Introdução: O câncer de pulmão está entre os principais tipos de neoplasias, sendo o adenocarcinoma o tipo histológico mais frequente. Atualmente, tem-se buscado marcadores de prognóstico para o carcinoma não de pequenas células. Objetivo: analisar os níveis da cinase reguladora extracelular (ERK) ativada em amostras histológicas de pacientes em estágios iniciais de adenocarcinoma de pulmão que foram submetidos a tratamento cirúrgico e suas correlações com dados clínicos e sobrevida. Material e métodos: foram selecionados aleatoriamente 36 pacientes com adenocarcinoma de pulmão nos estágios I e II submetidos a lobectomia pulmonar entre 1998 e 2004. Os pacientes foram divididos em dois grupos segundo os achados imuno-histoquímicos: pacientes com menos de 15% da positividade de células tumorais para ERK e pacientes com 15% ou mais de células positivas. Para comparação com os achados foi realizada análise de enriquecimento de base de dados de microarranjos (GSE29016, n = 72). Resultados: 21 pacientes (58%) apresentaram níveis da ERK ativada acima de 15% e 15 pacientes (43%) abaixo de 15%. Não foram observadas diferenças estatisticamente significativas para idade, sexo, tabagismo e índice de massa corporal entre os grupos estratificados para a ERK. O grupo de maior positividade (≥15%) apresentou menor sobrevida (P = 0,045). As análises de enriquecimento não demonstraram correlação da variação da expressão gênica de ERK em pacientes com adenocarcinoma quando comparados com a sobrevida nos estágios I e o grupamento dos estágios II e III. Conclusões: a alta positividade da ERK em células de amostras biológicas de pacientes com adenocarcinoma de pulmão está relacionada a tumores mais agressivos e com pior prognóstico. / Introduction: Lung cancer is among the most common types of neoplasias, and adenocarcinoma is the most frequent histological type. Currently, there is an extensive search for prognostic biomarkers of squamous nonsmall cell lung cancer. Objective: To analyze the correlation of clinical data and patient survival with the levels of activated extracellular regulatory kinase (ERK) in histological samples of surgically resected early stage lung adenocarcinoma. Methods: We randomly selected 36 patients with stage I or II lung adenocarcinoma who underwent pulmonary lobectomy between 1998 and 2004. Patients were divided into the following 2 groups according to immunohistochemical profile: a group with <15% ERK-positive tumor cells and a group with ≥15% ERK-positive tumor cells. For data comparison, an enrichment analysis of a microarray database was performed (GSE29016, n = 72). Results: Activated ERK levels were ≥15% and <15% in 21 (58%) and 15 (43%) patients, respectively. There were no statistically significant differences in age, sex, smoking history, and body mass index among the groups stratified by ERK levels. The survival rate was lower in the ERK ≥15% group than in the ERK <15% group (P = 0.045). Enrichment analyses showed no correlation between variations in gene expression of ERK in adenocarcinoma patients and survival rates in patients with stage I and combined stage II+III disease. Conclusions: High ERK positivity in cells from biological samples of lung adenocarcinoma is related with tumor aggressiveness and a poorer prognosis.

L’objectif principal de ce travail de thèse est de contribuer au développement d’un outil de diagnostic miniaturisé permettant d’identifier et de quantifier une combinaison de composés organiques volatils (COVs) présents dans l’haleine et qui sont considérés comme des marqueurs chimiques du cancer du poumon. Les principaux verrous scientifiques de ce projet sont liés aux très faibles concentrations de ces composés cibles (de l’ordre de quelques ppb) et également à la présence de nombreux autres composés chimiques qui sont naturellement présents dans l’haleine. La voie de développement proposée dans ce projet est d’utiliser un micro-capteur résistif à base de SnO2 associé à un micro-préconcentrateur et une micro-colonne chromatographique afin d’aboutir à un dispositif sélectif et présentant des limites de détection très basses. Dans un premier temps, plusieurs adsorbants ont été caractérisés en vue d’être utilisés dans le micro-préconcentrateur afin de concentrer les marqueurs du cancer du poumon. Les résultats ont permis de sélectionner deux types de zéolites (DaY et NaY) ainsi que des microsphères de carbone W5. Par la suite, les unités de préconcentration et de séparation des COVs ont été développées en s’appuyant sur la technologie silicium/verre disponible en salle blanche. La dernière étape de cette étude a concerné l’évaluation des performances du système d’analyse alors assimilable à un micro-chromatographe en phase gazeuse. Après avoir déterminé les conditions optimales d’élution et de préconcentration des COVs, le système miniaturisé a permis d’analyser une haleine artificielle constituée de trois COVs présents à des concentrations proches des celles mesurées dans l’haleine (toluène (24 ppb), propanol (21 ppb) et o-xylène (5 ppb)) même en présence des interférents majeurs de l’haleine (vapeur d’eau et dioxyde de carbone). / The main goal of this research is to develop a miniaturized diagnostic equipment in order to identify some volatile organic compounds present in exhaled breath and referred as lung cancer biomarkers. The main scientific and technical obstacles of this project are linked to the very low concentrations of these chemical compounds and the presence of high concentrations of H2O and CO2 naturally present in exhaled breath. To address these issues, we suggest to use a SnO2-based gas sensor combined with a micro-preconcentrator and a chromatographic micro-column in order to engineer a low detection limit system. First, some specific adsorbents have been characterized with a view to concentrate chemical biomarkers trough the micro-preconcentrator. In accordance with research findings, two types of zeolites (DaY and NaY) and one type carbonaceous microspheres (W5) have been selected. Then micro-preconcentrators and chromatographic micro-columns have been developed on silicon wafers by using clean room facilities. The last step of this study was to evaluate the performances of the analytical device. After determining optimal elution and pre-concentration conditions of each VOCs, the miniaturized system achieved the analysis of an artificial breath constituted of toluene (24 ppb), 1-propanol (21 ppb) and o-xylene in presence of high concentrations of water vapors and carbon dioxide.

As alterações genéticas mais frequentes em câncer de pulmão são mutações pontuais que ativam o oncogene KRAS. Embora estas mutações estejam causalmente relacionadas à oncogênese, até hoje diferentes abordagens para inibir as proteínas RAS diretamente não obtiveram sucesso. Portanto, para que melhores alvos terapêuticos para o câncer de pulmão se tornem disponíveis é necessário identificar os mecanismos moleculares ativados por KRAS que estão diretamente envolvidos com a aquisição de propriedades malignas importantes, como o desenvolvimento e a manutenção de um fenótipo tronco-tumoral pelas células iniciadoras de tumor (CITs). CITs, também conhecidas como células tronco-tumorais, são definidas como uma subpopulação de células tumorais capazes de se autorrenovar, iniciar a formação de tumores e sustentar o crescimento tumoral. O desenvolvimento de estratégias terapêuticas dirigidas a estas células é imprescindível para melhorar a eficácia da terapia antitumoral. Uma vez que KRAS está associada a manutenção de um fenótipo tronco-tumoral e ativa o fator de transcrição NF-kB através da quinase IKK&#946; para promover a tumorigênese pulmonar, nós hipotetizamos que a quinase IKK&#946; contribui para o fenótipo tronco-tumoral induzido por KRAS em câncer de pulmão. Nós utilizamos ensaios de formação de tumoresferas para enriquecer e avaliar a função de CITs das linhagens pulmonares positivas para KRAS A549 e H358. As células A549 e H358 formaram tumoresferas em cultura de baixa aderência e, quando comparadas às células derivadas da cultura aderente, as células oriundas da cultura de tumoresferas apresentaram maior crescimento clonogênico, maior expressão de genes associados ao fenótipo tronco por qPCR e maior atividade da quinase IKK&#946;. A inibição da atividade de IKK&#946; através de um inibidor farmacológico altamente específico (Composto A) diminuiu levemente a proliferação de células A549 e H358, sem resultar em morte celular significativa. Entretanto, a inibição da atividade ou da expressão de IKK&#946; por interferência de RNA reduziu a expressão de genes associados ao fenótipo tronco e diminuiu a formação de tumoresferas. A inibição da expressão de IKK&#946; em células A549 reduziu também a capacidade de autorrenovação de CITs. Estes resultados sugerem que IKK&#946; desempenha um papel importante na manutenção do fenótipo tronco-tumoral de CITs pulmonares induzidas por KRAS. Em seguida, nós demonstramos que a inibição da atividade de IKK&#946; afetou preferencialmente a proliferação celular e o crescimento clonogênico de células oriundas da cultura de tumoresfera, sugerindo que IKK&#946; desempenha um papel mais importante em CITs do que em células derivadas da cultura aderente. A análise por citometria de fluxo identificou que células derivadas da cultura de tumoresfera apresentam um enriquecimento para células CD24+ na linhagem A549 e células CD44+ na linhagem H358, sugerindo que estes possam ser marcadores promissores para purificação de CITs nestas linhagens. Adicionalmente, demonstramos, por ensaios de wound-healing de células A549 e H358, que a inibição da atividade de IKK&#946; reduziu a migração celular, uma outra uma propriedade aumentada em CITs. Além disso, mostramos que a atividade da quinase IKK&#946; em células A549 e H358 não depende das vias da MAPK ou PI3K/Akt. Interessantemente, a inibição combinada de IKK (um efetor downstream de KRAS) e de EGFR/ERRB2 (reguladores upstream de KRAS que ativam as vias MAPK e PI3K/Akt) reduziu de forma aditiva a formação de tumoresferas, proliferação e migração celular. Quando avaliados em conjunto, nossos resultados sugerem que a quinase IKK&#946; desempenha um papel importante na biologia de CITs pulmonares portadoras de KRAS oncogênica e que a inibição desta quinase sozinha ou em combinação com a inibição de outras vias pode representar uma estratégia terapêutica promissora a ser explorada para reduzir a recidiva e metástase no câncer de pulmão induzido por KRAS. / The most frequent genetic alterations in lung cancer are point mutations that activate the KRAS oncogene. Although these mutations are causally related to oncogenesis, different approaches to inhibit RAS proteins directly have not been successful to date. Therefore, for better therapeutic targets for lung cancer to become available, it is necessary to identify the molecular mechanisms activated by KRAS that are directly involved with important malignant features, such as the development and maintenance of a cancer stem-like phenotype by the tumour-initiating cells (TICs). TICs, also known as cancer stem cells, are defined as a subpopulation of tumour cells able to self-renew, promote tumour initiation, and sustain tumour growth. The development of therapeutic strategies to target these cells is imperative to improve the efficacy of antitumor therapy. Since KRAS is associated with the maintenance of a cancer stem-like phenotype and activates the transcription factor NF-kB through the IKK&#946; kinase to promote lung tumourigenesis, we hypothesised that IKK&#946; kinase contributes to the cancer stem-like phenotype induced by KRAS in lung cancer. We used tumoursphere formation assays to enrich and evaluate the function of TICs of KRAS-mutant cell lines A549 and H358. A549 and H358 cells formed tumourspheres in low adhesion culture and, when compared to cells grown in adherent culture, sphere-derived cells displayed increased clonogenic growth, higher expression of stemness genes by qPCR, and increased IKK&#946; kinase activity . Inhibition of IKK&#946; activity through a highly specific pharmacological inhibitor (Compound A) slightly decreased proliferation of A549 and H358 cells without inducing significant cell death. On the other hand, inhibition of IKK&#946; activity or expression by RNA interference reduced the expression of stemness genes and decreased tumoursphere formation. Inhibition of IKK&#946; expression in A549 cells also reduced TICs self-renewal . These results suggest that IKK&#946; plays an important role in maintaining the cancer stem-like phenotype of KRAS-driven lung TICs. Next, we demonstrated that IKK&#946; inhibition preferentially reduced cell proliferation and clonogenic growth of sphere-derived cells, suggesting that IKK&#946; plays a more important role in TICs than in adherent culture-derived cells. Flow cytometry analysis identified that sphere-derived cells display an enrichment for the surface marker CD24 in A549 cells and CD44 in H358 cells, indicating that these could be promising markers for the purification of TICs in these cell lines. Furthermore, we have shown by wound-healing assays of A549 and H358 cells that IKK&#946; inhibition reduced cell migration , another feature increased in TICs. In addition, we have shown that IKK&#946; activity in A549 and H358 cells does not depend on the MAPK or PI3K/Akt pathways. Interestingly, combined inhibition of IKK&#946; (a downstream effector of KRAS) and EGFR/ERBB2 (upstream regulators of KRAS that activate the MAPK and PI3K/Akt pathways) additively reduced tumoursphere formation, cell proliferation and migration. Taken together, our results suggest that IKK&#946; kinase plays an important role in the biology of KRAS-driven lung TICs, and that inhibition of this kinase alone or in combination with inhibition of other signalling pathways may represent a promising therapeutic strategy to be explored in order to reduce tumour recurrence and metastasis in KRAS-driven lung cancer.

Les analyses moléculaires et la classification des adénocarcinomes bronchiques ont conduit au développement de thérapies ciblées sélectives visant à améliorer le contrôle de la maladie et la survie des patients. ALK (anaplastic lymphoma kinase) est un récepteur tyrosine kinase de la famille des récepteurs de l'insuline. Des réarrangements chromosomiques impliquant le domaine kinase d’ALK sont présents dans environ 3 à 6% des patients atteints d'un adénocarcinome bronchique. La protéine de fusion provoque une activation du domaine kinase de manière constitutive et indépendante du ligand. Lorlatinib est un inhibiteur d’ALK de troisième génération avec une efficacité et une sélectivité optimale, ainsi qu’une pénétration élevée vers le système nerveux central. Lorlatinib peut vaincre la résistance induite par plus de 16 mutations secondaires dans le domaine kinase d’ALK acquises lors de la progression aux ALK TKI de première et deuxième générations. Le traitement par lorlatinib est donc efficace chez les patients préalablement traités par un ALK TKI de première ou deuxième génération, et est actuellement approuvé pour cette indication. Le spectre complet de mécanismes de résistance au lorlatinib chez les patients reste à élucider. Il a récemment été rapporté que l'acquisition séquentielle de deux mutations ou plus dans le domaine kinase, également appelées mutations composées, est responsable de la progression de la maladie chez environ 35% des patients traités par le lorlatinib, principalement en altérant sa liaison au domaine kinase d’ALK. Cependant, l’effet de ces mutations sur la sensibilité aux différents inhibiteurs d’ALK peut varier, et les autres mécanismes de résistance survenant chez la plupart des patients restent inconnus. Mon travail de thèse avait pour but d’explorer la résistance au lorlatinib chez des patients atteints d'un cancer du poumon ALK réarrangé par la mise en œuvre de biopsies spatiales et temporelles et le développement de modèles dérivés de patients. Dans le cadre de l’étude institutionnelle MATCH-R (NCT02517892), nous avons effectué un séquençage à haut débit de l’exome, de l’ARN et ciblé, ainsi qu’un séquençage des ctDNA afin d’identifier les mécanismes de résistance. Nous avons établi des lignées cellulaires dérivées de patients et caractérisé de nouveaux mécanismes de résistance et identifiés de nouvelles stratégies thérapeutiques in vitro et in vivo. Nous avons identifié trois mécanismes de résistance chez quatre patients avec des biopsies appariées. Nous avons étudié l'induction de la transition épithélio-mésenchymateuse (EMT) par l'activation de SRC dans une lignée cellulaire, dérivée d’un patient, exposée au lorlatinib. Les cellules mésenchymateuses étaient sensibles à l’inhibition combinée de SRC et d'ALK, montrant que même en présence d'un phénotype agressif, des stratégies de combinaison peuvent surmonter la résistance aux ALK TKI. Nous avons identifié deux nouvelles mutations composées du domaine kinase d’ALK, F1174L / G1202R, C1156Y / G1269A survenues chez deux patients traités par le lorlatinib. Nous avons développé des modèles de cellules Ba / F3 exprimant les mutations simples et composées pour étudier leur effet sur la résistance au lorlatinib. Enfin, nous avons caractérisé un nouveau mécanisme de résistance provoqué par la perte de fonction de NF2 au moment de la progression du lorlatinib par l’utilisation de PDX et de lignées cellulaires dérivées de patients, et par CRISPR / CAS9 knock-out de NF2. Nous avons constaté que l'activation de mTOR par la perte de fonction de NF2 provoquait la résistance au lorlatinib et qu'elle pouvait être surmontée par le traitement avec des inhibiteurs de mTOR.Cette étude montre que les mécanismes de résistance au lorlatinib sont plus divers et complexes que prévu. Nos résultats démontrent également comment les études longitudinales de la dynamique tumorale permettent de déchiffrer la résistance aux TKI et d'identifier des stratégies thérapeutiques. / The molecular study and classification of lung adenocarcinomas has led to the development of selective targeted therapies aiming to improve disease control and survival in patients. The anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor from the insulin tyrosine kinase receptor family, with a physiologic role in neural development. Gene rearrangements involving the ALK kinase domain occur in ~3-6% of patients with lung adenocarcinoma. The fusion protein dimerizes leading to transactivation of the ALK kinase domain in a ligand-independent and constitutive manner. Lorlatinib is a third generation ALK inhibitor with high potency and selectivity for this kinase in vitro and in vivo, and elevated penetrance in the central nervous system. Lorlatinib can overcome resistance mediated by over 16 secondary kinase domain mutations occurring in 13 residues upon progression to first - and second - generation ALK TKI. In addition, treatment with lorlatinib is effective for patients who have been previously treated with a first and a second generation or a second generation ALK TKI upfront and is currently approved for this indication. The full spectrum of biological mechanisms driving lorlatinib resistance in patients remains to be elucidated. It has been recently reported that the sequential acquisition of two or more mutations in the kinase domain, also referred as compound mutations, is responsible for disease progression in about 35% of patients treated with lorlatinib, mainly by impairing its binding to the ALK kinase domain. However, the effect of these compound mutations on the sensitivity to the repertoire of ALK inhibitors can vary, and other resistance mechanisms occurring in most patients are unknown. My PhD thesis aimed at exploring resistance to lorlatinib in patients with ALK-rearranged lung cancer through spatial and temporal tumor biopsies and development of patient-derived models. Within the institutional MATCH-R study (NCT02517892), we performed high-throughput whole exome, RNA and targeted next-generation sequencing, together with plasma sequencing to identify putative genomic and bypass mechanisms of resistance. We developed patient-derived cell lines and characterized novel mechanisms of resistance and personalized treatment strategies in vitro and in vivo. We characterized three mechanisms of resistance in four patients with paired biopsies. We studied the induction of epithelial-mesenchymal transition (EMT) by SRC activation in a patient-derived cell line exposed to lorlatinib. Mesenchymal cells were sensitive to combined SRC and ALK co-inhibition, showing that even in the presence of an aggressive and challenging phenotype, combination strategies can overcome ALK resistance. We identified two novel ALK kinase domain compound mutations, F1174L/G1202R, C1156Y/G1269A, occurring in two patients treated with lorlatinib. We developed Ba/F3 cell models harboring single and compound mutations to study the differential effect of these mutations on lorlatinib resistance. Finally, we characterized a novel mechanism of resistance caused by NF2 loss of function at the time of lorlatinib progression through the development of patients derived PDX and cell lines, and in vitro validation of NF2 knock-out with CRISPR/CAS9 gene editing. Downstream activation of mTOR was found to drive lorlatinib resistance by NF2 loss of function and was overcome by providing treatment with mTOR inhibitors.This study shows that mechanisms of resistance to lorlatinib are more diverse and complex than anticipated. Our findings also emphasize how longitudinal studies of tumor dynamics allow deciphering TKI resistance and identifying reversing strategies.

Contexte : Bien que le tabagisme soit de loin le facteur de risque majeur de cancer de poumon, les expositions professionnelles, qui représentent la 2eme étiologie majeure de ces cancers, méritent un intérêt particulier. Peu d'information existe actuellement sur le risque de cancer du poumon associé aux expositions professionnelles aux solvants. Ces substances sont largement utilisées dans différentes industries et certaines professions comme les peintres par exemple, particulièrement exposés aux solvants, sont reconnues à risque de cancer du poumon, sans identification de l'agent cancérogène en cause.Objectifs : L'objectif de mon travail de thèse est l'évaluation du rôle de l'exposition à différents solvants organiques (groupés en solvants chlorés, carburants et solvants pétroliers, solvants oxygénés) sur le risque du cancer du poumon. Dans un premier temps nous avons étudié l'effet de l'exposition professionnelle aux solvants chlorés; ensuite l'analyse a intégré l'exposition aux deux autres familles de solvants, toujours en tenant en compte des principaux facteurs de risque de cancer du poumon, y compris le tabac et l'exposition professionnelle à l'amiante.Population et méthode: Ce travail est basé sur les données d'une large étude de type cas-témoins en population générale, ICARE. Des données détaillées sur l'activité professionnelle vie entière ont été recueillis. Au total, 2,926 cas de cancer de poumon (2,276 hommes et 650 femmes) et 3,555 témoins (2,780 hommes et 775 femmes) ont été inclus. L'exposition aux solvants a été évaluée par des matrices emplois-expositions, élaborés séparément par chaque substance par des hygiénistes industriels. En outre, l'exposition à certains solvants était directement déclarée par les sujets dans le questionnaire. Nous avons d'abord suivi une approche d'analyse statistique classique par régression afin d'étudier le rôle des solvants chlorés. Lors de l'analyse qui intégrait les trois familles des solvants, nous avons suivi une approche méthodologique complexe multidimensionnelle. La méthode bayésienne profil regression nous a permis de contourner les limites de l'analyse classique, dues à la forte multicollinéarité qui existe entre les solvants.Résultats et conclusions: L'analyse focalisée sur les solvants chlorés a suggéré une association entre le cancer du poumon et l'exposition professionnelle au perchloroethylene (PCE), en particulier chez les femmes. En revanche, aucune association avec le risque de cancer du poumon n'a pu être mise en évidence par la seconde analyse intégrant les trois familles de solvants (chlorés, pétroliers et oxygénés). Cependant, parce que nous avons augmenté la spécificité de la définition de l'exposition le PCE n'était pas inclus dans les solvants étudiés, nous empêchant de confirmer ou infirmer l'association mise en évidence précédemment. Bien que les solvants ne soient pas associés au cancer du poumon, nous avons identifié deux groupes de sujets avec un risque de cancer du poumon supérieur à la moyenne. L'un de ces groupes incluait notamment une forte concentration de peintres. Notre analyse ne plaide pas en faveur du rôle des solvants pour expliquer cet excès. Malgré l'absence d'association entre l'exposition aux solvants et le risque de cancer du poumon l'approche Bayésienne utilisée nous a permis d'analyser l'exposition conjointe à un nombre relativement important de substances, ce qui est une situation assez courante en matière d'expositions professionnelles. Notre souhait est maintenant grâce à cette nouvelle méthodologie de nous pencher sur l'étude du rôle des expositions conjointes de substances reconnues cancérigènes pour le poumon comme l'amiante, la poussière de silice et les gaz d'échappement du moteur diesel, toutes trois retrouvés très fréquemment dans l'industrie de la construction par exemple. / Context: The principal risk factor for lung cancer is by far tobacco smoke, however occupational exposures deserve a special interest because they are the 2nd main etiology of this cancer. Little information is currently available on the relationship between lung cancer risk and occupational exposure to solvents that are widely used in several industries. Certain jobs such as painters, who are particularly exposed to solvents, are known to be at increased risk of lung cancer, but the causative agent is still unknown or unproven.Objectives: The aim of the present work was the evaluation of the role of occupational exposure to different organic solvents on lung cancer risk. Initially we studied the effect of occupational exposure to chlorinated solvents; then the analysis integrated exposure to two other solvent families, namely fuels and petroleum solvents and oxygenated solvents, always taking into account the main risk factors for lung cancer, including cigarette smoking and occupational exposure to asbestos.Population and methods: This work was based on a large population-based case-control study, ICARE. Detailed data on the lifelong occupation was collected. A total of 2,926 cases of lung cancer (2,276 men and 650 women) and 3,555 controls (2,780 men and 775 women) were included. Exposure to solvents was assessed by job exposure matrices, developed separately for each substance by industrial hygienists. In addition, exposure to certain solvents was directly reported by subjects in the questionnaire. First we followed a classical statistical regression analysis approach to investigate the role of chlorinated solvents. In the analysis that included three families of solvents, we followed a multidimensional complex methodological approach. Bayesian profile regression method enabled us to overcome traditional analysis limits due to high multicollinearity between solvents.Results and conclusions: The analysis focused on chlorinated solvents suggested an association between lung cancer and occupational exposure to perchloroethylene (PCE), especially in women. While, no association with lung cancer risk could be demonstrated in the second analysis, incorporating the three families of solvents (chlorinated, petroleum and oxygenated). However, as we had increased the specificity of exposure definition, PCE was not included in the studied solvents anymore, preventing us from confirming or denying the association suggested above. Although solvents were not associated with lung cancer, we identified two groups of subjects whose risk of lung cancer was higher than the average. In particular, one of these groups included a high concentration of painters, however our analysis does not advocate the role of solvents to explain this excess. Despite the lack of association between solvent exposure and risk of lung cancer, the Bayesian approach allowed us to analyze the effect of joint exposure to a relatively large number of substances, which is a fairly common situation in occupational exposures. Thanks to this innovative method, our intention is now to look at the role of joint exposure to substances recognized as carcinogenic for the lung such as asbestos, silica dust and diesel engine exhaust, that are frequently found for instance in construction industry.

[ES] El cáncer de pulmón es el tipo de cáncer más frecuentemente diagnosticado y la principal causa de muerte debida a cáncer en el mundo, con sólo un 15% de pacientes con una supervivencia mayor a 5 años tras el diagnóstico. La resección quirúrgica es el tratamiento estándar para los pacientes en estadios tempranos con un buen ECOG, pero el 75% de los pacientes son diagnosticados en estadios avanzados, cuando la intervención quirúrgica no es posible y entre un 35% y un 50% de los pacientes operados recaen tras una cirugía aparentemente exitosa. En los últimos años, se han logrado importantes avances en el desarrollo de la inmunoterapia y de tratamientos contra mutaciones conductoras, pero muchos pacientes todavía desarrollan resistencia, progresan y mueren. Esta resistencia terapéutica ha sido asociada a las células madre tumorales (CMTs), una población tumoral con propiedades de célula madre capaz de sobrevivir a las terapias convencionales y regenerar el tumor incluso cuando son indetectables. En esta tesis doctoral, se establecieron cultivos primarios de pacientes de cáncer de pulmón no microcítico (CPNM) resecados, usando ensayos de formación de tumoresferas para el enriquecimiento en CMTs y condiciones de adherencia para los controles. Las tumoresferas derivadas de pacientes mostraron capacidad de autorenovación y crecimiento exponencial ilimitado, alta resistencia a agentes quimioterápicos, capacidad de invasión y diferenciación in vitro y un elevado potencial tumorigénico in vivo. Usando PCR cuantitativa, se analizaron los perfiles de expresión de los cultivos y se determinó que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 e ITGA6 eran los genes más diferencialmente expresados entre tumoresferas y células adherentes. Los análisis de inmunoblot e inmunofluorescencia confirmaron que las proteínas codificadas por estos genes se encuentran aumentadas en tumoresferas de los pacientes con adenocarcinoma y mostraron patrones de expresión y localización diferencial entre éstas y los controles en adherencia. El valor pronóstico de los genes significativamente sobreexpresados en tumoresferas fue evaluado in silico en una cohorte de 661 pacientes con CPNM procedente del TCGA. De todos ellos, CDKN1A, SNAI1 y ITGA6 mostraron estar relacionados con el pronóstico de los pacientes de acuerdo a un análisis de regresión de Cox y fueron seleccionados para construir una firma de expresión génica, denominada firma de CMTs. Los análisis de supervivencia por Kaplan-Meier mostraron que los pacientes con valores elevados de la firma tienen una supervivencia global (SG) menor para la cohorte completa de CPNM [37,7 vs. 60,40 meses, p = 0,001] y para la subcohorte de adenocarcinoma (ADC) [36,6 vs. 53,5 meses, p = 0,003], pero no para la de los epidermoides. Además, el análisis multivariante mostró que la firma de CMTs es un marcador pronóstico independiente para la SG de los pacientes en la cohorte completa [hazard ratio (HR): 1,498; intervalo de confianza (IC) 95%, 1,167-1,922; p = 0,001] y la subcohorte de ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Esta firma fue también analizada en un grupo independiente de 245 pacientes procedentes del Consorci Hospital General Universitari de València, confirmando su valor pronóstico en los pacientes con ADC [42,90 vs. no alcanzado (NA) meses, p = 0,020]. En resumen, nuestros hallazgos aportan información pronóstica relevante para los pacientes con ADC de pulmón y establecen las bases para el desarrollo de nuevos tratamientos. / [CAT] El càncer de pulmó és el tipus de càncer més diagnosticat i la principal causa de mort deguda a càncer en el món, amb només un 15% de pacients amb una supervivència major a 5 anys després del diagnòstic. La resecció quirúrgica és el tractament estàndard per als pacients en estadis primaris amb un bon ECOG, però el 75% dels pacients són diagnosticats en estadis avançats, quan la intervenció quirúrgica no és possible i entre un 35% i un 50% dels pacients operats recauen després d'una cirurgia aparentment satisfactòria. En els últims anys, s'han aconseguit importants avanços en el desenvolupament de la immunoteràpia i de tractaments contra mutacions conductores, però molts pacients encara desenvolupen resistència, progressen i moren. Aquesta resistència a les teràpies ha estat relacionada amb les cèl·lules mare tumorals (CMTs), una població tumoral amb propietats de cèl·lula mare capaç de sobreviure a les teràpies convencionals i regenerar el tumor fins i tot quan són indetectables. En aquesta tesi doctoral, es van establir cultius primaris de pacients de càncer de pulmó no microcític (CPNM) ressecats, usant assajos de formació de tumoresferes per a l'enriquiment en CMTs i condicions d'adherència per als controls. Les tumoresferes derivades de pacients van mostrar capacitat d'autorenovació, creixement exponencial il·limitat, alta resistència a agents quimioteràpics, capacitat d'invasió i diferenciació in vitro i un elevat potencial tumorigènic in vivo. Usant PCR quantitativa, es van analitzar els perfils d'expressió dels cultius i es va determinar que NANOG, NOTCH3, CD44, CDKN1A, SNAI1 i ITGA6 eren els gens més diferencialment expressats entre tumoresferes i cèl·lules adherents. Les anàlisis de immunoblot i immunofluorescència van confirmar que les proteïnes codificades per aquests gens es troben augmentades en tumoresferes dels pacients amb adenocarcinoma i van mostrar patrons d'expressió i localització diferencial entre aquestes i els controls en adherència. El valor pronòstic dels gens significativament sobreexpressats en tumoresferes va ser avaluat in silico en una cohort de 661 pacients amb CPNM procedent del TCGA. De tots ells, CDKN1A, SNAI1 i ITGA6 van mostrar estar relacionats amb el pronòstic dels pacients d'acord a una anàlisi de regressió de Cox i van ser seleccionats per a construir una signatura d'expressió gènica, denominada signatura de CMTs. Les anàlisis de supervivència per Kaplan-Meier van mostrar que els pacients amb valors elevats de la signatura tenen una supervivència global (SG) menor per a la cohort completa de CPNM [37,7 vs. 60,40 mesos, p = 0,001] i per a la subcohort d'adenocarcinoma (ADC) [36,6 vs. 53,5 mesos, p = 0,003], però no per a la dels escamosos. A més, l'anàlisi multivariant va mostrar que la signatura de CMTs és un marcador pronòstic independent per a la SG dels pacients en la cohort completa [hazard ratio, (HR): 1,498; interval de confiança (IC) 95%, 1,167-1,922; p = 0,001] i la subcohort d'ADC [HR: 1,869; IC 95%, 1,275-2,738; p = 0,001]. Aquesta signatura va ser també analitzada en un grup independent de 245 pacients procedents del Consorci Hospital General Universitari de València, confirmant el seu valor pronòstic en els pacients amb ADC [42,90 vs. no arribat (NA) mesos, p = 0,020]. En resum, els nostres resultats aporten informació pronòstica rellevant per als pacients amb ADC de pulmó i estableixen les bases per al desenvolupament de nous tractaments. / [EN] Lung cancer is the most commonly diagnosed type of cancer and the leading cause of cancer-related death worldwide, with approximately 15% of patients surviving 5 years after diagnosis. Curative surgery is the standard of care for early-stage patients with a good performance status, but 75% are diagnosed at advances stages, when surgery is not possible, and 35-50% of the resected patients relapse after an apparently successful surgical treatment. Significant advances in the development of therapies against driver mutations and immune-based treatments for these patients have been achieved in recent years, but many patients still develop treatment resistance, progress, and die. The high resistance against these therapies has been associated to cancer stem-like cells (CSCs), a population with stem properties which is able to survive after conventional treatments and regenerate tumor even when are undetectable. In this thesis, primary cultures from early-stage non-small cell lung cancer (NSCLC) patients were established, using sphere-forming assays for CSCs enrichment and adherent conditions for the control counterparts. Patient-derived tumorspheres showed self-renewal and unlimited exponential growth potentials, resistance against chemotherapeutic agents, invasion and differentiation capacities in vitro, and superior tumorigenic potential in vivo. Using RTqPCR, gene expression profiles were analyzed, and NANOG, NOTCH3, CD44, CDKN1A, SNAI1, and ITGA6 were selected as the best contributors to distinguish tumorspheres from adherent cells. Immunoblot and immunofluorescence analyses confirmed that proteins encoded by these genes were consistently increased in tumorspheres from adenocarcinoma patients and showed differential localization and expression patterns. The prognostic role of genes significantly overexpressed in tumorspheres was evaluated in silico in a cohort of 661 NSCLC patients from TCGA. Based on a Cox regression analysis, CDKN1A, SNAI1 and ITGA6 were found to be associated with prognosis and used to calculate a gene expression score, named CSCs score. Kaplan-Meier survival analysis showed that patients with high CSCs score have shorter overall survival (OS) in the entire cohort [37.7 vs. 60.4 months, p = 0.001] and in the adenocarcinoma (ADC) subcohort [36.6 vs. 53.5 months, p = 0.003], but not in the squamous cell carcinoma one. Multivariate analysis indicated that this gene expression score is an independent biomarker of prognosis for OS in both, the entire cohort [hazard ratio (HR): 1.498; 95% confidence interval (CI), 1.167-1.922; p = 0.001], and the ADC subcohort [HR: 1.869; 95% CI, 1.275-2.738; p = 0.001]. This score was also analyzed in an independent group of 245 patients from Consorci Hospital General Universitari de València, confirming its prognostic value in the ADC subtype [42.90 vs. not reached (NR) months, p = 0.020]. In conclusion, our findings provide relevant prognostic information for lung ADC patients and the basis for developing novel therapies. / Herreros Pomares, A. (2020). Tumorspheres as an in vitro model for cancer stem-like cell characterization in non-small cell lung cancer. Prognostic implications [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/137036 / TESIS

In the age of big data, thoughtful management and harmonization of clinical metadata and sample processing in translational research is a critical for effective data generation, integration, and analysis. These steps enable the cutting edge discoveries and enhance overall conclusions that may come from complex multi-omic translational research studies. The focus of my thesis has been on harmonizing the clinical metadata collected as part of the lung Pre Cancer Genome Atlas (PCGA) in addition to expanding the use of banked samples. The lung PCGA study included longitudinal collected samples and data from participants in a high-risk lung cancer-screening program at Roswell Park Comprehensive Cancer Center (Roswell) in Buffalo, NY. Clinical metadata for this study was collected over many years at Roswell and subsets of this data were shared with Boston University Medical Campus (BUMC) for the lung PCGA study. During the study, additional clinical metadata was acquired and shared with BUMC to complement the analysis of genomic profiling of DNA and RNA, as well as protein staining of tissue. With regards to the PCGA study, my thesis has two aims: 1) Curate the clinical metadata from received from Roswell during the PCGA study to enhance both its accessibility to current investigators and collaborators and reproducibility of results 2) Test methods to isolate DNA from remnant samples to expand the use of banked samples for genomic profiling. We hypothesized that the accomplishment of these goals would allow for increased use of the clinical metadata, enhanced reproducibility of the results, and expansion of samples available for DNA sequencing The clinical metadata received from Roswell was consolidated into a singular source that is continually updated and available for export for future research use. These metadata management efforts led to increased use among the members of our laboratory and collaborators working with the lung PCGA cohort. Additionally, the curation of metadata has allowed for improved analysis, reproducibility, and increased awareness of the current inventory of remaining samples. During the process of lung PCGA clinical metadata curation, physical inventory of the remaining samples revealed remnant organic phase samples. Therefore, in addition to my work associated with clinical metadata, the second goal of my thesis focuses on DNA isolation from remnant banked biological samples from the lung PCGA cohort. In the first phase of the lung PCGA, nucleic acid isolation of RNA was intended to be collected exclusively from fresh frozen endobronchial biopsy samples, and formalin-fixed paraffin embedded (FFPE) biopsy samples were to be used for DNA isolation. DNA isolation from the FFPE samples was unsuccessful. However, from the RNA isolation, the remaining organic phase was banked and could potentially serve as a source of DNA. The organic phase of this isolation contained cell debris, proteins, and, as previously mentioned, DNA. We hypothesized that current protocols for organic phase DNA isolation might yield adequate quantities of DNA for genomic profiling. Utilizing immortalized cell culture lines to establish methodology, numerous organic phase DNA isolation protocols were tested. During subsequent validation using the remaining organic phase samples from the lung PCGA cohort, the protocol yielded varied results, suggesting that further optimization to increase DNA purity is required. The ability to isolate DNA from these valuable samples will enhance progress in the lung PCGA study. The aims of this thesis involving curation of clinical metadata and generation of additional DNA samples for DNA profiling has had significant impact on the PCGA study and future expansions of this work.

京都大学 / 0048 / 新制・課程博士 / 博士(社会健康医学) / 甲第21701号 / 社医博第92号 / 新制||社医||10(附属図書館) / 京都大学大学院医学研究科社会健康医学系専攻 / (主査)教授 平井 豊博, 教授 小西 靖彦, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Public Health / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22005号 / 医博第4519号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 萩原 正敏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第12803号 / 論医博第2075号 / 新制||医||1001(附属図書館) / 80847 / (主査)教授 伊達 洋至, 教授 武藤 学, 教授 川上 浩司 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM