Nierenzellkarzinome (NZKs) gelten als stark immunogene Tumore. Dies ist insbesondere auf die Infiltration durch verschiedene Immunzellpopulationen, wie T-Lymphozyten und Natürliche Killer (NK)-Zellen, sowie das klinische Ansprechen auf immuntherapeutische Strategien zurückzuführen. Bisher existieren jedoch nur sehr wenige Studien zur Rolle von humanen nativen dendritischen Zellen (DCs) in NZK-Geweben und über die Tumor-vermittelte Modulation dieser DCs. DCs nehmen als professionelle Antigen-präsentierende Zellen eine zentrale Schlüsselrolle bei der Induktion und Aufrechterhaltung der angeborenen sowie adaptiven Immunantwort ein. Daher wurde im Rahmen dieser Arbeit erstmals der Effekt von klarzelligen NZKs auf den Phänotyp sowie die immunmodulatorischen Fähigkeiten von 6-sulfo LacNAc+ (slan)DCs evaluiert. SlanDCs, welche eine große Subpopulation humaner Blut-DCs darstellen, sind neben der Sekretion großer Mengen proinflammatorischer Zytokine dazu befähigt, Tumorzellen direkt zu lysieren. Des Weiteren sind slanDCs in der Lage, die antitumoralen Effekte von NK-Zellen zu fördern und CD4+ T-Helfer-Zellen sowie Tumor-reaktive CD8+ T-Lymphozyten effizient zu stimulieren. Angesichts dieser proinflammatorischen Eigenschaften können slanDCs wesentlich an einer Tumor-gerichteten Immunantwort beteiligt sein. Auf dieser Grundlage erfolgte im Rahmen der vorliegenden Arbeit der immunhistochemische Nachweis von slanDCs in klarzelligen NZK-Geweben. Im Vergleich zu Tumor-freiem Nierengewebe trat in den primären Tumorgeweben eine erhöhte Zahl infiltrierender slanDCs auf. Zudem wurde die Präsenz von slanDCs in Lymphknoten- sowie Fernmetastasen von NZK-Patienten beobachtet. Weiterführende Untersuchungen an frischen klarzelligen NZK-Geweben demonstrierten, dass NZK-infiltrierende slanDCs einen unreifen Phänotyp ausprägen und Interleukin-10 produzieren. Ausgehend von diesen Erkenntnissen erfolgten funktionelle Analysen, bei denen der Einfluss der kommerziell erhältlichen klarzelligen NZK-Linien ACHN und Caki-1 sowie der primären klarzelligen NZK-Linien MZ1257RC und MZ2877RC auf bedeutende immunmodulatorische Fähigkeiten von slanDCs untersucht wurde. In diesem Zusammenhang zeigte sich, dass NZK-Zellen effektiv in der Lage sind, sowohl die slanDC-vermittelte Proliferation von CD4+ und CD8+ T-Lymphozyten, als auch die slanDC-induzierte Differenzierung naïver CD4+ T-Lymphozyten in proinflammatorische T-Helfer 1-Zellen zu inhibieren. Darüber hinaus wurde demonstriert, dass NZK-Zellen das Potenzial von slanDCs zur Aktivierung von NK-Zellen hemmen. Untersuchungen der zugrunde liegenden Mechanismen zeigten, dass die funktionelle Inhibition von slanDCs durch klarzellige NZK-Zellen über membranständige Moleküle vermittelt wird. Die im Rahmen dieser Dissertation gewonnenen Erkenntnisse weisen darauf hin, dass NZKs die Ausreifung sowie wesentliche funktionelle Eigenschaften von DCs inhibieren. Dies deutet auf einen neuen Immunescape-Mechanismus klarzelliger NZKs hin, welcher auf einer Tumorzell-vermittelten Generierung von tolerogenen slanDCs basiert und eine unzureichende Aktivierung der angeborenen sowie adaptiven Tumor-gerichteten Immunantwort zur Folge hat. Diese neuen Erkenntnisse können einen Beitrag zu einem besseren Verständnis der Interaktion von NZKs mit nativen humanen DCs leisten und die Konzeption neuer therapeutischer Strategien ermöglichen, welche auf einer Verstärkung der antitumoralen Eigenschaften von DCs beruhen.

Nierenzellkarzinome (NZKs) gelten als stark immunogene Tumore. Dies ist insbesondere auf die Infiltration durch verschiedene Immunzellpopulationen, wie T-Lymphozyten und Natürliche Killer (NK)-Zellen, sowie das klinische Ansprechen auf immuntherapeutische Strategien zurückzuführen. Bisher existieren jedoch nur sehr wenige Studien zur Rolle von humanen nativen dendritischen Zellen (DCs) in NZK-Geweben und über die Tumor-vermittelte Modulation dieser DCs. DCs nehmen als professionelle Antigen-präsentierende Zellen eine zentrale Schlüsselrolle bei der Induktion und Aufrechterhaltung der angeborenen sowie adaptiven Immunantwort ein. Daher wurde im Rahmen dieser Arbeit erstmals der Effekt von klarzelligen NZKs auf den Phänotyp sowie die immunmodulatorischen Fähigkeiten von 6-sulfo LacNAc+ (slan)DCs evaluiert. SlanDCs, welche eine große Subpopulation humaner Blut-DCs darstellen, sind neben der Sekretion großer Mengen proinflammatorischer Zytokine dazu befähigt, Tumorzellen direkt zu lysieren. Des Weiteren sind slanDCs in der Lage, die antitumoralen Effekte von NK-Zellen zu fördern und CD4+ T-Helfer-Zellen sowie Tumor-reaktive CD8+ T-Lymphozyten effizient zu stimulieren. Angesichts dieser proinflammatorischen Eigenschaften können slanDCs wesentlich an einer Tumor-gerichteten Immunantwort beteiligt sein. Auf dieser Grundlage erfolgte im Rahmen der vorliegenden Arbeit der immunhistochemische Nachweis von slanDCs in klarzelligen NZK-Geweben. Im Vergleich zu Tumor-freiem Nierengewebe trat in den primären Tumorgeweben eine erhöhte Zahl infiltrierender slanDCs auf. Zudem wurde die Präsenz von slanDCs in Lymphknoten- sowie Fernmetastasen von NZK-Patienten beobachtet. Weiterführende Untersuchungen an frischen klarzelligen NZK-Geweben demonstrierten, dass NZK-infiltrierende slanDCs einen unreifen Phänotyp ausprägen und Interleukin-10 produzieren. Ausgehend von diesen Erkenntnissen erfolgten funktionelle Analysen, bei denen der Einfluss der kommerziell erhältlichen klarzelligen NZK-Linien ACHN und Caki-1 sowie der primären klarzelligen NZK-Linien MZ1257RC und MZ2877RC auf bedeutende immunmodulatorische Fähigkeiten von slanDCs untersucht wurde. In diesem Zusammenhang zeigte sich, dass NZK-Zellen effektiv in der Lage sind, sowohl die slanDC-vermittelte Proliferation von CD4+ und CD8+ T-Lymphozyten, als auch die slanDC-induzierte Differenzierung naïver CD4+ T-Lymphozyten in proinflammatorische T-Helfer 1-Zellen zu inhibieren. Darüber hinaus wurde demonstriert, dass NZK-Zellen das Potenzial von slanDCs zur Aktivierung von NK-Zellen hemmen. Untersuchungen der zugrunde liegenden Mechanismen zeigten, dass die funktionelle Inhibition von slanDCs durch klarzellige NZK-Zellen über membranständige Moleküle vermittelt wird. Die im Rahmen dieser Dissertation gewonnenen Erkenntnisse weisen darauf hin, dass NZKs die Ausreifung sowie wesentliche funktionelle Eigenschaften von DCs inhibieren. Dies deutet auf einen neuen Immunescape-Mechanismus klarzelliger NZKs hin, welcher auf einer Tumorzell-vermittelten Generierung von tolerogenen slanDCs basiert und eine unzureichende Aktivierung der angeborenen sowie adaptiven Tumor-gerichteten Immunantwort zur Folge hat. Diese neuen Erkenntnisse können einen Beitrag zu einem besseren Verständnis der Interaktion von NZKs mit nativen humanen DCs leisten und die Konzeption neuer therapeutischer Strategien ermöglichen, welche auf einer Verstärkung der antitumoralen Eigenschaften von DCs beruhen.

Introdução: Apesar da identificação de fatores prognósticos clínicos e patológicos, muitos pacientes portadores de carcinoma de células renais (CCR) apresentam metástases ao diagnóstico e outros irão desenvolver recorrência local ou à distância durante o seguimento. Novos fatores prognósticos e de origem molecular têm sido avaliados no CCR, destacando-se o PTEN como um dos principais genes envolvidos na carcinogênese renal. Objetivos: Avaliar os fatores clínicos e anatomopatológicos mais significativos nas taxas de sobrevida, identificar a frequência de hipermetilação do gene PTEN através da técnica do pirosequenciamento, o impacto da hipermetilação do gene nas taxas de sobrevida global (SG) e livre de doença (SLD), como também, a associação da presença de hipermetilação com os principais fatores prognóticos. Material e métodos: Foram avaliados 137 pacientes portadores de CCR submetidos a tratamento cirúrgico do tumor primário entre 1997 e 2009. Foram considerados os dados epidemiológicos, clínicos, anatomopatológicos, de estadiamento (TNM 2004) e os obtidos da reação de pirosequenciamento. Resultados: O tempo de seguimento médio foi de 32,3 meses e mediana de 28,8 meses. Considerando o estadimento clínico, foram fatores independentes para a SG: idade (p<0,01), ASA (p=0,02), margens cirúrgicas (p=0,04), grau de Fuhrman (p=0,01), estádio clínico (p<0,001) e subtipo histológico (p<0,01). No modelo múltiplo a SLD foi influenciada únicamente pelo estádio clínico (p<0,001). Dos 137 casos analisados, hipermetilação do gene foi detectada em cinco casos (3,6%). Devido a baixa freqüência detectada optou-se por não realizar a associação da metilação do PTEN com os fatores prognósticos. Em relação às taxas de SG e SLD, de acordo com o perfil de hipermetilação do PTEN, não houve a ocorrência de nenhum evento, ou seja, morte, morte por CCR ou recorrência da doença para os cinco casos que apresentavam hipermetilação. Conclusões: A hipermetilação do xv PTEN foi detectada com baixa frequência, sugerindo a participação de outros genes ou mecanismos moleculares diferentes da metilação na inativação deste gene frequentemente envolvido na carcinogênese renal. As taxas de sobrevida não foram influenciadas pelo perfil de hipermetilação do PTEN, permanecendo o estadiamento clínico do TNM como a principal variável determinante da evolução e do risco de recidiva pelo CCR / Introduction: Despite the identification of clinical and pathological prognostic factors, many patients with renal cell carcinoma (RCC) have metastases at diagnosis and others will develop local or distant recurrence during follow-up. New prognostic factors and of molecular origin have been evaluated in RCC, highlighting PTEN, one of the main genes involved in renal carcinogenesis. Objetives: To assess the most significant clinical and pathological factors in survival rates, and identify the frequency of hypermethylation of the PTEN gene by the pyrosequencing technique, the impact of gene hypermethylation on overall survival (OS) rates and disease free interval (DFS), as well as associating presence of hypermethylation with main prognostic factors. Methods: We evaluated 137 patients with RCC that underwent surgical treatment of primary tumor between 1997 and 2009. We considered the epidemiological, clinical, pathological, staging (TNM 2004) data and those obtained from pyrosequencing. Results: Mean follow-up was of 32.3 months and the median of 28.8 months. Considering the clinical TNM stage, the OS was influenced in the multiple model by age (p < 0.01), ASA (p = 0.02), surgical margins (p = 0.04), Fuhrman´s grade (p = 0,01), clinical stage (p <0.001) and cell subtype (p < 0.01). DFS were influenced in multivariate analysis only by presence of clinical stage (p <0.001). Of the 137 cases examined, gene hypermethylation was detected in five cases (3,6%). Because of this low frequency perceived, we elected not to carry out the association of PTEN methylation with prognostic factors. Regarding OS and DFS rates, according to the hypermethylation of PTEN profile, no event occurred, that is to say death, death from RCC or disease recurrence in the five cases with hypermethylation. Conclusions: Hypermethylation of PTEN was detected with low frequency suggesting involvement of other genes or different molecular mechanisms of methylation upon inactivation of this gene, frequently involved in renal xvii carcinogenesis. Survival rates were not influenced by the hypermethylation of PTEN profile, with clinical TNM staging remaining as the main determinant for development and risk of RCC recurrence

Ce travail avait deux objectifs: 1) définir des groupes de patients (pts) susceptibles de bénéficier d’un conseil génétique par l’identification de facteurs prédictifs de l’existence d’une mutation du gène CDKN2A, un des gènes majeurs de prédisposition au mélanome, dans les familles ne comportant que deux cas (Fam_2 cas). 2) la caractérisation épidémiologique et clinique d’entités particulières du mélanome dans l’objectif secondaire de contribuer à l’identification de gènes de prédisposition à ces entités. Les 2 entités étudiées étaient le mélanome cutané (MC) associé au cancer du rein (CR) et les mélanomes muqueux de la sphère ano-génitale (MMAG).Les populations d’étude sont une collection de 293 pts atteints de MC recrutés de façon consécutive sans connaissance à priori de l'histoire familiale et la collection française MELARISK qui comprend ≥ 3000 sujets prélevés appartenant à des familles à cas multiples de mélanomes ou ayant un MC survenant dans un contexte particulier (association à un autre cancer, topographie rare, survenue avant l’âge de 20 ans, MC multiples sporadiques). Nous avons étudié l'effet de 3 facteurs prédictifs potentiels sur la présence d’une mutation de CDKN2A dans une famille en fonction du nombre de pts atteints dans une famille (2 pts versus ≥3 pts). L’étude a été menée dans 483 familles françaises comprenant 387 Fam_2 cas, et 96 familles avec ≥3 pts atteints de mélanome (Fam_3+ cas). Les facteurs étudiés dans la famille un à un puis conjointement étaient : l’âge médian <50 ans au diagnostic de MC, la survenue de ≥1 cas de MC primitifs multiples (MPM) et la survenue de ≥1 cas de cancer du pancréas (CPCP). La fréquence des mutations était plus élevée dans les Fam_3+ cas (32%) que dans les Fam_2 cas (13%). Alors qu’un âge jeune au diagnostic et la survenue de ≥ 1 MPM étaient associés à la présence de mutations de CDKN2A dans les Fam_2 cas, un âge jeune au diagnostic ainsi que la présence de ≥1 cas de CP était associé significativement aux mutations de CDKN2A dans les Fam_3+ cas. L’étude a montré que les caractéristiques cliniques associées aux mutations de CDKN2A varient, en France, pays de faible incidence de mélanome, en fonction du degré d’agrégation familiale. L’identification de facteurs prédictifs de mutations de CDKN2A dans les Fam_2 cas a contribué à définir des sous-groupes de familles (âge jeune au diagnostic, survenue de MPM) dans lesquels la fréquence des mutations de CDKN2A est supérieure à 20% et auxquels il est légitime de proposer un test génétique. L’analyse des deux séries de pts MM+CR et MMAG a permis d’identifier, en les comparant à la série de MC recrutés de manière consécutive, leurs caractéristiques cliniques et histologiques. Dans ces deux séries, nos résultats ont mis en évidence un contexte de prédisposition héréditaire en partie indépendant de CDKN2A. L’étude de l’association MC et CR chez un même patient a eu deux conséquences pratiques: pour les cliniciens ces résultats suggèrent l’intérêt d’un examen dermatologique en cas de CR et l’intérêt de l’échographie abdominale dans le bilan initial d’un MC pour le dépistage du CR; pour la recherche en génétique, cette série a contribué à l’identification d’une mutation germinale dans le gène MITF qui augmente le risque de développer un MC, un CR ou l’association des deux cancers et qui a des propriétés biologiques intéressantes. L’étude des MMAG a montré que ces mélanomes pouvaient être associés à des MC chez un même malade et/ou survenir dans un contexte familial de mélanome. Le corollaire clinique de ces résultats est que l’examen dermatologique de dépistage ou de surveillance doit être à la fois cutané et muqueux dans un contexte familial de mélanome et qu’en cas de MMAG un examen dermatologique des apparentés doit être proposé comme c’est la règle dans les MC. L’absence de mutation de CDKN2A dans ces localisations muqueuses incite à entreprendre des études génétiques pour identifier les gènes impliqués. / This thesis had two main objectives: 1) To define groups of patients which may benefit from genetic counseling by identifying predictors of mutations of the CDKN2A gene, a major gene predisposing to cutaneous melanoma (CM) in families with only two cases. 2) Epidemiological and clinical characterization of specific entities of melanoma with the secondary objective of contributing to the identification of susceptibility genes for these entities. Coexistence of CM with renal cell carcinoma and mucosal anogenital melanomas were studied.The study populations are a collection of 293 melanoma patients that were ascertained systematically and the French collection MELARISK which is a collection including over 3000 subjects drawn from families with multiple cases of melanoma or melanoma occurring in a particular context (association with another cancer, rare locations, occurrence before the age of 20, multiple sporadic melanomas).We investigated association of three clinical features with the presence of a CDKN2A mutation in a family by extent of CM family clustering (2 versus ≥3 CM patients among first-degree relatives in a family).The study was conducted in 483 French families including 387 families with two melanoma patients, and 96 families with three or more patients with melanoma. The factors examined individually and in a joint analysis in a family were: median age at diagnosis <50 years, ≥1 patient in a family with multiple primary melanomas (MPM) or with pancreatic cancer. The frequency of CDKN2A mutations was higher in F3+ families (32%) than in F2 families (13%). While early age at melanoma diagnosis and occurrence of MPM in ≥1 patient were significantly associated with the risk of a CDKN2A mutation in F2 families, early age at melanoma diagnosis and occurrence of pancreatic cancer in a family were significantly associated with CDKN2A mutations in F3+ families. Thus this study showed that clinical features associated with CDKN2A mutations vary, in France, a country of low incidence of melanoma, according to the degree of familial clustering. Identifying predictors of CDKN2A mutations in families with two melanoma cases has helped to define subgroups of families (early age at CM diagnosis, and/or ≥1 MPM patient) in which the frequency of CDKN2A mutations is above 20% such that these subgroups of F2 families should be offered genetic testing.The analysis of two series of patients, either patients with melanoma coexisting with renal cell carcinoma or patients with anogenital mucosal melanoma identified their clinical and histological features by comparing them to a series of melanomas that were ascertained systematically. In both series, our results suggested a genetic predisposition at least partly independent of CDKN2A. The study of the c renal cell carcinoma; coexistence of CM and renal cancer in the same patient had two practical consequences for clinicians: it suggests the interest of a dermatologic screening visit in patients with renal cell carcinoma and that abdominal ultrasonography or computed tomography scanning performed at the initial workup and during the follow-up of patients with CM may be of value for the early detection of renal cancer. Regarding genetic research, this series has contributed to the identification of a germline mutation in the MITF gene that increases the risk of developing melanoma, renal cancer or both cancers and has interesting biological properties. The study of anogenital melanoma has shown that these melanomas could be associated with cutaneous melanoma in the same patient and it has also shown a high frequency of family history of melanoma associating mucosal and CM suggesting a shared genetic predisposition. Consequently dermatological screening or monitoring must include examination of both skin and mucosa in families with multiple cases of CM; and in case of a mucosal melanoma, a dermatological examination should be offered to relatives. The genetic mechanism has to be identified

O carcinoma de célula renal (RCC) subtipo célula clara é o câncer mais letal e prevalente do sistema urinário. O diagnóstico deste tipo de câncer frequentemente é tardio em conseqüência da falta de sintomas perceptíveis aos pacientes. Um dos objetivos deste trabalho é a identificação de novos marcadores moleculares para diagnóstico precoce, o que ajudaria a diminuir a mortalidade em função de complicações resultantes do avanço da doença. Outro objetivo é a identificação de um conjunto de marcadores moleculares de prognóstico, de modo à prever com acurácia a evolução clínica da doença e, por conseqüência, o tempo de sobrevida do paciente. As modificações transcricionais associadas à carcinogênese e à progressão do câncer de rim ainda não foram completamente elucidadas. Além dos oncogenes e genes supressores de tumor, RNAs não-codificadores (ncRNAs) recentemente foram apontados como importantes reguladores da expressão gênica em humanos, e podem ter um papel importante na transformação maligna do câncer de rim. Para analisar a expressão gênica de ncRNAs e de genes codificadores para proteína foram utilizados dois microarranjos desenvolvidos por nosso grupo, enriquecidos em sondas para ncRNAs. Uma das plataformas possui 4 mil sondas de cDNA, das quais 822 sondas são para ncRNAs mapeando em regiões intrônicas. Outra possui 44 mil elementos e combina sondas de oligonucleotídeos (60-mer) intrônicas e exônicas de um mesmo locus genômico. Análises estatísticas foram feitas com a ferramenta Significance Analysis of Microarrays (q &#8804; 0,05) combinadas ou com a técnica de \"patient leave-one-out\" (genes com presença em 8 100% dos subconjuntos), ou alternativamente com o teste discriminante de Golub (p &#8804; 0,01 ou p < 0,05). Com a plataforma de 4 mil sondas foram estudadas 30 amostras de tecido renal de 18 pacientes com RCC subtipo célula clara. Um conjunto de 36 ncRNAs foi identificado como diferencialmente expresso entre amostras tumorais e não-tumorais. Uma assinatura adicional de 265 genes codificadores de proteínas foi identificada, indicando possíveis novos marcadores moleculares. Uma análise estatística supervisionada com dados de 16 pacientes identificou uma assinatura de ncRNAs correlacionada com sobrevida de 5 anos, formada por 27 ncRNAs com significativa expressão alterada em pacientes livres da doença em comparação com pacientes que morreram em função da doença. Uma assinatura adicional de 64 genes codificadores de proteínas também foi identificada como significativamente correlacionada com o acompanhamento clínico dos pacientes. Com a plataforma de 44 mil sondas foram analisados 17 pacientes, com amostras pareadas de tecido renal tumoral e não-tumoral agrupadas em 8 pools, sendo 4 de amostras tumorais e 4 de não-tumorais. Um conjunto de 66 ncRNAs parcialmente intrônicos antisenso e outro de 52 ncRNAs totalmente intrônicos antisenso foram identificados como diferencialmente expressos. Identificamos um subconjunto de 28 ncRNAs totalmente intrônicos antisenso e senso cuja expressão do gene codificador de proteína do mesmo locus estava simultaneamente alterada. Estes dados apontam para possíveis redes de regulação da expressão gênica dos ncRNAs em câncer. A extensa lista de ncRNAs e de genes codificadores para proteína identificados neste estudo podem ser promissores marcadores moleculares de carcinoma renal subtipo célula clara. / Renal cell carcinoma (RCC) is the most common malignancy of the adult kidney, and the clear cell subtype is the most prevalent and lethal cancer of the urinary system. Late diagnosis for this type of cancer is frequent, usually as a consequence of the lack of symptoms. One of the objectives of the present work is the identification of new molecular markers for the early diagnosis, which would help decrease mortality that develops as a function of disease progression. Another objective is the identification of a set of prognosis molecular markers, so as to accurately predict the clinical outcome of the disease, and consequently, patient survival. Transcriptional changes associated to carcinogenesis and to kidney cancer progression have not been entirely elucidated. Besides oncogenes and tumor suppressor genes, non-coding RNAs (ncRNAs) have been recently indicated as important regulators of gene expression in humans, and could have an important role in the malignant transformation in renal cancer. In order to measure ncRNA and protein-coding gene expression we have used two microarray platforms developed by our group, which are enriched in ncRNA probes. One of the platforms has 4 thousand cDNA probes, of which 822 are for ncRNAs that map to intronic regions. Another has 44 thousand elements and combines 60-mer oligonucleotide probes for intronic and exonic regions from the same genomic locus. Statistical analyses have been performed with the Significance Analysis of Microarrays tool (q &#8804; 0.05) combined with a patient leave-one-out approach (genes present in 100% of the sub-sets), or alternatively with Golubs discriminant test (p &#8804; 0.01 or p < 0.05). 11 With the 4-thousand probes platform we studied 30 samples from renal tissue of 18 RCC patients with clear cell subtype. A set of 36 ncRNAs has been identified as differentially expressed between tumor and non-tumor tissue. An additional signature of 265 protein-coding genes has been identified, indicating possible new molecular markers. A supervised statistical analysis with data from 16 patients has identified a ncRNA signature correlated to 5-year survival outcome, comprised of 27 ncRNAs with significantly altered expression in diseasefree patients compared to patients who died from cancer within the 5-year follow-up. An additional 64-gene signature of protein-coding genes has been identified as significantly correlated to clinical outcome. With the 44-thousand probes platform we have analyzed 17 patients, with paired tumor and non-tumor samples grouped into 8 pools, of which 4 were from tumor and 4 from nontumor samples. A set of 66 partially intronic antisense ncRNAs and another of 52 totally intronic antisense ncRNAs have been identified as differentially expressed between tumor and non-tumor tissue. A sub-set of 28 totally intronic antisense or sense ncRNAs were identified as having a simultaneous change in expression of the protein-coding gene from the same locus. Overall, the data point to a possible ncRNA regulatory network in cancer. The extensive lists of ncRNAs and of protein-coding genes identified in the present study can be seen as promising molecular markers of RCC from the clear-cell subtype.

Introdução: com o uso generalizado de métodos de imagem, aumentou-se a detecção de lesões renais como achados incidentais. Tais lesões podem ser tanto benignas, tais como os angiomiolipomas, e outras malignas, como os carcinomas de células renais, portanto torna-se importante um método não-invasivo com boa acurácia para sua distinção . A ultrassonografia é pouco específica para este fim. A tomografia computadorizada e a ressonância magnética são os métodos mais utilizados na caracterização de lesões incidentais renais. Na Ressonância Magnética, o uso de sequências convencionais ponderadas em T2W e T1W, antes e após a administração endovenosa de contraste paramagnético, mostrou-se pouco eficaz para este fim. Técnicas quantitativas associadas às imagens convencionais, tais como a oposição de fase e a restrição a difusão da água, vêm sendo estudadas, devido ao potencial para melhorar a caracterização não-invasiva, evitando nefrectomias parciais ou totais, e outras intervenções invasivas por lesões benignas. Objetivos: avaliar a acuidade diagnóstica de técnicas combinadas de ressonância magnética para diferenciação de angiomiolipomas pobres em gordura de lesões malignas do rim. Métodos: pacientes que obtiveram o diagnóstico histológico das lesões renais entre os anos de 2010 e 2014 e que realizaram exame pré-operatório. As lesões foram estudadas, colocando-se um ROI (region of interest, no inglês) na maior parte da lesão e córtex renal normal, evitando-se área de não-lesões, calculando a intensidade de sinal nas seqüências T1W in e out-phase, T2W, o Wash in, Wash out relativo e absoluto das lesões e o cálculo absoluto do sinal da lesão no ADC (coeficiente de difusão aparente, do inglês). Os resultados foram obtidos na forma de índices padronizados pelo córtex renal e baço, pelas fases pré e pós-contraste, e de forma absoluta pelo ADC. Os resultados foram confrontados com o diagnóstico final e feito associações estatísticas para observar a relevância. Resultados:Foram estudadas 85 lesões em 74 pacientes, sendo 40 do gênero masculino e 34 do feminino. O cálculo do teor de gordura se mostrou ineficaz para distinção entre CCRs e AMLpg; o índice de intensidade de sinal em T2W, lesão/córtex normal foi útil na diferenciação dos CCRs CC de AMLpg. Outro parâmetro importante foi a cálculo de wash out relativo que se mostrou mais acentuado no AMLpg que em todos os subtipos de CCRs estudados e da medida do ADC médio, que apresentou valores maiores nos casos de CCR CC, em comparação com os outros subtipos e com os AMLpg. Conclusão: As técnicas combinadas de RM, principalmente o índice de sinal T2W da lesão, Wash out relativo e IS do ADC, associados a dados epidemiológicos são viáveis, quando utilizados em conjunto, para a diferenciação de lesões malignas renais dos angiomiolipomas, podendo ter implicações na conduta terapêutica, com redução do número de nefrectomias por lesão benignas. / Introduction: With the widespread use of imaging methods, detection of incidental renal masses has steadily increased in recent years, and these may be either benign, such as angiomyolipoma, or malignant, such as renal cell carcinomas. Therefore, it is important to have a method that allows accurate characterization. Ultrassonography is not very specific for this purpose. CT and MRI are the methods used in the characterization of renal incidental lesions. In MRI, the use of conventional sequences, such as T1W and T2W before and after intravenous administration of paramagnetic contrast media, has proved ineffective for this purpose. Quantitative techniques associated with conventional images, such as chemical shift and diffusion weighted imaging (DWI), have played a key role in this differentiation, which aims to improve characterization, avoiding partial or total nephrectomy, and other invasive interventions for benign lesions. Objectives: to evaluate the diagnostic accuracy of combined techniques of MRI to differentiate fat-poor angiomyolipoma from renal cell carcinomas. Methods: Patients who had a histological diagnosis of renal lesions between 2010 and 2014 and underwent pre-operative exam. An ROI (region of interest)cwas placed in most of the lesion and normal renal cortex, avoiding area of non lesions, by calculating the signal intensity in all sequences, in and out phase T1W, T2W, and the wash in, wash out, relative and absolute, of the lesions and estimation of the ADC. The results were obtained in the form of standardized indices for renal cortex and spleen, the pre- and post-contrast phases, and absolute values for ADC. Results were confronted with the final diagnosis and statistical analysis to observe the relevancy. Results: the estimation of intracellular fat content was ineffective for characterization, while the T2W signal intensity index was used for differentiation between CCRs clear cells from fat-poor AML. Another important parameter was the \"wash out\", which was more prominent for AMLpg. ADC values was higher for CCR CC. Conclusion: We concluded that the combined techniques of MRI mainly T2W signal ratio, \"Wash out\" and ADC values, when used in association and correlated with epidemiological data may be feasible for the differentiation among fat-poor angiomyolipomas and renal malignancies, with important therapeutics implications, reducing unnecessary nephrectomies for benign lesions.

Les vitamines B et les facteurs liés au métabolisme monocarboné (C1) aident à maintenir la synthèse de l'ADN, régulent l'expression des gènes, et peuvent affecter le risque de cancer. L'objectif général de cette thèse est d'étudier l'importance des biomarqueurs du C1 dans l'étiologie de trois cancers distincts qui diffèrent dans leur force d'association avec le tabagisme. Les articles inclus dans cette thèse ont été conduits au sein de deux études prospectives : l'étude prospective européenne sur la nutrition et le cancer (EPIC) et le consortium de cohortes du cancer du poumon (LC3). Dans l'article 1, nous avons étudié la relation entre les biomarqueurs du C1 et l'incidence ainsi que le pronostic du cancer de la sphère oto-rhino-laryngée (ORL) et de l'oesophage au sein de l'étude EPIC. Nous avons observé que les sujets ayant des concentrations élevées d'homocystéine avaient un risque accru de développer un cancer de la sphère ORL. Dans l'article 2, nous avons examiné la relation entre les biomarqueurs du C1 et l'incidence ainsi que le pronostic du carcinome à cellules rénales (CCR) au sein de l'étude EPIC. Nous avons constaté que les participants ayant des concentrations de vitamine B6 élevées avaient une diminution du risque de CCR avec un effet dose-réponse ainsi qu'une amélioration de la survie post-diagnostic. Dans l'article 3, nous avons étudié si les biomarqueurs du C1 sont associés au risque de cancer du poumon au sein de l'étude LC3. Dans l'ensemble, nous avons mis en exergue une faible association inverse, sans tendance claire, entre les concentrations de vitamine B6 et de folate et le risque du cancer du poumon. La principale conclusion de nos études est que les concentrations élevées de vitamine B6 sont associées à un risque plus faible de développer un CCR, et également à un meilleur pronostic chez les patients atteints de cette pathologie. Davantage d'études sont nécessaires afin d'évaluer si la vitamine B6 exerce une influence causale sur l'étiologie et la mortalité du CCR, ou si d'autres facteurs métaboliques sont impliqués / B-vitamins and factors related to one-carbon metabolism (OCM) pathway help to maintain DNA synthesis and regulate gene expression and may affect cancer risk. The overarching aim of this thesis is to investigate the importance of OCM biomarkers in the etiology of three distinct cancer sites that differed in their strength of association with smoking. Papers included in this thesis were conducted within two prospective studies, the European Prospective Investigation into nutrition and Cancer (EPIC) study and the Lung Cancer Cohort Consortium (LC3). In paper 1, we investigated if OCM biomarkers are associated with incidence and survival of cancer of the head and neck and esophagus in the EPIC study. We observed that subjects with higher concentrations of homocysteine had increased risk of developing head and neck cancer. In paper 2, we investigated if OCM biomarkers are associated with incidence and survival of renal cell carcinoma (RCC) in the EPIC study. We observed that study subjects with elevated vitamin B6 concentrations had lower risk of RCC in a dose-response fashion and improved survival following diagnosis. In paper 3, we investigated whether OCM factors are associated with lung cancer risk in the LC3 study. Overall, we observed a weak inverse association, with no clear trend, between concentrations of vitamin B6 and folate and risk of lung cancer. The most important conclusion is that elevated vitamin B6 concentrations are associated with lower risk of developing RCC, and also better prognosis among RCC cases. Further studies are warranted to evaluate if vitamin B6 exerts a causal influence on RCC etiology and mortality, or if other metabolic factors are involved

Aufgrund ihres sehr hohen Metastasierungsrisikos weisen Patienten mit klarzelligem Nierenzellkarzinom (kzNZK) eine sehr hohe Sterblichkeit auf. Mit den zurzeit zur Verfügung stehenden klinischen Parametern kann der Krankheitsverlauf der Patienten nach der operativen Entfernung des Primärtumors nur unzureichend vorhergesagt werden. Um das Nachsorge- und Therapieregime der Patienten zu optimieren, muss die Vorhersagegenauigkeit der bestehenden Prognosemodelle durch molekulare Marker erhöht werden. Um geeignete Gene für eine Abschätzung von Metastasierungsrisiko und krankheitsfreiem Überleben (DFS) zu identifizieren, wurden genomweite Expressionsanalysen sowohl an Lungenmetastasen (n = 24) als auch an Primärtumoren (n = 24) des kzNZK vorgenommen. Durch Vergleich von Metastasensubgruppen, die sich nach unterschiedlich langen DFS entwickelt hatten bzw. Primärtumoren, die nach unterschiedlich langen DFS Metastasen bedingten, wurden tumorintrinsische DFS-assoziierte Expressionsmuster identifiziert. Weiterhin wurden Gene identifiziert, deren Expression sich zwischen Primärtumoren unterschied, die im Krankheitsverlauf manifeste Metastasen bedingten und solchen, die dies nicht taten. Die differenzielle Expression funktionell interessanter, teilweise auch in anderen publizierten Microarraystudien an kzNZK bestätigter Gene wurde im Folgenden mittels quantitativer Polymerasekettenreaktion (qPCR) validiert. Anschließend wurde die Assoziation ausgewählter Gene mit klinischen Parametern und dem Überleben der Patienten untersucht. Ein von klinischen Parametern unabhängiger Einfluss auf den Krankheitsverlauf der Patienten wurde dabei für EDNRB und PECAM1 auf Expressionsebene (qPCR; n = 86) sowie für TSPAN7 auf Proteinebene (Immunhistochemie an „Tissue Microarrays“; n = 106) belegt. EDNRB und PECAM1 waren signifikant höher exprimiert in Primärtumoren mit günstigen klinischen Parametern (TNMI/II, G1/2, V0, N0/M0). TSPAN7 war vorwiegend in den Gefäßen der primären kzNZK nachweisbar; eine signifikant höhere Zahl TSPAN7-positiver Gefäße war ebenfalls in Tumoren mit günstigen klinischen Parametern zu verzeichnen (pT1/2, TNMI/II, N0). Überlebensanalysen zeigten ein signifikant längeres DFS für Patienten mit einer hohen im Vergleich zu solchen mit einer geringen EDNRB-Expression und für Patienten, die in beiden untersuchten Gewebestanzen der „Tissue Microarrays“ TSPAN7-positive Gefäße aufwiesen im Vergleich zu Patienten mit nur einer oder keiner TSPAN7-gefäßpositiven Stanze. Für Patienten mit einer hohen im Vergleich zu solchen mit einer geringen EDNRB- bzw. PECAM1-Expression oder mit zwei im Vergleich zu keiner oder einer TSPAN7-gefäßpositiven Gewebestanze war zudem ein signifikant längeres tumorspezifisches Überleben (TSS) zu verzeichnen. Mit Hilfe multivariater Cox-Regressionsanalysen wurde eine unabhängige günstige prognostische Relevanz für EDNRB auf das DFS sowie für EDNRB, PECAM1 und TSPAN7 auf das TSS nachgewiesen. Somit sind diese molekularen Faktoren geeignet, um die Genauigkeit der bestehenden und ausschließlich auf klinischen Parametern basierenden Prognosemodelle zu erhöhen. Für eine Abschätzung von DFS und Metastasierungsrisiko erscheint dabei insbesondere EDNRB geeignet. / Patients with clear cell renal cell carcinoma (ccRCC) have an extremely poor prognosis due to their high risk of metastases. Currently used clinico-patological parameters are insufficient for reliable prediction of metastatic risk and disease free survival (DFS) after surgical resection of the primary tumor. Molecular markers are strongly needed to improve outcome prediction, and thus to optimize the follow up and treatment schedule for patients with ccRCC. To identify genes which are suitable for the prediction of metastatic risk and DFS, genome-wide expression analyses were performed on pulmonary metastases (n = 24) and primary tumors (n = 24) obtained from patients with ccRCC. Tumor-intrinsic DFS-associated expression patterns were observed by comparing subgroups of metastases, which had developed within different DFS as well as primary tumors, which had caused metastases after different DFS. Furthermore, genes differentially expressed in primary tumors, which caused macroscopic metastases and tumors, which did not were identified. The differential expression of genes with a potential function in metastatic spread, which has in part been identified in independent published microarray studies as well, were validated by quantitative polymerase chain reaction (qPCR). Moreover, an independent prognostic impact on the survival of ccRCC patients was observed for the EDNRB und the PECAM1 gene expression (qPCR; n = 86) as well as for the TSPAN7 protein level (immunohistochemistry on tissue microarrays; n = 106). Primary tumors of patients with favourable clinico-pathological parameters (TNMI/II, G1/2, V0, N0/M0) showed a significantly higher EDNRB und PECAM1 gene expression than those with unfavorable parameters. TSPAN7 was predominantly detected in blood vessels of ccRCC tissues. In patients with favourable clinico-pathological parameters (pT1/2, TNMI/II, N0) a significantly higher number of TSPAN7-positive vessels was observed. Using survival analyses, a significantly longer DFS was observed for patients with a high compared to those with a low EDNRB expression as well as for patients with TSPAN7-positive vessels in both cores compared to no or one of the both cores investigated on tissue microarrays. A significantly longer TSS was observed for patients with a high EDNRB or PECAM1 expression as well as for patients with TSPAN7-positive vessles in both tissue cores investigated. Furthermore, EDNRB was an independent prognostic factor for the DFS of the patients; EDNRB, PECAM and TSPAN7 had an independent prognostic impact on the TSS. Therefore, these molecular markers are suitable to improve the accuracy of outcome prediction based on clinico-pathological parameters in ccRCC. For the prediction of DFS and metastatic risk EDNRB is particularly interesting.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser. Även tryckt utgåva.

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