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Identification de biomarqueurs de sensibilité et de résistance aux inhibiteurs de tyrosine kinase dans les cellules tumorales circulantes de patients atteints de cancers bronchiques non à petites cellules - Cas des remaniements ALK et ROS1

Abstract : 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.
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Emma Pailler. Identification de biomarqueurs de sensibilité et de résistance aux inhibiteurs de tyrosine kinase dans les cellules tumorales circulantes de patients atteints de cancers bronchiques non à petites cellules - Cas des remaniements ALK et ROS1. Cancer. Université Paris Saclay (COmUE), 2016. Français. ⟨NNT : 2016SACLS410⟩. ⟨tel-02375325⟩

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