Single cell DNA-seq depicts clonal evolution of multiple driver alterations in osimertinib resistant patients

International audienceBackground: The development of targeted agents, such as osimertinib for EGFR-mutated non-small-cell lung cancer (NSCLC), has drastically improved patient outcome, but tumor resistance eventually always occurs. In osimertinibresistant NSCLC, the emergence of a second molecular driver alteration (such as ALK, RET, FGFR3 fusions or BRAF, KRAS mutations) has been described. Whether those alterations and the activating EGFR mutations occur within a single cancer cell or in distinct cell populations is largely debated. Patients and methods: Tumor sequencing was used to identify the acquired resistance mechanisms to osimertinib in the MATCH-R trial (NCT0251782). We implemented single-cell next-generation sequencing to investigate tumor heterogeneity on patient's frozen tissues in which multiple alterations have been identified. Patient-derived models, cell lines, and patient-derived xenografts were exposed to specific inhibitors to investigate combination treatment strategies. Results: Among the 45 patients included in MATCH-R who progressed on osimertinib, 9 developed a second targetable alteration (n ¼ 2 FGFR3-TACC3, n ¼ 1 KIF5B-RET, n ¼ 1 STRN-ALK fusions; n ¼ 2 BRAF V600E , n ¼ 1 KRAS G12V , n ¼ 1 KRAS G12R , n ¼ 1 KRAS G12D mutations). Single-cell analysis revealed that the two driver alterations coexist within one single cancer cell in the four patients whose frozen samples were fully contributive. A high degree of heterogeneity within samples and sequential acquisitions of molecular events were highlighted. A combination treatment concomitantly targeting the two driver alterations was required on the corresponding patient-derived models to restore cell sensitivity, which was consistent with clinical data showing efficacy of brigatinib in the patient with ALK fusion after progression to osimertinib and crizotinib administered sequentially. Conclusions: Distinct molecular driver alterations at osimertinib resistance coexist with initial EGFR mutations in single cancer cells. The clonal evolution of cancer cell populations emphasized their heterogeneity leading to osimertinib relapse. Combining two targeted treatments is effective to achieve clinical benefit