Radiation therapy (RT) plays a crucial role in the management of cancer, however, current standards of care have yet to account for patient specific radiation sensitivity. Raman spectroscopy (RS) is a promising technique for radiobiological studies as a way to measure radiation responses in biological samples and could provide a method for monitoring and predicting radiation response in patients. The work in this dissertation gives way to significant advances in the implementation of RS for applications in radiation oncology. Specifically, instrumentation improvements for clinical implementation of RS were achieved through the investigation and development of Raman microfluidic systems. Unique magnesium fluoride based microfluidic systems were engineered and evaluated for applications in radiobiological studies. These systems were found to yield superior spectral quality over traditional microfluidic designs. Furthermore, in order to assert RS as a key technique for clinical monitoring and prediction of radiation responses, human non-small cell lung cancer (NSCLC) and breast adenocarcinoma tumour xenograft models were investigated for Raman signatures of radiation response. These studies found that RS can identify unique and distinct signatures of radiation response in tumours, that can be tracked over time. In particular, NSCLC tumours were found to have key radiation induced modulations in cell cycle and metabolic linked spectral features- including glycogen. Breast adenocarcinoma tumours were found to exhibit distinct fluctuations in spectral features linked to cell cycle as well as protein content. In the case of NSCLC, radiation response signatures were found to be linked to tumour regression and hypoxic status of the tumour- a key factor that dictates radiation resistance in the disease. This work provides the first application of RS to measure radiation response signatures of tumours irradiated \textit{in vivo}. These results show that RS is a versatile technique that can offer insight into radiation induced molecular changes that are unique to the type of cancer and can be monitored over several days following radiation exposure. Together with improved instrumentation for radiobiological studies using microfluidics, the work presented in this dissertation further emphasizes the key role RS can have in radiation oncology and personalization of RT. / Graduate / 2019-08-21

O câncer de pulmão é responsável por aproximadamente 13% do total de casos de neoplasias malignas e por cerca de 1.4 milhões de mortes por ano em todo mundo. Esta neoplasia apresenta-se sob dois principais subtipos: câncer de pulmão de pequenas células (CPPC) e câncer de pulmão de não-pequenas células (CPNPC). Cerca de 85% dos casos de câncer de pulmão são do tipo CPNPC. Os sinais e sintomas são secundários ao crescimento do tumor primário, ao comprometimento lobo-regional, à disseminação à distância, ou são secundários às síndromes paraneoplásicas. Essas características refletem diretamente sobre as taxas de mortalidade; de cada 100 novos casos, 80 são inoperáveis e a maioria morre dentro de 3 anos. Isso significa que, apesar dos diversos avanços no diagnóstico e tratamento, o prognóstico do câncer de pulmão permanece sendo extremamente ruim, com sobrevida média de 10 meses, e cumulativa total em 5 anos de aproximadamente 12%. Atualmente, o prognóstico e a decisão terapêutica de pacientes com câncer de pulmão é baseada no TNM, Embora esse seja o procedimento considerado padrãoouro entre os profissionais de saúde, ele não leva em consideração características biológicas do tumor. Nesse contexto, a identificação de biomarcadores para câncer pode agregar importantes informações ao já estabelecido sistema TNM e resultar em tratamentos mais eficientes e em menores taxas de mortalidade. Existem 5 fases distintas que conceitualizam o desenvolvimento de um biomarcador tumoral. Através dessas fases consecutivas, é possível que se desenvolvam ferramentas úteis para triagem populacional, capazes de serem implementadas na rotina clínica para predição de desfecho do paciente, resposta terapêutica e monitoramento da doença. O presente projeto avaliou o valor prognóstico dos principais genes citados na literatura como potenciais biomarcadores para CPNPC, e verificou-se que nenhum deles apresentou significância na correlação estatística que indica poder prognóstico. Além disso, identificamos e validamos o papel prognóstico da cofilina-1 por meio de dados de microarranjo e quantificação de seu imunoconteúdo em biópsias de CPNPC. Para tanto, fizemos uso de meta-análise de bancos de dados e análise densitométrica das reações imuno-histoquímicas, seguida de correlação com dados de grau de diferenciação tumoral, classificação histológica, sexo, idade e desfecho relativo a cada caso. Além disso, desenvolvemos um método de baixo custo, fácil execução e ampla aplicação e reproducibilidade, capaz de quantificar a proteína em amostras biológicas, com potencial para ser implementado na rotina clínica e aplicamos esse método em uma coorte restrospectiva de CPNPC. Confirmamos assim o papel prognóstico da cofilina-1. Estes achados seguem a lógica das fases de desenvolvimento de um biomarcador e representam um grande passo no seu processo de validação. / Lung cancer accounts for approximately 13% of all malignant tumor cases and for about 1.4 million deaths per year worldwide. This cancer has two main subtypes: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC). About 85% of cases of lung cancer are NSCLC type. The signs and symptoms are secondary to the primary tumor growth, to regional lobe commitment and distant spread, or are secondary to paraneoplastic syndromes. These features reflect directly on mortality rates; 80 in every 100 new cases are inoperable and most die within 3 years. This means that, despite many advances in diagnosis treatment, the prognosis of lung cancer remains extremely poor, with median survival of 10 months, and total cumulative survival in 5-year of approximately 12%. Currently, prognosis and therapeutic decisions in patients with lung cancer is based on TNM. Although this procedure is considered gold standard among health professionals, it does not take into account the biological characteristics of the tumor. In this context, the identification of cancer biomarkers may add important information to the already established TNM system and result in better treatments and lower mortality rates. There are five distinct phases that conceptualize a tumor biomarker development of. Through these successive phases, it is possible to develop useful tools for population screening, capable of implementation in clinical practice for prediction of patient outcome, therapeutic response and disease monitoring. This project evaluated the prognostic value of major genes mentioned in literature as potential biomarkers for NSCLC and found that none of them showed statistical significance in the correlation that indicates prognostic power. It also identified and validated the prognostic role of cofilin-1 by microarray data and quantification of their immunocontent in biopsies of NSCLC. For this purpose, we used data metaanalysis and immunohistochemical reactions densitometric analysis, followed by correlation with data from tumor grade, histological classification, sex, age and outcome for each case. In addition, we developed a low-cost protocol, of easy implementation and wide application and reproducibility, able to quantify the protein in biological samples, with the potential to be implemented in clinical practice. We applied this method in a retrospective cohort of NSCLC and confirm the prognostic role of cofilin-1. These findings follow the logical phases of biomarker development and represent a major step in its validation process.

O câncer de pulmão é responsável por aproximadamente 13% do total de casos de neoplasias malignas e por cerca de 1.4 milhões de mortes por ano em todo mundo. Esta neoplasia apresenta-se sob dois principais subtipos: câncer de pulmão de pequenas células (CPPC) e câncer de pulmão de não-pequenas células (CPNPC). Cerca de 85% dos casos de câncer de pulmão são do tipo CPNPC. Os sinais e sintomas são secundários ao crescimento do tumor primário, ao comprometimento lobo-regional, à disseminação à distância, ou são secundários às síndromes paraneoplásicas. Essas características refletem diretamente sobre as taxas de mortalidade; de cada 100 novos casos, 80 são inoperáveis e a maioria morre dentro de 3 anos. Isso significa que, apesar dos diversos avanços no diagnóstico e tratamento, o prognóstico do câncer de pulmão permanece sendo extremamente ruim, com sobrevida média de 10 meses, e cumulativa total em 5 anos de aproximadamente 12%. Atualmente, o prognóstico e a decisão terapêutica de pacientes com câncer de pulmão é baseada no TNM, Embora esse seja o procedimento considerado padrãoouro entre os profissionais de saúde, ele não leva em consideração características biológicas do tumor. Nesse contexto, a identificação de biomarcadores para câncer pode agregar importantes informações ao já estabelecido sistema TNM e resultar em tratamentos mais eficientes e em menores taxas de mortalidade. Existem 5 fases distintas que conceitualizam o desenvolvimento de um biomarcador tumoral. Através dessas fases consecutivas, é possível que se desenvolvam ferramentas úteis para triagem populacional, capazes de serem implementadas na rotina clínica para predição de desfecho do paciente, resposta terapêutica e monitoramento da doença. O presente projeto avaliou o valor prognóstico dos principais genes citados na literatura como potenciais biomarcadores para CPNPC, e verificou-se que nenhum deles apresentou significância na correlação estatística que indica poder prognóstico. Além disso, identificamos e validamos o papel prognóstico da cofilina-1 por meio de dados de microarranjo e quantificação de seu imunoconteúdo em biópsias de CPNPC. Para tanto, fizemos uso de meta-análise de bancos de dados e análise densitométrica das reações imuno-histoquímicas, seguida de correlação com dados de grau de diferenciação tumoral, classificação histológica, sexo, idade e desfecho relativo a cada caso. Além disso, desenvolvemos um método de baixo custo, fácil execução e ampla aplicação e reproducibilidade, capaz de quantificar a proteína em amostras biológicas, com potencial para ser implementado na rotina clínica e aplicamos esse método em uma coorte restrospectiva de CPNPC. Confirmamos assim o papel prognóstico da cofilina-1. Estes achados seguem a lógica das fases de desenvolvimento de um biomarcador e representam um grande passo no seu processo de validação. / Lung cancer accounts for approximately 13% of all malignant tumor cases and for about 1.4 million deaths per year worldwide. This cancer has two main subtypes: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC). About 85% of cases of lung cancer are NSCLC type. The signs and symptoms are secondary to the primary tumor growth, to regional lobe commitment and distant spread, or are secondary to paraneoplastic syndromes. These features reflect directly on mortality rates; 80 in every 100 new cases are inoperable and most die within 3 years. This means that, despite many advances in diagnosis treatment, the prognosis of lung cancer remains extremely poor, with median survival of 10 months, and total cumulative survival in 5-year of approximately 12%. Currently, prognosis and therapeutic decisions in patients with lung cancer is based on TNM. Although this procedure is considered gold standard among health professionals, it does not take into account the biological characteristics of the tumor. In this context, the identification of cancer biomarkers may add important information to the already established TNM system and result in better treatments and lower mortality rates. There are five distinct phases that conceptualize a tumor biomarker development of. Through these successive phases, it is possible to develop useful tools for population screening, capable of implementation in clinical practice for prediction of patient outcome, therapeutic response and disease monitoring. This project evaluated the prognostic value of major genes mentioned in literature as potential biomarkers for NSCLC and found that none of them showed statistical significance in the correlation that indicates prognostic power. It also identified and validated the prognostic role of cofilin-1 by microarray data and quantification of their immunocontent in biopsies of NSCLC. For this purpose, we used data metaanalysis and immunohistochemical reactions densitometric analysis, followed by correlation with data from tumor grade, histological classification, sex, age and outcome for each case. In addition, we developed a low-cost protocol, of easy implementation and wide application and reproducibility, able to quantify the protein in biological samples, with the potential to be implemented in clinical practice. We applied this method in a retrospective cohort of NSCLC and confirm the prognostic role of cofilin-1. These findings follow the logical phases of biomarker development and represent a major step in its validation process.

O câncer de pulmão é responsável por aproximadamente 13% do total de casos de neoplasias malignas e por cerca de 1.4 milhões de mortes por ano em todo mundo. Esta neoplasia apresenta-se sob dois principais subtipos: câncer de pulmão de pequenas células (CPPC) e câncer de pulmão de não-pequenas células (CPNPC). Cerca de 85% dos casos de câncer de pulmão são do tipo CPNPC. Os sinais e sintomas são secundários ao crescimento do tumor primário, ao comprometimento lobo-regional, à disseminação à distância, ou são secundários às síndromes paraneoplásicas. Essas características refletem diretamente sobre as taxas de mortalidade; de cada 100 novos casos, 80 são inoperáveis e a maioria morre dentro de 3 anos. Isso significa que, apesar dos diversos avanços no diagnóstico e tratamento, o prognóstico do câncer de pulmão permanece sendo extremamente ruim, com sobrevida média de 10 meses, e cumulativa total em 5 anos de aproximadamente 12%. Atualmente, o prognóstico e a decisão terapêutica de pacientes com câncer de pulmão é baseada no TNM, Embora esse seja o procedimento considerado padrãoouro entre os profissionais de saúde, ele não leva em consideração características biológicas do tumor. Nesse contexto, a identificação de biomarcadores para câncer pode agregar importantes informações ao já estabelecido sistema TNM e resultar em tratamentos mais eficientes e em menores taxas de mortalidade. Existem 5 fases distintas que conceitualizam o desenvolvimento de um biomarcador tumoral. Através dessas fases consecutivas, é possível que se desenvolvam ferramentas úteis para triagem populacional, capazes de serem implementadas na rotina clínica para predição de desfecho do paciente, resposta terapêutica e monitoramento da doença. O presente projeto avaliou o valor prognóstico dos principais genes citados na literatura como potenciais biomarcadores para CPNPC, e verificou-se que nenhum deles apresentou significância na correlação estatística que indica poder prognóstico. Além disso, identificamos e validamos o papel prognóstico da cofilina-1 por meio de dados de microarranjo e quantificação de seu imunoconteúdo em biópsias de CPNPC. Para tanto, fizemos uso de meta-análise de bancos de dados e análise densitométrica das reações imuno-histoquímicas, seguida de correlação com dados de grau de diferenciação tumoral, classificação histológica, sexo, idade e desfecho relativo a cada caso. Além disso, desenvolvemos um método de baixo custo, fácil execução e ampla aplicação e reproducibilidade, capaz de quantificar a proteína em amostras biológicas, com potencial para ser implementado na rotina clínica e aplicamos esse método em uma coorte restrospectiva de CPNPC. Confirmamos assim o papel prognóstico da cofilina-1. Estes achados seguem a lógica das fases de desenvolvimento de um biomarcador e representam um grande passo no seu processo de validação. / Lung cancer accounts for approximately 13% of all malignant tumor cases and for about 1.4 million deaths per year worldwide. This cancer has two main subtypes: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC). About 85% of cases of lung cancer are NSCLC type. The signs and symptoms are secondary to the primary tumor growth, to regional lobe commitment and distant spread, or are secondary to paraneoplastic syndromes. These features reflect directly on mortality rates; 80 in every 100 new cases are inoperable and most die within 3 years. This means that, despite many advances in diagnosis treatment, the prognosis of lung cancer remains extremely poor, with median survival of 10 months, and total cumulative survival in 5-year of approximately 12%. Currently, prognosis and therapeutic decisions in patients with lung cancer is based on TNM. Although this procedure is considered gold standard among health professionals, it does not take into account the biological characteristics of the tumor. In this context, the identification of cancer biomarkers may add important information to the already established TNM system and result in better treatments and lower mortality rates. There are five distinct phases that conceptualize a tumor biomarker development of. Through these successive phases, it is possible to develop useful tools for population screening, capable of implementation in clinical practice for prediction of patient outcome, therapeutic response and disease monitoring. This project evaluated the prognostic value of major genes mentioned in literature as potential biomarkers for NSCLC and found that none of them showed statistical significance in the correlation that indicates prognostic power. It also identified and validated the prognostic role of cofilin-1 by microarray data and quantification of their immunocontent in biopsies of NSCLC. For this purpose, we used data metaanalysis and immunohistochemical reactions densitometric analysis, followed by correlation with data from tumor grade, histological classification, sex, age and outcome for each case. In addition, we developed a low-cost protocol, of easy implementation and wide application and reproducibility, able to quantify the protein in biological samples, with the potential to be implemented in clinical practice. We applied this method in a retrospective cohort of NSCLC and confirm the prognostic role of cofilin-1. These findings follow the logical phases of biomarker development and represent a major step in its validation process.

Submitted by Edineia Teixeira (edineia.teixeira@unioeste.br) on 2018-02-23T18:26:15Z No. of bitstreams: 2 Jose_santos2017.pdf: 2362954 bytes, checksum: 3cd8da60eb3efb9e2ed240ef51fa9a23 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-02-23T18:26:15Z (GMT). No. of bitstreams: 2 Jose_santos2017.pdf: 2362954 bytes, checksum: 3cd8da60eb3efb9e2ed240ef51fa9a23 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-08-01 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Lung cancer is highly lethal and smoking is an important risk factor. Non-small cell tumor is the most common, less aggressive type of lung carcinoma and treatment involves surgery, chemotherapy, and radiation therapy. However, late diagnosis, due to the absence of signs and symptoms in the early stages of the disease, makes the survival rate low. In this sense, the search for new chemical substances with characteristics of selectivity, effectiveness and low toxicity have been investigated in the treatment of cancer. Studies show that sericin, a protein extracted from silkworm cocoons, exhibits anti-tumor and anticarcinogenic activity in colon and skin cancer cells, stimulating apoptosis and disrupting the cell cycle, and protecting normal cells against oxidative stress and lipid peroxidation . Thus, the therapeutic potential of sericin has raised interest in evaluating its effect on non-small cell lung cancer cell line. The studies were conducted in culture, where the effects of silk protein were evaluated: on cell viability, by neutral red assays and tetrazolium - MTT cytotoxicity; in the apoptotic potential, with annexin-5 and Alexa Fluor® and Propidium Iodide assays; and in cell migration by the Wound Healing model assay. Low doses of sericin were able to increase lysosomal viability, reduce mitochondrial viability, increase apoptosis and cell migration, while high doses of sericin exponentially reduced cell migration and did not alter the rate of apoptosis / necrosis of cancer cells. Sericin is a biomaterial that causes biological effects on the cell line tested, and can be used to increase lysosomal viability, reduce mitochondrial function, increase apoptosis at low doses and inhibit high-dose cell migration. / O câncer de pulmão apresenta alta letalidade e o fumo se constitui como um importante fator de risco. O tumor de células não pequenas é o tipo de carcinoma pulmonar mais comum, menos agressivo e o tratamento envolve cirurgia, quimioterapia e radioterapia. Entretanto, o diagnóstico tardio, em função da ausência de sinais e sintomas nos estágios iniciais da doença, faz com que a taxa de sobrevivência seja baixa. Nesse sentido, a busca de novas substâncias químicas com características de seletividade, de efetividade e de baixa toxicidade tem se refletido em pesquisas que investigam o uso dessas substâncias no tratamento do câncer. Estudos mostram que a sericina, proteína extraída dos casulos do bicho-da-seda, apresenta atividade antitumoral e anticarcinogênica em células cancerosas de cólon e de pele, estimulando a apoptose e interrompendo o ciclo celular, além de proteger células normais contra estresse oxidativo e peroxidação lipídica. Assim, o potencial terapêutico da sericina suscitou o interesse em avaliar seu efeito em linhagem celular de câncer de pulmão do tipo células não pequenas. Os estudos foram conduzidos em cultura, em que foram avaliados os efeitos da proteína da seda: na viabilidade celular, por meio de ensaios pelo vermelho neutro e de citotoxicidade com o tetrazólio - MTT; no potencial apoptótico, com ensaios com anexina-5 e Alexa Fluor® e Iodeto de Propídio; e na migração celular, ensaio com o modelo Wound Healing. Baixas doses de sericina foram capazes de aumentar a viabilidade lisossomal, reduzir a viabilidade mitocondrial, aumentar a apoptose e a migração celular, enquanto altas doses de sericina reduziram exponencialmente a migração celular e não alteraram a taxa de apoptose/necrose das células cancerosas. A sericina é um biomaterial que provoca efeitos biológicos na linhagem celular testada, e pode ser utilizada para aumentar a viabilidade lisossomal, reduzir a função mitocondrial, aumentar a apoptose em baixas doses e inibir a migração celular em doses altas.

No description available.

京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第23121号 / 人健博第83号 / 新制||人健||6(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 椎名 毅, 教授 藤井 康友, 教授 平井 豊博 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Depuis peu, l’immunothérapie a émergé comme une nouvelle stratégie chez les patients atteint de Cancer Bronchique Non à Petites Cellules (CBNPC), confirmant ainsi le rôle du système immunitaire dans cette maladie. Malgré ces nouveaux traitements (thérapies ciblées, immunothérapie), les taux de réponses restent faibles avec un impact modeste sur la survie globale. Des biomarqueurs sont donc nécessaire pour définir les populations cibles de ces traitements. Une des pistes explorées est le statut immunitaire des patients, en effet celui-ci a un impact pronostic et pourrait influencer la réponse aux traitements standards tels que la chimiothérapie, les thérapies ciblées et même l’immunothérapie. Parmi les cellules du système immunitaire, les cellules Natural Killer (NK) auraient un rôle effecteur dans le CBNPC. Il est maintenant clairement établit que les cellules NK favorisent la mise en place d’une immunité adaptative fonctionnelle et efficace. Ainsi une altération des fonctions NK pourrait être un mécanisme associé à l’échappement à l’immunité adaptative de la tumeur. Dans notre première étude, nous avons mis en évidence que les exosomes de cellules dendritiques stimulaient les cellules NK via NKp30, cette activité étant associée à un gain de survie sans progression chez des malades inopérables porteurs d’un CBNPC avancé. Notre second projet a révélé, pour la première fois, un rôle pronostic des transcrits de NCR3 (gène de NKp30) chez des patients naïfs de tout traitement. L’activation des cellules NK via NKp30 pourrait être une stratégie efficace d’immunomodulation chez les patients atteints de CBNPC avancé. Ces travaux confirment le rôle important des cellules NK dans le CBNPC avancé. / Recently, immunotherapy has emerged as a new strategy in Non-Small Cell Lung Cancer (NSCLC) patients, confirming the key role of the immune system in this disease. Despite these new treatments (targeted therapies, immunotherapy), response rates remain low with a modest impact on overall survival. Biomarkers are needed to define the target population of these treatments. One of the options explored is the immune status; indeed the immune status of cancer patients has a prognosis impact and may influence the response to standard treatments such as chemotherapy, targeted therapies and even immunotherapy. Among the immune cells, Natural Killer cells (NK) have an effector role in NSCLC. It is now established that NK cells can promote a functional and efficient adaptive immunity. Therefore, an impaired NK functions could be a mechanism associated with the escape from adaptive immunity of the tumor. In our first study, we demonstrated that exosomes from dendritic cells stimulated NK cells through NKp30, this activity is being associated with improved survival in advanced NSCLC. Our second project has revealed, for the first time, a independent prognostic role of NCR3 transcript (NKp30 gene) for naïve advanced NSCLC. Activation of NK cells via NKp30 could be an effective strategy for immunomodulation in advanced NSCLC patients. These studies confirm a major role of NK cells in advanced NSCLC.

Les résultats insuffisants de la radiochimiothérapie conventionnelle dans les cancers bronchiques non à petites cellules (CBNPC) ont motivé l’évaluation d’une nouvelle stratégie de modulation pharmacologique de la radiosensibilité tumorale basée sur les modifications épigénétiques. Pour cela nous avons évalué la combinaison de la radiothérapie et d’un nouvel pan-inhibiteur des histones déacétylases (HDACi), l’abexinostat en préclinique in vitro et in vivo sur deux modèles de CBNPC puis en phase clinique précoce chez l’homme dans le cadre d’un essai de phase I. Nous avons d’abord montré que l’abexinostat augmente la radiosensibilité des cellules de CBNPC de manière dépendante de la séquence thérapeutique en normoxie et en hypoxie en augmentant l’apoptose caspase dépendante ainsi que les cassures doubles brins radio-induites et en réduisant la signalisation et la réparation de ces dommages de l’ADN. L’abexinostat potentialise également le retard de croissance tumorale induit par la radiothérapie in vivo dans des xénogreffes sous-cutanées de CBNPC avec un profil de toxicité acceptable. Nous avons également montré pour la première fois que la triple combinaison de radiothérapie, abexinostat et cisplatine potentialise le retard de croissance tumorale in vivo.Les premiers résultats in vitro et in vivo confortant le rationnel pour la combinaison abexinostat-radiothérapie nous avons réalisé étude de phase I exploratoire d’escalade de dose combinant l’abexinostat à la radiothérapie palliative hypofractionnée standard délivrant 30y en 10 fractions pour des tumeurs solides métastatiques. Parmi les 58 patients traités, d’âge médian 61 ans (20-82), on note 71% de stade M1 et 88% de patients ayant déjà reçu des traitements préalables par chimiothérapie et/ou radiothérapie et/ou chirurgie. La dose recommandée pour un essai de phase 2 que nous avons établie est de 90mg/m². Sur les 51 patients évaluables, on observe un taux de réponse globale de 7,8% (1 réponse complète (RC) et 3 réponses partielles (RP)) et un taux de réponse locorégionale de 11,8% (1 RC et 5 RP) avec un suivi médian de 16 semaines. Les patients présentant des lésions (cibles ou non) cérébrales ont présenté des taux de réponse encourageant avec notamment un patient en RC. Nous avons retrouvé peu d’effets secondaires de grade ≥3, les plus fréquents étant la thrombopénie (17,2%), la lymphopénie (12,1%) et l’hypokaliémie (6,9%). Au total, 6 patients (10%) ont interrompu leur traitement du fait des effets secondaires. Nous n’avons pas observé de prolongation de l’intervalle QTc de grade ≥3 et il n’y a pas eu d’interruption de traitement en rapport avec cet effet secondaire. Dans l’ensemble nos données in vitro et in vivo montrent une potentialisation de l’effet antitumoral par la combinaison d’abexinostat et radiothérapie. Chez les patients présentant des tumeurs solides avancées l’abexinostat oral en combinaison à la radiothérapie est bien toléré. Cette combinaison pourrait avoir un potentiel particulièrement intéressant dans le traitement des métastases cérébrales.De plus nos travaux précliniques suggèrent pour la première fois un effet prometteur d’une triple combinaison avec HDACi, cisplatine et radiothérapie qui justifie de plus amples investigations et pourrait guider de nouvelles stratégies thérapeutiques dans les CBNPC.Nos travaux s’inscrivent dans une stratégie de recherche translationnelle et montrent l’importance de la recherche préclinique pour les études d’association aux rayonnements ionisants. Seuls un développement préclinique rationnel et un développement clinique méthodique permettront l’émergence de combinaisons modulant la radiosensibilité tumorale de manière suffisamment efficace pour modifier nos standards de traitement et améliorer le pronostic de nos patients. / Insufficient results of conventional chemoradiotherapy in non-small cell lung carcinomas (NSCLC) have encouraged assessment of new pharmacological strategies for modulation of radiosensitization based on epigenetic changes. We have investigated the combination of radiotherapy and a novel pan-histone deacetylase inhibitor (HDACi), abexinostat in vitro and in vivo in two NSCLC models and in an early phase clinical trial. Our findings demonstrate that abexinostat enhances radiosensitivity of NSCLC cells in a time dependent way in vitro in normoxia and hypoxia increasing radio-induced caspase dependent apoptosis and persistent DNA double strand breaks associated with decreased DNA damage signaling and repair. Interestingly, abexinostat potentiates tumor growth delay in vivo in combined modality treatments associating not only abexinostat and irradiation but also in the triplet combination of abexinostat, irradiation and cisplatin.We conducted an exploratory phase 1, dose-escalation study of abexinostat in combination with standard hypofractionated radiotherapy (30Gy in 10 fractions) in patients with advanced solid tumors treated in a palliative setting. Among 58 treated patients, the median age was 61.5 years (range, 20-82); 47% of the patients had M1 stage disease, and 95% had received previous chemotherapy alone or chemotherapy in combination with surgery and/or radiotherapy. The recommended phase 2 dose was determined to be 90 mg/m2. Of the 51 patients evaluable for response, best overall response was 8% (1 complete response [CR], 3 partial responses [PRs]), and best loco-regional response was 12% (1 CR and 5 PRs) at a median follow-up of 16 weeks. Of note, patients with target or non-target brain lesions showed encouraging responses, with 1 patient achieving a best loco-regional response of CR. Treatment-emergent grade ≥3 adverse events (AEs) were few, with most common being thrombocytopenia (17%), lymphopenia (12%), and hypokalemia (7%). Six patients (10%) discontinued treatment due to AEs. No grade ≥3 prolongation of the QTc interval was observed, with no treatment discontinuations due to this AE.Altogether, our data demonstrate in vitro and in vivo a potentiation of anti-tumor effect by abexinostat in combination with irradiation in NSCLC. Oral abexinostat combined with radiotherapy was well tolerated in patients with advanced solid tumors. The combination may have potential for treatment of patients with brain lesions.Moreover, our work suggest for the first time to our knowledge promising triple combination opportunities with HDACi, irradiation and cisplatin which deserves further investigations and could be of major interest in the treatment of NSCLC.Our studies which are part of a translational research strategy highlight the importance of preclinical investigations in the area of radiotherapy and drug combination research. Only rationale preclinical development and methodologically well conducted clinical development will allow new standards of treatment to emerge and improve patient’s prognostic.

Les cellules tumorales circulantes (CTC) représentent un large champ de recherche susceptible de fournir des informations tant cliniques que fondamentales. Les CTC proviennent de tumeurs primitives ou métastatiques et représentent une population hétérogène de cellules très rares dans le flux sanguin. Leur caractérisation moléculaire est un défi technologique qui requiert des méthodes très sensibles et spécifiques. Dans les cancers bronchiques non à petites cellules (CBNPC), les CTC ont un véritable intérêt car les biopsies tumorales ne permettent pas toujours de réaliser les analyses moléculaires nécessaires au choix du traitement. De plus, elles ne sont probablement pas représentatives de l’hétérogénéité tumorale.L’objectif de ma thèse a été de rechercher dans les CTC de patients atteints de CBNPC porteurs du remaniement de gène ALK, des anomalies génomiques connues pour être des biomarqueurs de sensibilité et de résistance aux thérapies ciblant cet oncogéne, ainsi qu’à caractériser les CTC porteuses de ces anomalies. La première partie du projet a consisté au développement d’une méthode d’hybridation in situ de l’ADN (fluorescent in situ hybridization, FISH) adaptée à un système d’enrichissement des CTC par filtration, la FA-FISH (filter adapted FISH) (brevet PCT/FR2011/052688). Nous avons ensuite développé une approche de microscopie semi-automatisée permettant la digitalisation et l’analyse de ces CTC enrichies par filtration (Pailler, BMC Cancer, 2016). Dans la seconde partie du projet, nous avons mis en œuvre cette méthode et montré pour la première fois qu’il est possible d’identifier le remaniement de gène ALK dans les CTC de patients porteurs de ce réarrangement dans la tumeur (Pailler, J Clin Oncol, 2013). Les CTC remaniées présentent un unique réarrangement de type break-apart, y compris chez des patients présentant exclusivement une autre forme de réarrangement dans la tumeur, et un phénotype mésenchymateux. Cette observation nous a amené à émettre l’hypothèse que ces CTC ont acquis des propriétés migratoires et d’invasivité, et pourraient résulter d’une forte sélection clonale. Nous avons ensuite étendu cette observation aux patients porteurs du remaniement ROS1 et rapporté pour la première fois la détection de ce remaniement dans des CTC (Pailler, Ann Oncol, 2015). Dans la troisième partie du projet, nous avons émis l’hypothèse que certaines sous-populations de CTC anormales pour le gène ALK, mesurées avant et à deux mois de traitement par le crizotinib, pourraient prédire l’évolution clinique des patients traités. Dans une cohorte élargie de patients, nous avons montré que l’évolution sous crizotinib du nombre de CTC présentant exclusivement des gains de copies natives du gène ALK est un biomarqueur « surrogate » d’efficacité du traitement pouvant permettre d’identifier les patients qui ont un risque élevé de progresser rapidement (Pailler, soumis). Finalement, dans la quatrième partie du projet, nous avons recherché dans des CTC des mutations de résistance aux inhibiteurs de ALK. Nous avons mis au point des technologies permettant de caractériser phénotypiquement, isoler et analyser moléculairement (séquençages ciblés et d’exomes) des CTC à l’échelle de cellule unique. Les expériences sur lignées cellulaires ont permis de valider les approches et les analyses d’échantillons de patients sont en cours.Dans ce travail, nous montrons qu’il est possible de caractériser des anomalies génomiques dans les CTC de patients porteurs du remaniement ALK à différentes étapes de leur maladie, et ainsi d’identifier des biomarqueurs de sensibilité et d’efficacité à une thérapie ciblée. Ce travail ouvre des perspectives sur la personnalisation des traitements qui pourraient reposer sur l’analyse génomique non invasive des CTC. Il apporte en outre des éléments nouveaux sur les caractéristiques biologiques des CTC chez ces patients, certaines étapes du processus métastatique et la diversité génomique de ces cancers. / Circulating tumor cells (CTCs) are a broad field of research which may provide both clinical and basic information. CTCs migrate from primitive or metastatic tumors and represent a heterogeneous population of very rare cells in the blood stream. The molecular characterization of CTCs is a technical challenge requiring highly sensitive and specific methods. Because tumor biopsies are invasive and in some cases associated with risk in non-small-cell lung cancer (NSCLC), CTCs may offer an attractive option to analyze tumor genomic alterations and detect molecular biomarkers. CTCs could provide a more comprehensive picture of the tumor content than single tumor biopsies.The aim of my thesis was to characterize genomic abnormalities in CTCs from ALK-rearranged NSCLC patients and identified biomarkers of sensitivity and resistance to targeted therapies. The first part of the project consisted in the development of a fluorescent in situ hybridization (FISH) method adapted to CTCs enriched by filtration, the FA-FISH (filter-adapted-FISH) (patent PCT/FR2011/052688). Then, we developed a method for the semi-automated microscopy of filtration enriched CTCs (Pailler, BMC Cancer, 2016). In the second part of my project, using this method, we provided the first proof-of-concept that ALK-rearrangement can be detected in CTCs of patients with ALK-rearranged NSCLC (Pailler, J Clin Oncol, 2013). We showed that CTCs from these patients harbor a unique ALK break-apart rearrangement, including patients presenting another form of rearrangement in the biopsy, and a mesenchymal phenotype. This suggests that these CTCs may arise from a clonal selection of tumor cells that have acquired invasive and migratory properties and possibly the potential to drive metastatic progression. Then, we characterized CTCs from patients with ROS1-rearranged NSCLC and reported for the first time the detection of ROS1-rearrangement in CTCs (Pailler, Ann Oncol, 2015). In the third part of the project, we evaluated whether CTCs with abnormal ALK-FISH patterns monitored under crizotinib (baseline and early sampling at 2 months) may inform on treatment benefit in a cohort of ALK-rearranged patients treated by crizotinib. In an extended cohort of patients, the dynamic change in the numbers of CTCs with a gain of ALK-native copies was associated with the progression-free survival and thus may be a surrogate biomarker for crizotinib efficacy (Pailler, submitted). These results show that the molecular analysis of CTCs performed under treatment could help to stratify patients at risk of early resistance to crizotinib. Finally, in the last part of my project, we sought to evaluate whether CTCs could be used for identifying resistance mutations to ALK inhibitors. We developed technologies to characterize, isolate and molecularly (targeted sequencing and exome sequencing) analyze CTCs at the single cell level. Experiments on cell lines allowed to validate these technical processes; Experiments on patient samples are ongoing.In this work, we characterize genomic abnormalities present in CTCs from ALK-rearranged patients at different stages of the disease and identify biomarkers of sensitivity and efficacy to targeted therapies. Our results provide new perspectives on the potential of CTCs for personalizing treatments in NSCLC patients. Furthermore, our findings may offer new insights on the biological characteristics of CTCs in ALK-rearranged patients, their overall role in the metastatic progression and the genomic diversity of these cancers.