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Introduction: Circulating tumor cells (CTCs) are considered as a “liquid biopsy” that allows the assessment of tumor changes over time. Tumor cells may escape from the immune system through, among others, the activation of PD-1/PD-L1 axis. Targeting these molecules with monoclonal antibodies has shown encouraging results at many types of cancers including NSCLC. In the current study we investigated the expression of PD-1/PD-L1 molecules on the CTCs isolated from NSCLC patients treated with Nivolumab. Methods: CTCs were isolated based on their size using the ISET platform from 27 patients treated with Nivolumab before treatment, after 1 cycle and after 3 cycles. CTCs were detected after staining with Giemsa and immunofluorescence (IF) double staining using either Cytokeratin (A45-B/B3) (CK7)/PD-1 or Cytokeratin (A45-B/B3)(CK7)//PD-L1 antibodies and analysis with the ARIOL system. Spiking experiments using the NSCLC H460, H1299, HCC827 and SKMES cell lines in normal blood were used to evaluate the detection method. Results: Giemsa evaluation in Nivolumab-treated patients at baseline (25 evaluable samples), after the 1st (9 evaluable samples) and after the 3rd (8 evaluable patients) cycle of treatment showed that CTCs could be detected in 48% (12/25), 33.3% (3/9) and 50% (4/8) of patients, respectively. IF could also reveal the presence of CK-positive cells in 48% (12/25), 22% (2/9) and 75% (6/8) patients, respectively. PD-1 (+) CTCs were detected in 33.3% (4/12) of patients at baseline, in, 0% after the 1 and 16.7% (1/6) of patients after the 3rd cycle whereas PD-L1(+) CTCs in 33.3% (4/12), 0% and 16.7% (1/6),patients, respectively. The expression of PD-1 at baseline was associated with poorer OS (p=0.022) and PFS (p=0.011), while the expression of PD-L1 was associated with shorter PFS (p=0.011). Multivariate analysis revealed that the presence of CK-positive cells is an independent prognostic factor for OS (p=0.028) Conclusion: CTCs from patients with NSCLC express PD-1 and PD-L1 molecules and their expression seems to be associated with the clinical outcome of patients treated with nivolumab.

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