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Purpose: The use of circulating tumor cells (CTC) as "liquid biopsy" is limited by the very low yield of CTCs available for subsequent analyses. Most in vitro approaches rely on small sample volumes (5-10 mL)., Experimental Design: Here, we used a novel approach, the GILUPI CellCollector, which enables an in vivo isolation of CTCs from peripheral blood. In total, 50 lung cancer patients were screened in two subsequent device applications before and after therapy (n = 185 applications)., Results: By in vivo isolation, 58% (108/185) of the patients were positive for ≥1 CTC (median, 5 CTCs; range, 1-56 cells) as compared with 27% (23/84; range, 1-300 cells) using the FDA-cleared CellSearch system. Furthermore, we could show that treatment response during therapy was associated with significant decreases in CTC counts (P = 0.001). By dPCR, mutations in the KRAS and EGFR genes relevant for treatment decisions could be detected in CTCs captured by in vivo isolation and confirmed in the primary tumors of the same patients., Conclusions: In vivo isolation of CTCs overcomes blood volume limitations of other approaches, which might help to implement CTC-based "liquid biopsies" into clinical decision making. Clin Cancer Res; 22(9); 2197-206. ©2015 AACR., (©2015 American Association for Cancer Research.)