Although tyrosine kinase inhibitors (TKIs) can produce high objective response rates in molecularly-defined patients, their long-term efficacy is limited by the development of resistance. In patients with non-small cell lung cancer (NSCLC) harboring activating mutations of EGFR (epidermal growth factor receptor) or ALK (anaplastic lymphoma kinase) rearrangement, a broad range of genetic alterations are observed at the time of acquired resistance to TKIs, including additional mutations in the drug target itself, activation of alternative signaling pathways, or still unidentified resistance mechanisms. Subjecting patients to serial tumor biopsies for identification of resistance mutations is invasive and in some cases associated with risk. With the expanding number of TKIs available, the development of new noninvasive approaches which can offer insight regarding the onset and mechanism of underlying resistance is becoming critical. Circulating tumor cells (CTC) are the main route of metastatic dissemination and may contain clones with tumorigenic potential. They represent a noninvasive source of tumor cells, easily accessible through simple blood sampling. Molecular characterization of CTC will assist the need to characterize TKI response by identifying predictive biomarkers of their sensitivity and/or resistance. The first objective of this project is to identify acquired resistance mutations to ALK inhibitors by analyzing CTC in ALK-rearranged NSCLC patients. This will be done by analyzing known mutations using targeted PCR and next generation sequencing (NGS), and new mutations using whole exome sequencing (WES). The second objective is to identify acquired resistance mutations to EGFR inhibitors by analyzing CTCs in EGFR-mutated NSCLC patients using the same techniques. Analysis of resistance mutations to TKIs will be conducted in single CTCs, collected by filtration at baseline and at disease progression, and identified/isolated phenotypically (epithelial, epithelial and mesenchymal) using a validated process involving laser microdissection of ISET filters. Data from CTCs at progression and at baseline will be compared to that of the corresponding tumor biopsy and circulating tumor DNA (ctDNA), collected at the same timepoints. The third part of the project aims to establish xenografts derived from CTCs (collected at resistance to TKIs) in immunocompromised mice, with subsequent characterisation using extensive phenotypic, genotypic, transcriptomic and functional (cancer stem cell/tumor-initiating cell capacity) analyses. The project will be carried out by three teams: the CTC team (INSERM U981, Gustave Roussy), the service of Thoracic Oncology (Gustave Roussy) and XenTech (SME). All have an internationally recognized expertise in their field of activity. Results from the project will contribute to the development of a non-invasive means to detect TKI resistance biomarkers in EGFR-mutated and ALK-rearranged NSCLC patients, and will inform future treatment strategies in NSCLC, potentially increasing the access of patients to personalized treatments. The molecular analysis of CTCs proposed by the project is expected to generate new knowledge on resistance mechanisms to EGFR and ALK inhibitors, identifying new therapeutic targets. It will allow a deeper understanding of the complexity and heterogeneity of EGFR-mutant and ALK-rearranged tumors, particularly the nature of resistance mechanisms to anti-cancer treatment. The functional analysis of CTCs in xenografts models will generate original data on the characteristics of CTCs with cancer stem cell (CSC) capacity, offering further insight into the optimal method of therapeutically targeting these cells.