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Supplemental material, Supplementary_material for CD8+ PD-1+ T-cells and PD-L1+ circulating tumor cells in chemotherapy-naïve non-small cell lung cancer: towards their clinical relevance? by Athanasios Kotsakis, Galatea Kallergi, Despoina Aggouraki, Zaharoula Lyristi, Filippos Koinis, Eleni Lagoudaki, Anastasios Koutsopoulos, Vassilis Georgoulias and Eleni-Kyriaki Vetsika in Therapeutic Advances in Medical Oncology

Background: Since tumor cells may escape from immune surveillance through the programmed cell death 1 (PD-1)/programmed death ligand (PD-L)1 axis, this study was designed in order to evaluate whether there is a correlation between the levels of PD-1+ and PD-L1+-expressing immune cells (ICs) and circulating tumor cells (CTCs) in patients with non-small cell lung cancer (NSCLC). Patients and methods: Peripheral blood was obtained from 37 chemotherapy-naïve patients with metastatic NSCLC before treatment. PD-1 and PD-L1 expression was evaluated (1) on ICs with anti-tumor function (CD4+ and CD8+ T-cells, B-cells, monocytes/dendritic cells) using flow cytometry, (2) on CTCs by immunofluorescence and (3) on cells from tumor tissues by immunohistochemistry. The levels of PD-1+ and PD-L1+-expressing ICs were correlated with progression-free survival (PFS). Results: The presence of PD-1+ CD8+ cells, with reduced interferon (IFN)-γ expression, but not other ICs, were positively correlated with PD-L1+ CTCs (p < 0.04). Increased percentages of PD-1+ CD8+ T-cells, were associated with a worse response to treatment (p = 0.032) and shorter PFS (p = 0.023) which, in multivariate analysis, was revealed as an independent predictor for decreased PFS [hazard ratio (HR): 4.1, p = 0.0007]. Conclusion: The results of the current study, for first time, provide evidence for a possible interaction between ICs and CTCs in NSCLC patients via the PD-1/PD-L1 axis and strongly support that the levels of PD-1+ CD8+ in these patients may be of clinical relevance.

Background: Programmed cell death-1 (PD-1), an inhibitory immune check-point, plays a pivotal role in tumor immune escape. The interaction of PD-1 with its ligand (PD-L1) results in T cells exhaustion, and the blockade of this interaction can partially restore T cell function. Recently, antibodies targeting PD-1 and PD-L1 have been approved for treatment of advanced Non Small Cell Lung Cancer (NSCLC). In this pilot study, we aimed to investigate the effect of anti-PD1 treatment or chemotherapy on the frequencies of circulating PD-1+ T cells and PD-L1+ immunosuppressive cells in NSCLC patients. Patients & Methods: Peripheral blood samples were collected from 35 advanced NSCLC patients before initiation of treatment and after 3 cycles. Twelve treatment-naïve patients received front-line chemotherapy, whereas 23 patients received anti-PD1 treatment in the second-line setting. Flow cytometry was used to quantify PD-1- and PD-L1-expressing immune cells. Changes in the frequencies of these cells were compared between the two settings and correlated with the clinical outcome. Results: Chemotherapy had no effect on the percentages of PD-1+CD4+ and PD-1+CD8+ T cells after 3 cycles, whereas there was a significant decrease in PD-1+CD4+ and PD-1+CD8+ T cells in patients who received 3 administrations of anti-PD1 antibody (p=0.007 and p=0.05, respectively). Moreover, the levels of PD-1-CD4+ (p=0.009) and PD-1-CD8+ (p=0.009) were augmented in response to anti-PD-1 therapy. The frequencies of both peripheral CD4+ Tregs (CD3+CD4+CD25highCD127-/lowCD152+FoxP3+) and granulocytic MDSCs (G-MDSC; CD14-CD15+CD33+CD11b+HLA-DR-Lin-) expressing PD-L1 were decreased following anti-PD1 therapy (p=0.01 and p=0.02, respectively). In contrast, chemotherapy affected only the PD-L1+CD4+ Tregs, but not the PD-L1+G-MDSC, by increasing their levels after 3 cycles (p=0.04). Anti-PD-1 treatment induced a superior reduction of the PD-1+CD4+, PD-1+CD8+ T cells, PD-L1+CD4+ Tregs and PD-L1+G-MDSCs percentages compared to the effect of first line chemotherapy (p=0.04, p=0.05, p=0.002 and p=0.01, respectively). Furthermore, a significant decrease in PD-1+CD8+ T cells, PD-L1+CD4+ Tregs and PD-L1+G-MDSCs after 3 doses of anti-PD-1 was observed in patients who experienced stable disease compared to baseline (p=0.006, p=0.05 and p=0.03, respectively). At the time of response evaluation to chemotherapy, the percentage of the PD-L1+CD4+ Tregs after 3 cycles was significantly inferior compared to baseline, in disease progressors (p=0.04). Conclusion: These data indicate that although chemotherapy affected the levels of PD-L1+CD4+ Tregs, anti-PD1 therapy seems to exert an effect on both PD1+ T cells and PD-L1+ immunosuppressive cells. Additional studies are needed in a larger cohort in order to document its impact on their clinical relevance in NSCLC patients. This study is ongoing and updated data will be presented at the meeting. Citation Format: Eleni-Kyriaki Vetsika, Galatea Kallergi, Despoina Aggouraki, Zaharoula Lyristi, Aristeidis Koukos, Despoina Kourougkiaouri, Filippos Koinis, Vassilis Georgoulias, Athanasios Kotsakis. Effect of anti-PD-1 therapy on immune cells in the peripheral blood of non-small cell lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2017-619