Evaluation of PD-L1/PD-1 on circulating tumor cells in patients with advanced non-small cell lung cancer

Abstract

Background: Circulating tumor cells (CTCs) could escape from the immune system through the programmed death-ligand 1 (PD-L1)/programmed cell death protein 1 (PD-1) axis leading to the development of metastasis. The current study investigated the expression of PD-1/PD-L1 on CTCs isolated from non-small cell lung cancer (NSCLC) patients treated with chemotherapy.

Patients and methods: CTCs were isolated from 30 chemo-naïve stage IV NSCLC patients before and after front-line chemotherapy using the ISET filtration platform. CTCs were detected by Giemsa and immunofluorescence (IF) staining. Samples were analyzed with the ARIOL system.

Results: Giemsa staining revealed that 28 (93.3%) out of 30 and 9 (81.8%) out of 11 patients had detectable CTCs at baseline and after the third chemotherapy cycle, respectively. Cytokeratin (CK)+/CD45- CTCs by IF could be detected in 17 of 30 (56.7%) patients at baseline and in 8 of 11 (72.7%) after the third chemotherapy cycle. Spearman analysis revealed a significant correlation (p = 0.001) between Giemsa-positive and IF-positive (CK+/CD45-) CTCs. At baseline, PD-1 and PD-L1 expression was observed in 53% and in 47% CK-positive patients, respectively. After the third treatment cycle the corresponding numbers were 13% and 63% respectively. Median progression-free survival (PFS) was significantly shorter in patients with >3 PD-1(+) CTCs at baseline compared with those with <3 PD-1(+) CTCs (p = 0.022) as well as in patients with >1 Giemsa-positive tumor cells (p = 0.025).

Conclusion: PD-1(+) and PD-L1(+) CTCs could be detected before and after front-line chemotherapy in patients with metastatic NSCLC. The presence of high PD-1(+) CTC numbers before treatment is associated with a poor patient clinical outcome.

Keywords: CTCs; NSCLC; PD-L1/PD-1.

Figure 1.

Quantification of PD-1 and PD-L1 in control cell lines. (A) Mean intensity per pixel of PD-1 expression in H460, H1299, HCC827 and SKMES cell lines, automatically quantified by ARIOL system. (B) Mean intensity per pixel of PD-L1 expression in H460, H1299, HCC827 and SKMES cell lines, automatically quantified by ARIOL system. PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.

Figure 2.

CTCs in NSCLC patients. (A) Representative Giemsa staining in patients’ samples. (B) Percentage of NSCLC patients with CTCs at baseline and after the third cycle as evaluated by IF and Giemsa staining. (C) (a–d): Representative ARIOL Images (magnification ×40) of CTCs stained with PD-1/CK/CD45. In the same patient, both [PD-1+(green)/CK+(orange)/CD45-(far red)]] and (PD-1-/CK+/CD45-) phenotypes were observed. (e–h): Representative ARIOL Images (magnification ×40) of CTCs stained for PD-L1 [(orange)/CK (green)/CD45(far red)] antibodies (magnification ×40). CK, cytokeratin; CTC, circulating tumor cell; IF, immunofluorescence; NSCLC, non-small cell lung cancer; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1.

Figure 3.

Expression of PD-1 and PD-L1 NSCLC patients. (A) Percentage of patients expressing CK(+)/PD-L1(+)/CD45(-) cells at baseline and after the 3rd cycle of treatment as compared to the CK-positive group of patients. (B) Percentage of patients expressing CK(+)/PD-L1(-)/CD45(-) at baseline and after the 3rd cycle of treatment as compared to the CK-positive group of patients. (C) Percentage of patients expressing CK(+)/PD-1(+)/CD45(-) cells at baseline and after the 3rd cycle of treatment as compared to the CK-positive group of patients. (D) Percentage of patients expressing CK(+)/PD-1(-)/CD45(-) cells at baseline and after the 3rd cycle of treatment as compared to the CK-positive group of patients.

Figure 4.

CTCs and clinical outcome. (A) Patients with >1 Giemsa(+) CTC at baseline experienced lower PFS. (B) Patients with >3 PD-1(+) CTCs at baseline experienced lower PFS. CTC, circulating tumor cell; PD-1, programmed cell death protein 1; PFS, progression-free survival.