e cura da infecção pelo HPV e realizamos uma metanálise para determinar a acurácia da telomerase nas lesões precursoras do câncer cervical. Métodos: O estudo de coorte foi iniciado em fevereiro de 2003. Foram coletados espécimes cervicais para citologia oncótica e para detecção do DNA HPV na entrada do estudo e no seguimento. O desfecho foi dividido em quatro categorias: (1) persistência, (2) conversão (3) cura. A quarta categoria (referência) eram mulheres negativas no início que permaneceram negativas. Foram usados o teste χ2 de Pearson, regressão logística multinomial e Kaplan- Meier para análise estatística. Para a metanálise foram incluídos estudos que comparavam o teste de telomerase (TRAP) e anatomopatológicos, obtidos por biópsias cervicais para diagnóstico de lesões cervicais. Resultados: A Incidência de HPV foi 12,3%. O HPV16 foi o tipo mais encontrado (18,6%), entre as 501 mulheres do estudo.Trinta e quatro mulheres (6,78%) ficaram persistentemente infectadas pelo HPV, estando essa categoria associada à idade da sexarca inferior a 21 anos (OR = 3,14, IC 95%, 1,43-6,87) e a quatro ou mais parceiros durante a vida (OR = 2,48 IC 95%, 1,14-5,41). No período mediano de 19 meses, 80,7 % das mulheres tinham curado o HPV, a cura foi significativamente associado à cor preta (OR= 3,44 IC 95%, 1,55-7,65), co-infecção com C. trachomatis no arrolamento (OR= 3,26, IC 95%, 1,85-5,76) e história de já ter realizado exame de Papanicolaou (OR= 3,48, IC 95%, 1,51- 8,00). Na metanálise dez estudos foram analisados, os quais incluíram 1069 mulheres. Para lesões intraepiteliais de baixo grau (LIEBG) vs. normal ou lesões benignas, encontrou-se uma positividade do teste da telomerase, sendo que o resultado da odds ratio para diagnóstico (DOR) foi de (DOR = 3,2, IC 95%,1,9-5,6). Nas lesões intraepiteliais de alto grau (LIEAG) vs LIEBG, normal ou benigna: (DOR = 5,8, IC 95%, 3.1-10). )]. Encontrou-se uma DORelevada de 8,1 (IC 95%: 3,2-20) nas lesões de câncer cervical vs LIEAG. Da mesma forma, nas lesões de câncer cervical vs. LIEBG, a razão de chance foi elevada, com uma DOR de 40,9 (IC 95%: 18,2-91). Conclusões: A persistência da infecção pelo HPV foi associada com a sexarca precoce e ao número de parceiros sexuais na vida, sugerindo que estratégias de orientação sexual podem modificar as taxas de persistência do HPV. A associação da cura do HPV com história prévia de realização de Papanicolaou salienta a importância de aprimorar os programas de rastreamento de câncer cervical. Futuros estudos da associação de infecções ginecológicas com cura da infecção pelo HPV são necessários. Na metanálise nossos dados suportam a corrente hipótese da atividade da telomerase como um evento precoce na carcinogênese e que poderia estar associado ao início e à progressão de lesões cervicais. / We analysed a cohort of women in Southern Brazil with the aim to identify epidemiological correlates for persistence and clearance of cervical HPV infection. A quantitative systematic review was performed to estimate the accuracy of telomerase assay in cervical lesions. Methods: A cohort study was started on February 2003. Cervical smears were collected to perform Pap cytology and HPV DNA detection at baseline and during the follow up. The outcome was constructed in four categories (1) persistence of HPV DNA; (2) conversion; (3) clearance of HPV. Pearson’s χ2 test, multinomial logistic regression and univariate analysis using the log-rank test were performed. Meta-analysis studies that evaluated the telomerase test (telomerase repeated amplification protocol) for the diagnosis of cervix lesions and compared it to paraffin-embedded sections as the diagnostic standard were included. Results: Incidence of HPV DNA: 12.3%. HPV16 was the most frequent type (18.6%) among 501 women in the study. Thirty-four women were persistently infected with HPV, which was associated with age below 21 years at first intercourse (OR 3.14, 95% CI, 1.43-6.87) and ≥ 4 sexual partners during lifetime (OR 2.48, 95% CI, 1.14-5.41). In a median period of 19 months, 80.7% of women had clearance of HPV, which was associated with black race (OR 3.44, 95% CI, 1.55-7.65), co-infection with C. trachomatis at baseline (OR 3.26, 95% CI, 1.85-5.76) and history of previous Pap smear (OR 3.48, 95% CI, 1.51-8.00). In meta-analysis ten studies were analyzed, which included 1,069 women. The diagnostic odds ratio (DOR) for a positive telomerase test for Lo-SIL vs. normal or benign lesions was 3.2 (95% CI, 1.9-5.6). The DOR for a positive telomerase test for Hi-SIL vs. Lo-SIL, normal or benign lesions was 5.8 (95% CI, 3.1-10). For cervix cancer vs. Hi-SIL, the DOR for a positive telomerase test was 8.1 (95% CI, 3.2-20.3) and for cervix cancer vs. Lo-SIL, normal or benign lesions, it was 40.9 (95% CI, 18.2-91). Conclusions: Persistence of HPV infection wasassociated with early age at first intercourse and number of sexual partners during lifetime, suggesting that strategies for sexual orientation may modify the rates of HPV persistence. The association of HPV clearance with a history of previous Pap smear screening highlights the importance of improving cervical screening programs. Further studies on the association of gynaecological infections with HPV clearance are needed. In meta-analysis our data support the current hypothesis that telomerase may activate an early event in cervical carcinogenesis, that could be associated with the initiation and progression of cervical lesions.

A história natural do câncer cervical invasivo começa com uma infecção produtiva pelo Papiloma vírus humana (HPV) na camada basal do epitélio. Infecções persistentes por vírus de HPVs de alto risco poderão provocar lesões que eventualmente darão origem ao carcinoma invasivo. Sabe-se que o infiltrado inflamatório pode ter um papel importante na evolução da doença. Neste trabalho, quantificamos e caracterizamos fenotipicamente linfócitos T, macrófagos e neutrófilos nas lesões precursoras assim como no carcinoma invasivo. Além disso, para determinar se existia alguma relação entre as populações foi realizada uma análise de correlação entres as populações descritas. Por outro lado, tentando determinar o efeito sistêmico do tumor avaliou-se a frequência de subtipos de monócitos circulantes e através de ensaios alogênicos foi avaliada a capacidade estimuladora das células dendríticas diferenciadas de monócitos circulantes. Os dados apresentados ajudarão a entender o papel que as células do sistema imune podem ter sobre a progressão da doença. / The natural history of cervical cancer begins with a human papilloma virus (HPV) infection of the cells of the basal layer of the epithelium. Persistent infection by high risk HPVs can originate precancerous lesions that may progress to invasive cancer. It has been established the role of the infiltrated inflammatory cells on the progression of the disease. In this work, the phenotype and the frequency of T lymphocytes, macrophages and neutrophils were characterized both in precursor lesions as in invasive carcinoma. In order to stablish a possible relation between the characterized cells, we made a correlation analysis. On the other hand, trying to determine the systemic effect of the tumor we evaluated the frequency of circulating monocyte subtypes and the capacity of dendritic cells to stimulate allogeneic T cells. The data presented will help to understand the role of the immune system cells on the progression of the disease.

Introdução: A incidência do carcinoma epidermóide de orofaringe (CEO) aumentou em todo o mundo nos últimos 30 anos. Estudos identificaram o papiloma vírus humano (HPV) como um fator de risco para essa neoplasia. Objetivos: O objetivo do presente estudo foi verificar a frequência do HPV em pacientes com CEO e em pacientes sem neoplasia maligna e avaliar a existência de uma diferença estatisticamente significativa na frequência do HPV entre os dois grupos. O objetivo secundário foi estudar a correlação entre a infecção pelo HPV e a localização do tumor na orofaringe, o estadiamento clínico e o grau de diferenciação tumoral. Métodos: Foi realizado um estudo de caso-controle com 59 pacientes com CEO e 54 pacientes sem neoplasia, no qual foram analisados os blocos de parafina contendo material tumoral e tecido não neoplásico. Foram analisadas respectivamente a frequência do HPV e sua atividade viral utilizando a técnica de hibridização in situ cromogênica (CISH) para HPV de baixo risco (BR) e alto risco (AR) e a expressão imunoistoquímica da proteína P16. Resultados: A frequência do HPV foi maior no grupo caso em comparação ao grupo controle quando utilizamos a expressão imunoistoquímica da proteína P16 como método de detecção isolado (OR=10,3; P<0,001) e quando utilizamos a CISH e a expressão imunoistoquímica da proteína P16 em conjunto (OR=21,4; P<0,001). A CISH isoladamente não mostrou uma diferença estatisticamente significativa na frequência do HPV entre os grupos estudados (P=0,572). A localização tumoral na orofaringe e o estadiamento clínico não mostraram correlação com a infecção pelo HPV em nenhum dos métodos utilizados, assim como o grau de diferenciação tumoral (P>0,20). Conclusão: Utilizando-se a técnica de imunoistoquímica para P16 isolada ou combinada com a técnica de CISH, observou-se uma maior positividade para o HPV no grupo de pacientes com CEO. A localização do tumor na orofaringe, o estadiamento clínico e o grau de diferenciação tumoral não tiveram correlação com a positividade para o HPV. / Introduction: The incidence of oropharyngeal squamous cell carcinoma (OSCC) has increased worldwide over the last 30 years. Studies have identified human papillomavirus (HPV) infection as a risk factor for OSCC. Objectives: To compare the frequency of HPV infection in patients with OSCC and patients with benign oral or oropharyngeal disease and ascertain whether a statistically significant difference in HPV frequency exists between these two groups. As a secondary objective, to assess potential correlations between HPV positivity, anatomic site of OSCC, tumor staging, and degree of tumor differentiation. Methods: Case-control study. The sample comprised 59 patients with OSCC and 54 non-OSCC controls who underwent surgery for benign oral or oropharyngeal conditions. Paraffin-embedded specimens from cases and controls were tested for HPV positivity by chromogenic in situ hybridization (CISH) for low-risk (LR) and high-risk (HR) HPV, and HPV activity was assessed by P16 immunohistochemistry (IHC). Results: The frequency of HPV positivity was higher in the case group than in the control group when assessed by P16 IHC alone (OR=10.3, P<0.001) or by CISH and P16 IHC in combination (OR=21.4, P<0.001). CISH alone did not detect any significant between-group difference in HPV frequency (P=0.572). Tumor site, staging, and differentiation did not correlate with HPV positivity with any of the methods employed (P>0.20). Conclusion: Using a P16 IHC assay alone or combined with CISH, the authors showed a higher rate of HPV positivity among patients with OSCC, as compared with patients with benign disease. Tumor site within the oropharynx, tumor stage, and degree of differentiation did not correlate with HPV positivity.

Orientadores: Sophie Françoise Mauricette Derchain, Cecilia Maria Roteli Martins / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-05T07:54:01Z (GMT). No. of bitstreams: 1 Gontijo_RenataClementino_D.pdf: 141945 bytes, checksum: 2c58c2feab74004e7047e409bad433a0 (MD5) Previous issue date: 2005 / Resumo: Introdução: Mulheres com resultado de citologia negativa e sem infecção pelo papilomavírus humano (HPV) têm teoricamente um risco quase nulo de terem uma lesão intra-epitelial escamosa cervical de alto grau ou câncer invasor. Porém, muitas mulheres com citologia negativa e colo uterino morfologicamente normal são infectadas pelo HPV, e o significado clínico desta infecção em relação ao risco de vir a apresentar anormalidades citológicas ou histológicas futuras ainda não está totalmente esclarecido. Objetivo: Investigar a incidência em 24 meses de alterações citológicas e histológicas cervicais segundo a detecção do HPV, em mulheres com citologia inicial normal, incluídas na coorte Latin American Screening study (LAMS). Material e métodos: Um grupo de 365 mulheres com resultado de citologia normal e resultado de Captura Híbrida II (CH II) para HPV de alto risco oncogênico positivo e negativo, foram seguidas por 24 meses em Campinas e São Paulo. Todas as mulheres responderam a um questionário referente aos fatores sociodemográficos e reprodutivos, e foram submetidas à coleta de material para citologia oncológica e CH II. As mulheres com pelo menos um exame positivo e uma amostra aleatória de 10% de mulheres com ambos os testes negativos foram convocadas para colposcopia com biópsia, se necessário, e seguimento semestral com citologia e colposcopia. Foram comparadas as mulheres com infecção pelo HPV com aquelas não infectadas, segundo as características sociodemográficas e reprodutivas, utilizando-se o cálculo do risco relativo (RR) e a análise de regressão logística em stepwise com intervalo de confiança (IC) de 95%. Foram calculados também a taxa de incidência e o RR com IC de 95% de desenvolver anormalidades citológicas ou histológicas durante o seguimento. Tomou-se como padrão-ouro a colposcopia. Quando a colposcopia foi normal ou quando a biópsia apresentou cervicite, as mulheres foram consideradas como diagnóstico negativo. As mulheres cuja biópsia foi compatível com neoplasia intra-epitelial cervical (NIC) grau 1 ou mais foram consideradas como diagnóstico positivo. Resultados: A incidência de lesões de baixo e alto graus na citologia foi maior entre as mulheres com resultado de CH II positivo, tanto aos 12 quanto aos 24 meses de seguimento. Até 12 meses de seguimento, mulheres com CH II de rastreamento positivo apresentaram um RR significativamente maior de lesões de baixo (1,4; IC 95% 1,1-1,7) e alto (1,5; IC 95% 1,4-1,7) graus na citologia. O RR para lesão de alto grau aumentou para 1,7 (IC 95% 1,5-1,9) naquelas acompanhadas por 24 meses. Em relação aos resultados histológicos, a incidência de NIC 1, 2 e 3 também foi maior entre as mulheres com resultado de CH II positivo, tanto aos 12 quanto aos 24 meses de seguimento. As mulheres com CH II positivo apresentaram um RR de 1,5 (IC 95% 1,4-1,6) para NIC 2 e 3 durante o seguimento até 12 meses e este RR aumentou para 1,7 (IC 95% 1,5-1,9) naquelas seguidas até 24 meses. Conclusão: O teste para detecção do HPV associado à citologia pode selecionar entre as mulheres com citologia normal aquelas com maior risco de lesão cervical subseqüente / Abstract: Introduction: Women with normal baseline cytology and non-infected by Human papillomavirus (HPV) have, in theory, no risk to develop a high-grade cervical intraepithelial lesion or cancer. However, many women with normal cytology and with morphologically normal uterine cervix are HPV infected, and, the clinical significance of this infection regarding to the risk of devoloping cytological or histological abnormalities in the future are not totally clear yet. Purpose: To investigate the incidence of cytological and histological cervical lesions in a 24 months follow-up, according to HPV detection among women with baseline normal cytology result, in a subgroup of women included in the Latin American Screening study (LAMS). Study design: A group of 365 women with normal Pap smear whatever the Hybrid Capture (HC) II test result were followed for 24 months at Campinas e São Paulo (Brazil). They answered a questionnaire regarding sociodemographic and reproductive factors and were submitted to a clinical exam, including Pap smear and HC II. Women with at least one positive result and a 10% random sample of women with both tests negative were referred to colposcopy and followed with cytology and colposcopy in a six-month interval. Women with positive and negative HPV test were compared regarding sociodemographic and reproductive factors using relative risk (RR) and stepwise logistic regression analysis calculated with 95% confidence interval (CI). Also the incidence rate and RR of developing any cytological or histological abnormality during the follow-up were calculated within 95% confidence limits. Colposcopy was considered as the gold standard. When colposcopy result was normal or biopsy result was cervicitis it was considered as negative diagnosis. Women with histologic diagnosis of cervical intraepithelial neoplasia (CIN) 1 or higher were considered as positive diagnosis. Results: Incidence of low and high-grade cytological lesion was higher in women with positive HPV testing than in women with negative HPV testing after 12 and 24 months of follow-up. In up to 12 months of follow-up, women with baseline positive HPV test had a significantly higher proportion of low-grade (1.4; 95% CI 1.1-1.7) and high -grade (1.5; 95%CI 1.4-1.7) cytological lesion. The RR for high-grade lesion increased to 1.7 (95%CI 1.5-1.9) for those followed-up in up to 24 months. For histological outcomes, the incidence of CIN 1, 2 or 3 was also higher in women with positive HPV testing than in women with negative HPV testing after 12 and 24 months of follow-up. Women with positive HPV test had a higher RR of CIN 2 and 3 (1.5; 95%CI 1.4-1.6) during the follow-up in up to 12 months and the RR increased to 1.7 (95%CI 1.5-1.9) for those followed-up in up to 24 months. Conclusions: HPV test is useful in addition to cytology to select from women with normal cytology those who are at highest risk for underlying cervical lesion / Doutorado / Tocoginecologia / Doutor em Tocoginecologia

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-15T13:30:32Z No. of bitstreams: 2 Dissertação - Larisse Silva Dalla Libera - 2016.pdf: 2593854 bytes, checksum: 6b47c9f7567c45f8cd463c53b5b7b129 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-09-15T13:31:04Z (GMT) No. of bitstreams: 2 Dissertação - Larisse Silva Dalla Libera - 2016.pdf: 2593854 bytes, checksum: 6b47c9f7567c45f8cd463c53b5b7b129 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-09-15T13:31:04Z (GMT). No. of bitstreams: 2 Dissertação - Larisse Silva Dalla Libera - 2016.pdf: 2593854 bytes, checksum: 6b47c9f7567c45f8cd463c53b5b7b129 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-03-29 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Anal cancer incidence is increasing significantly, especially when associated with the Human papillomavirus (HPV). The objective of this study was to evaluate the clinicopathological aspects of anal cancers and their association with HPV detection and genotypic distribution. It was a retrospective epidemiological study, with the use of the clinicopathological secondary data and molecular analyses of specimens of anal cancers embedded in paraffin blocks, diagnosed in a reference institution in the treatment of cancer at the Centro-Oeste region in a period of ten years (2000-2010). It was initially consulted a list of 140 cases of anal cancers but only 81 were included in the study. Molecular analysis included the amplification and detection of a viral genome fragment, by using PCR (polymerase chain reaction), and genotyping by reverse hybridization, using the commercial kit INNO LiPA. Data analysis was performed by descriptive statistics, univariate analysis and survival. According to the cases evaluated (n 81), 53.1% were more than 61 years of age, and the majority (63%) were females; 51.9% (n 42) presented squamous cells carcinoma (SCC). Lymph node metastases were described in 25.9%, distant metastases in 8.6% being the liver and the lung the most affected organs, the death was recorded in 45 cases (55.6%). HPV DNA was positive in 69% of cases. In the analysis by histologic type, 88.1% of squamous cell carcinomas (SCC), and 43.8% of adenocarcinomas was positive for viral DNA. The ECC was associated with HPV (p 0.0001) as well as females (p 0.01). Multiple infections were detected in 14.3% of the cases. The most prevalent genotypes were HPV16, HPV33 and HPV18. HPV 16 was found in 100% of multiple infections. After five years, overall survival was 44.3% for the group, the prognostic factors worse were female sex (p 0.008), squamous cell carcinoma (p 0.01) and the presence of distant metastasis (p 0. 01). Survival was not influenced by the presence of HPV (p 0.54). The aggressiveness of anal cancer described in this study reinforces the need of prevention strategies for this type of disease, including the HPV vaccine. / A incidência do câncer anal vem aumentando significativamente, principalmente quando associado ao Papilomavírus humano (HPV). O objetivo deste estudo foi avaliar os aspectos clínicopatológicos dos cânceres anais e suas associações com a detecção e distribuição genotípica do HPV. Trata-se de um estudo epidemiológico transversal retrospectivo, com a utilização dos dados clínicopatológicos secundários e análises moleculares de espécimes de cânceres anais contidos em blocos de parafina, diagnosticados em uma instituição de referência no tratamento de câncer na região Centro-Oeste em um período de dez anos (2000-2010). Inicialmente foi consultada uma lista com 140 casos de cânceres anais mas apenas 81 cumpriram os critérios de inclusão e exclusão e foram incluídos no estudo. A análise molecular incluiu a amplificação de um fragmento do genoma viral por PCR (reação em cadeia da polimerase) e hibridização reversa, com o uso do kit comercial INNO LiPA. A análise dos dados foi feita por estatística descritiva, análise univariada e de sobrevida. Do número de casos avaliados (n 81), 53,1% tinham mais que 61 anos de idade e a maior parte (63%) foram mulheres; 51,9%(n 42) apresentavam câncer anal de células escamosas (CCE). Metástases linfonodais foram descritas em 25,9%, metástases a distância em 8,6% sendo o fígado e o pulmão os órgãos mais acometidos e o óbito foi registrado em 45 casos (55,6%). A detecção de DNA do HPV foi positiva em 69% dos casos. Na ánalise por tipo histológico, 88,1% dos carcinomas de células escamosas (CCE) e 43,8% dos adenocarcinomas foram positivos para o DNA viral. O CCE foi associado ao HPV (p 0,0001) assim como o sexo feminino (p 0,01). Infecções múltiplas foram detectadas em 14,3% dos casos. Os genótipos mais prevalentes foram o HPV16, 33 e 18. O HPV 16 esteve presente em 100% das infecções múltiplas. A sobrevida global em 60 meses foi de 44,3%, os fatores de pior prognósticos foram o sexo feminino (p 0,008), o carcinoma de células escamosas (p 0,01) e a presença de metástase à distância (p 0,01). A sobrevida não foi influenciada pela presença do HPV (p 0,54). A agressividade dos cânceres anais descritos neste trabalho reforça a necessidade de estratégias de prevenção para esses cânceres, incluindo a vacina contra HPV.

Submitted by JÚLIO HEBER SILVA (julioheber@yahoo.com.br) on 2016-11-21T15:56:00Z No. of bitstreams: 2 Dissertação - Keila Patrícia Almeida de Carvalho - 2016.pdf: 1984915 bytes, checksum: 0d6b814fa8ededd81b851b12fb3ff2f8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-11-21T20:25:40Z (GMT) No. of bitstreams: 2 Dissertação - Keila Patrícia Almeida de Carvalho - 2016.pdf: 1984915 bytes, checksum: 0d6b814fa8ededd81b851b12fb3ff2f8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-11-21T20:25:40Z (GMT). No. of bitstreams: 2 Dissertação - Keila Patrícia Almeida de Carvalho - 2016.pdf: 1984915 bytes, checksum: 0d6b814fa8ededd81b851b12fb3ff2f8 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-10-14 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Introduction. Squamous cell carcinoma is the most common type of cervical cancer, followed by endocervical adenocarcinoma. Tumor staging is important to evaluate prognosis. Cervical infection by one of the oncogenic types of HPV, especially HPV 16 and 18 is a pre-requisite for developing invasive cancer. Epigenetics alterations, as DNA methylation, are well-known carcinogenic mechanisms. WIF1 is a tumor suppressor gene which silencing by methylation helps in neoplastic progression. Objective. The goal of this study was to evaluate staging, HPV genotypes and WIF1 methylation in cervical cancer and to test association between these variables with age, prognosis and survival. Methods. It was included 95 cases obtained in Araujo Jorge Hospital (Goiânia/ GO): 73 of invasive squamous cell carcinoma and 22 of invasive endocervical adenocarcinoma. DNA was extracted from paraffin-embedded biopsies using phenol-chloroform. HPV detection and genotyping were conducted using the kit INNO- LiPA HPV GENOTYPING EXTRA® (Innogenetics™). Methylation of WIF1 gene was evaluated by methylation-specific PCR (MSP). Odds Ratio was used to calculate statistical association. The calculation of survival used the Kaplan-Meier method and the log-hank test was used to compare means of survival and prognostic factors. A value of p<0.05 was considered statically significant. Results. There was significant association between tumor stages III and IV and worse prognosis (OR = 7.32). The 5-year overall survival was 79.1% and it was significant higher in cases with tumor stages I and II (p = 0.001). Women between 50 and 60 years had more chances having tumors in stages III and IV (OR = 0.15). Although, considering mean and median ages of women included, those over 51 years were more likely having tumor stages III and IV (OR = 5.92). No association was found between worse prognosis or lower survival and histological type, HPV infection and WIF1 methylation. Conclusion. Worse prognosis and lower survival was determined by tumor stages III and IV. / Introdução. O tipo histológico mais comum do câncer do colo do útero é o carcinoma de células escamosas, seguido pelos adenocarcinomas endocervicais. O estadiamento tumoral é importante para avaliar o prognóstico. A infecção da cérvice por um dos genótipos oncogênicos de HPV, especialmente os HPV 16 e 18, é pré-requisito para o desenvolvimento do câncer invasor. Alterações epigenéticas, como a metilação do DNA, são mecanismos conhecidos de carcinogênese. O gene WIF1 é supressor tumoral e seu silenciamento por metilação auxilia na progressão neoplásica. Objetivo. O objetivo desse estudo foi avaliar o estadiamento, genótipos de HPV e a metilação do gene WIF1 e testar a associação entre estas variáveis e a idade, o prognóstico e a sobrevida de mulheres com câncer do colo do útero. Métodos. Foram incluídos 95 casos obtidos no Hospital Araújo Jorge (Goiânia/GO) sendo 73 de carcinomas escamosos invasores e 22 de adenocarcinomas endocervicais invasores. O DNA foi extraído com fenol-clorofórmio dos materiais de biópsia incluídos em parafina e a detecção e genotipagem de HPV foram realizadas utilizando-se o kit INNO- LiPA HPV GENOTYPING EXTRA® (Innogenetics™). A análise de metilação do gene WIF1 foi realizada através da técnica PCR específica para metilação (MSP). As associações estatísticas foram feitas com cálculo de Odds Ratio e a sobrevida foi calculada pelo método de Kaplan-Meier, sendo a comparação das médias de sobrevida e os fatores prognósticos analisada pelo teste log-rank. Um valor de p<0,05 foi considerado estatisticamente significativo. Resultados. Houve associação estatisticamente significante entre neoplasias diagnosticadas nos estágios III e IV e pior prognóstico (OR = 7,32). A sobrevida global em cinco anos foi de 79,1% e foi significativamente maior nos casos com estágios I e II (p = 0,001). Mulheres na faixa etária entre 50 e 60 anos idade mostraram-se com maior chance de serem portadoras de câncer do colo do útero em estágios I e II (OR = 0,15). Contudo, considerando a média e mediana da idade das mulheres incluídas, aquelas com idade acima dos 51 anos tiveram mais chance de serem diagnosticadas com câncer em estágios III e IV (OR = 5,92). Não houve associação significante entre tipo histológico, infecção por HPV ou metilação do gene WIF1 e pior prognóstico ou menor sobrevida. Conclusão. Pior prognóstico e menor sobrevida das mulheres foram determinados pelo estadiamento tumoral em III e IV.

O carcinoma epidermóide de cabeça e pescoço (CECP) é um problema de saúde relevante no mundo, por sua prevalência e agressividade. Os papéis do tabaco e do álcool estão bem definidos na sua etiologia, mas uma minoria crescente dos pacientes acometidos pela doença não é tabagista ou etilista. A infecção pelo papilomavirus humano (HPV) parece ser responsável por parte desses casos. Sugere-se que o CECP que acomete pacientes não tabagistas e não etilistas (NTNE) tenha processos carcinogênicos e evolução clínica distintos daqueles de pacientes tabagistas e etilistas (TE). Os objetivos desse estudo foram verificar se os aspectos demográficos, clínicos e histopatológicos de pacientes com CECP são diferentes conforme os hábitos tabágico e etílico; verificar se os CECPs de pacientes NTNE e TE diferem em relação à positividade para HPV; e comparar a sobrevida específica pela doença e a expressão de marcadores biológicos em amostras tumorais e de mucosa normal do trato aerodigestório de pacientes NTNE e de pacientes TE. Para tanto, realizamos estudo transversal de pacientes com carcinomas epidermóides de cavidade oral (exceto lábio), orofaringe, laringe e hipofaringe, prospectivamente incluídos em um banco de tumores de CECP de 2001 a 2009, pelo grupo de pesquisa multi-institucional GENCAPO. Seus dados demográficos, clínicos e patológicos foram analisados conforme os hábitos de tabagismo e etilismo. Através de análise de pareamento, pacientes NTNE e TE foram comparados em relação à sobrevida, à positividade para o HPV no tumor por reação em cadeia da polimerase (PCR) e aos marcadores imunohistoquímicos p53, FHIT, Ki-67, VEGF, EGFR e p16 tanto em amostras tumorais como de epitélio não tumoral. Dos 1633 pacientes selecionados, 80 eram NTNE (4,9%), 1374 TE (84,1%), 140 tabagistas atuais ou no passado mas não etilistas (TNE:8,6%) e 39 apenas etilistas atuais ou no passado, mas não tabagistas (NTE:2,4%). O grupo de pacientes NTNE constituiu-se principalmente por mulheres, preferencialmente idosas, com tumores da cavidade oral, diferentemente de pacientes tabagistas e/ou etilistas (p<0,001). Considerando os diferentes padrões de hábitos, pacientes TE eram geralmente mais jovens (p<0,001), pacientes TNE apresentaram proporcionalmente mais tumores de laringe (p<0,001) e pacientes NTNE apresentaram menor número de parentes de primeiro grau tabagistas (p<0,001). Observou-se um provável efeito do álcool na ocorrência de metástases ganglionares (p<0,001), enquanto o tabaco pareceu relacionar-se a menor grau de diferenciação tumoral à histologia e a menores índices de massa corpórea (p<0,001). Detectou-se a presença de material genético do HPV em 32,8% dos tumores de pacientes NTNE. Desses tumores positivos para o HPV, metade apresentou também hiperexpressão pelo p16. Foi observado menor risco de óbito pela doença em pacientes NTNE quando apresentavam expressão tumoral intensa do p16 (p=0,011 / RR = 0,07; IC 0,01-0,55) e menor sobrevida se apresentavam marcação pelo FHIT em camada basal de margem não tumoral (p= 0,031). Ao comparar pacientes NTNE e TE, não se observaram diferenças na sobrevida específica pela doença ou na positividade para o HPV de forma independente de sexo, idade, sítio tumoral, grau de diferenciação, variante morfológica, estádio T e presença de metástases linfonodais. Pacientes NTNE apresentaram marcação nuclear pelo FHIT em camada basal menos frequentemente do que pacientes TE (p=0,021) / Head and neck squamous cell carcinoma (HNSCC) is a major health problem worldwide, due to its prevalence and aggressiveness. The role of tobacco and alcohol in its etiology is well established; however, a growing minority of patients with HNSCC neither smokes nor consumes alcohol. Human Papillomavirus (HPV) infection seems to be responsible for some of these cases. It is suggested that HNSCC affecting non smoking and non drinking (NSND) patients has different carcinogenesis and outcomes than those in smoking and drinking (SD) subjects. The objectives of this study were to test if demographic, clinical and pathological aspects of patients with HNSCC vary according to smoking and drinking habits; to test if HNSCC in NSND and SD patients differ in terms of HPV positivity; and to compare NSND and SD patients with HNSCC according to survival and biomarkers in tumor and mucosal samples of the aerodigestive tract. We conducted a cross-sectional study of patients with oral cavity (lips excluded), oropharynx, larynx and hypopharynx tumors prospectively included in a multi-institutional HNSCC tumor bank - GENCAPO, from January 2001 to February 2009. Demographic, clinical and pathological data were analyzed in regards of smoking and drinking habits. Using matched-pair analysis, we compared NSND and SD patients in relation to disease-free survival, HPV positivity through polymerase chain reaction (PCR) and Immunohistochemical staining of p53, FHIT, Ki-67, VEGF, EGFR and p16 biomarkers in tumor and mucosal samples. From 1633 patients, 80 were NSND (4.9%), 1374 SD (84.1%), 140 current or past smokers, but non drinkers (SND:8.6%) and 39 current or past drinkers, but non smokers (NSD:2.4%). NSND patients were most frequently women, remarkably elderly, with oral cavity cancers more commonly than the other groups (p<0.001).Comparing to the other groups, SD patients were younger (p<0.001); SND patients were more frequently affected by larynx tumors (p<0.001) and NSND patients had fewer smoking first degree relatives (p<0.001). We observed that alcohol may influence the presence on node metastasis (p<0.001) whereas tobacco may be related to less differentiated tumors and lower body mass indexes (p<0.001). We found HPV DNA in 32.8% of tumors of NSND subjects. Half of the HPV positive tumors were also positive for p16 staining. A lower risk of death from disease was observed among NSND patients with intense p16 staining (p=0.011 / RR = 0.07; CI 0.01-0.55) and lower survival rates in patients with positive nuclear staining for FHIT at the basal layer of mucosal epithelium (p=0.031). We found no differences in disease-free survival of NSND and SD patients in an independent manner of gender, age, tumor site, differentiation grade at histology, pathological variants, T stage and the presence of node metastasis. NSND patients presented less frequently with nuclear staining for FHIT at the basal layer of mucosal epithelium than SD patients (p=0.021)

Human papillomavirus (HPV) is a double-stranded DNA virus, typically infecting mucosal or cutaneous epithelial keratinocytes. Today, more than 118 different HPV types have been formally described. Sexual transmission of mucosal HPVs is very common and generally asymptomatic, but HPV infection can be associated with benign lesions such as condylomata acuminata or, in rare cases, with malignant lesions such as cervical cancer. Two prophylactic vaccines are currently available in Europe, protecting against HPV-16 and HPV-18 (Cervarix&63720;) or against HPV-6, HPV-11, HPV-16 and HPV-18 (Gardasil&63720;). In order to assess the impact of the vaccination program, it is mandatory to obtain geographically widespread date on the baseline HPV prevalence and type distribution in cervical samples from women, presenting or not, normal or abnormal cytologic or histologic results. We undertook an epidemiological study in the Capital Region of Brussels to determine the HPV prevalence and type-distribution in 1526 cervical samples of women presenting a cytology within normal limits (WINL), atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intra-epithelial lesions (LSIL), high-grade squamous intra-epithelial lesions (HSIL) or invasive cervical cancer (ICC). The HPV prevalence was 10.8% (95%CI: 8.8-12.8) for NILM, 34.5% (95%CI: 28.3-40.8) for ASC-US, 54.0% (95%CI: 47.4-60.6) for LSIL and 100% for HSIL and ICC. With an HPV-16 and HPV-18 prevalence of 63.3% (95%CI: 44.1-67.7) and 73.5% (95%CI: 63.0-84.0) in mono-infected HSIL and ICC, respectively, HPV 16/18 L1 VLP vaccines would be expected to significantly reduce the management and treatment of women suffering from HSIL and ICC in the Capital Region of Brussels. We also detected HPV-30, HPV-53, HPV-66 and HPV-68 in mono-infected HSIL and ICC samples, possibly providing arguments for the reconsideration of the carcinogenicity of these types. <p>Vertical transmission of HPV was also previously reported, but in most cases one could not exclude a placental contamination by HPV positive cells from an infected birth canal. In order to confirm that the placenta can be infected with HPV, we analysed residual cells from 35 transabdominally obtained placental samples from pregnant women undergoing chorionic villous sampling for screening of suspected foetal abnormalities and found that two samples were positive for HPV-16 and HPV-62, respectively. The clinical importance of these results remains to be elucidated, but the previously observed association between placental HPV infection and pregnancy loss might gain further in importance. HPV gene regulation in placental trophoblastic cells has not been studied so far. We studied the HPV-16 early gene expression regulation in transiently transfected monolayer cultured trophoblastic cells with an HPV-16 long control region (LCR) driven reporter plasmid. We observed important differences in constitutive HPV-16 LCR activities between trophoblastic cell lines and could identify progesterone as an important inducer of HPV-16 early gene expression. Steroid hormones are induced during pregnancy and could therefore lead to an enhanced expression of the E5, E6 and E7 proteins upon placental HPV infection. Since these proteins were previously shown to affect trophoblast adhesion, survival, migration and invasion, their enhanced expression might eventually contribute to pregnancy loss. We furthermore found that the transcription of episomally maintained HPV-16 is not regulated by E2 or E1, but by E5, E6 and/or E7. <p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

Les carcinomes épidermoïdes des voies aéro-digestives supérieures (VADS) se situent au sixième rang des cancers les plus fréquents dans le monde. Ces tumeurs sont liés à deux facteurs de risque : l’intoxication éthylo-tabagique (80% des cas) et l’infection de l’épithélium des VADS par les papillomavirus humain (HPV) à haut risque oncogène (20% des cas). Ces derniers définissent une sous-population de patients de meilleur pronostic. Une des hypothèses actuellement étudiées, afin d’expliquer la survie améliorée des patients HPV positifs, serait une hypoxie moindre dans ces tumeurs. En effet, les tumeurs des VADS sont fréquemment hypoxiques, et l’hypoxie intratumorale est un facteur de mauvais pronostic. Dans une première partie de cette thèse, nous avons entrepris une caractérisation moléculaire de l’hypoxie intratumorale dans les tumeurs humaines oropharyngées en fonction du statut HPV. Il apparaît que les tumeurs HPV positives présentent un statut hypoxique moindre comparées aux tumeurs HPV négatives. Ces tumeurs se caractérisent également par une abondante vascularisation intratumorale, qui pourrait être à l’origine de ce statut hypoxique moindre. Dans une deuxième partie, nous avons étudié l’adaptation à l’hypoxie de la lignée cellulaire HPV négative SQ20B et la lignée cellulaire HPV positive SCC90. De plus, des modèles de xénogreffes ont été établis à partir de ces mêmes lignées cellulaires et ont été analysés du point de vue de l’hypoxie intratumorale. De façon comparable aux tumeurs HPV positives, les xénogreffes obtenus à partir de la lignée SCC90 montre un statut hypoxique réduit comparés aux xénogreffes SQ20B. Les deux lignées cellulaires s’adaptent également différemment en hypoxie in vitro. La réponse à l’hypoxie dans la lignée SCC90 semble plus dynamique. En effet, la lignée SCC90 tente de s’adapter et de répondre à cet environnement hypoxique en induisant de fort niveau d’expression de gènes comparée à la lignée SQ20B. / Head and neck squamous cell carcinoma (HNSCC) represents the sixth most common malignancy worldwide. The major risk factors for HNSCC identified are tobacco use and alcohol consumption (80% of all HNSCC), which seem to have a synergistic effect. A subgroup of HNSCCs (20% of cases), particularly those of the oropharynx, is caused by infection with high-risk types of human papillomavirus (HPV). Human papillomavirus HPV-related oropharyngeal squamous cell carcinoma defines a distinct clinical subgroup of head and neck cancer patients with improved prognosis. Currently, one of the several hypothesis studied to account for their improved survival outcomes could be a distinct hypoxia status compared to their HPV-negative counterpart. Indeed, tumour hypoxia is common in solid tumours including head and neck tumours, and hypoxia is a well-known poor prognosis factor. In first part of this thesis, we have performed a molecular characterisation of tumor hypoxia on cohort of oropharyngeal tumours according to HPV status of the patients. The results support the hypothesis that HPV-related tumours display a lesser hypoxia status compared to HPV-negative oropharyngeal tumours. These HPV-related tumours also characterize by an abundant tumour vascularisation, which could be responsible for a lesser hypoxia status. In a second part, we have studied the ability of the adaptation to hypoxia of the HPV-positive SCC90 cell line and HPV-negative SQ20B cell line. Furthermore, HPV-positive and HPV-negative HNSCC xenograft models have been established and have been analysed about tumor hypoxia. Similar to HPV-related HNSCC, tumours-derived HPV positive cell lines display a reduced hypoxic status compared to tumours-derived HPV negative cell lines. The two cell lines adapt also differently to in vitro hypoxia. In the HPV-positive cell line, the hypoxia response pathways could be more dynamics. Indeed, SCC90 cell lines attempt to adapt and to reply to hypoxic environment inducing highly expression of all of the hypoxia related genes compared to SQ20B cell lines.

Abstract High risk human papilloma virus (hrHPV) infection is a necessary but not a sufficient cause of cervical cancer. In Finland, since 1990 the incidence of cervical cancer has increased among women younger than 40 years of age despite a nationwide screening programme. In this thesis, the overall objective is to address the role of possible, earlier hrHPV epidemic in this increased incidence of cervical cancer. The target population includes all fertile-aged women in Finland during 1983–2006. The actual study population comprised all women with a minimum of two pregnancies within five years and under 32 years of age in 1983–1997 and under 29 years of age in 1995–2003 identified from the Finnish Maternity Cohort (FMC). From this subpopulation, two subcohorts were selected for hrHPV antibody analysis by random sampling stratified by age and calendar time. All cases of cervical cancer diagnosed for women under 50 years of age during 1983–2002 and 1986–2006 were identified from the Finnish Cancer Registry. The case-cohort design, used for estimating population attributable fractions (PAF) associated with hrHPV, included the cases of cervical cancer and the first subcohort of FMC. A steady annual increase of 0.7% per year in the incidence of HPV16 was estimated to have taken place in Finland from 1983 to 1997 among the 23–31-year-old women with at least two pregnancies. The estimated seroprevalence of HPV16 increased from 17% to 24%, respectively. The PAF of hrHPV exposures in squamous cell carcinoma of the uterine cervix (SCC) was estimated as 73% (95% CI: 13% to 93%). For 26–31-year-old women born in the 1960s and 1970s the incidence of SCC was roughly double compared with women born in the late 1950s. Mathematical modelling indicated that changes in the sexual behaviour partly accounted for the increase seen in the incidence of cervical cancer in the 1990s. The findings of this thesis indicate that growth in the background exposure to HPV16 preceded the increase of incidence of cervical cancer in Finland. At younger birth cohorts, the increase of the incidence of SCC is visible among fertile-aged women in Finland. Whether overall screening starting at 25 years of age, higher participation rate for cervical screening or HPV vaccination of early adolescents is the future solution to lowering the incidence of cervical cancer among young women remains to be seen. / Tiivistelmä Ihmisen papilloomaviruksen (HPV), erityisesti korkean riskin tyypin (hrHPV), aiheuttama infektio on kohdunkaulan syövän välttämätön, mutta ei riittävä syytekijä. Suomessa vuoden 1990 jälkeen kohdunkaulan syövän ilmaantuvuus on valtakunnallisesta seulonnasta huolimatta noussut alle 40-vuotiailla naisilla. Tämän väitöskirjan tavoitteena on osoittaa, mikä rooli mahdollisella aiemmalla hrHPV-epidemialla on kyseiseen kohdunkaulan syövän ilmaantuvuuden kasvuun. Tutkimuksen kohdeväestöön kuuluvat kaikki lisääntymisikäiset suomalaiset naiset. Varsinainen tutkimusväestö koostui kaikista vuosina 1983–97 alle 32-vuotiaana ja vuosina 1995–2003 alle 29-vuotiaana kaksi kertaa raskaana olleista naisista, jotka identifioitiin Suomen äitikohortista (FMC). Tästä joukosta valittiin satunnaisotannalla kaksi alikohorttia hrHPV-laboratorioanalyysejä varten. Kaikki vuosina 1983–2002 ja 1986–2006 kohdunkaulan syöpädiagnoosin alle 50-vuotiaana saaneet naiset poimittiin Suomen syöpärekisteristä. Tapaus-kohorttiasetelma, jota käytettiin hrHPV altistukseen liittyvien väestösyyosuuksien (PAF) estimoinnissa, sisälsi kohdunkaulan syöpätapaukset ja ensimmäisen alikohortin. Suomalaisten 23–31 -vuotiaiden, vähintään kahdesti raskaana olleiden, naisten vuosittainen HPV16-ilmaantuvuus kasvoi tasaisesti 0.7&#160;% per vuosi ajanjaksolla 1983–1997. Vastaavasti HPV16:n vallitsevuus kasvoi 17 prosentista 24 prosenttiin. Kohdunkaulan levyepiteelisyövän hrHPV-altistukseen liittyvän PAF:n estimoitiin olevan 73&#160;% (95&#160;%:n luottamusväli 13–93&#160;%). Levyepiteelisyövän ilmaantuvuus oli suunnilleen kaksinkertainen 1960- ja 1970-luvulla syntyneillä naisilla, heidän ollessaan 26–31 -vuotiaita, verrattuna 1950-luvulla syntyneisiin samanikäisiin naisiin. Matemaattisen mallinnuksen tulosten perusteella kohdunkaulan syövän ilmaantuvuuden nousu 1990- luvulla selittyy ainakin osittain sukupuolikäyttäytymisen muutoksilla. Tämän väitöskirjan tulokset osoittavat, että kasvanut HPV16-virukselle altistuminen edelsi kohdunkaulan syövän ilmaantuvuuden nousua Suomessa. Levyepiteelisyövän ilmaantuvuuden nousu nuorimmissa syntymäkohorteissa on nähtävissä lisääntymisikäisillä naisilla Suomessa. Tulevaisuudessa nähdään, onko seulonnan aloittaminen 25-vuotiaana, korkeampi seulontaan osallistumisosuus vai nuorten aikuisten HPV-rokottaminen ratkaisu nuorten naisten kohdunkaulan syövän ilmaantuvuuden vähentämiseksi.