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This study evaluated whether tumor expression of programmed death ligand 1 (PD-L1) could predict the response of EGFR-mutated NSCLC to EGFR tyrosine kinase inhibitor (TKI) therapy.We retrospectively evaluated patients who received EGFR-TKIs for advanced NSCLC at the Guangdong Lung Cancer Institute between April 2016 and September 2017 and were not enrolled in clinical studies. The patients' EGFR and PD-L1 statuses were simultaneously evaluated.Among the 101 eligible patients, strong PD-L1 expression significantly decreased objective response rate, compared with weak or negative PD-L1 expression (35.7% versus 63.2% versus 67.3%, p = 0.002), and shortened progression-free survival (3.8 versus 6.0 versus 9.5 months, p0.001), regardless of EGFR mutation type (19del or L858R). Furthermore, positive PD-L1 expression was predominantly observed among patients with de novo resistance rather than acquired resistance to EGFR-TKIs (66.7% versus 30.2%, p = 0.009). Notably, we found a high proportion of PD-L1 and cluster of differentiation 8 (CD8) dual-positive cases among patients with de novo resistance (46.7%, 7 of 15). Finally, one patient with de novo resistance to EGFR-TKIs and PD-L1 and CD8 dual positivity experienced a favorable response to anti-programmed death 1 therapy.This study revealed the adverse effects of PD-L1 expression on EGFR-TKI efficacy, especially in NSCLC patients with de novo resistance. The findings indicate the reshaping of an inflamed immune phenotype characterized by PD-L1 and CD8 dual positivity and suggest potential therapeutic sensitivity to programmed death 1 blockade.

Introduction Inhibition of programmed cell death-1 (PD-1) and its ligand programmed death ligand 1 (PD-L1) by using an immune checkpoint inhibitor has emerged as a promising immunotherapy for NSCLC. The correlation of PD-L1 expression in tumor cells with treatment outcomes has been reported in many pivotal trials; however, the relationship remains unclear. Here, we demonstrate that those patients with both high density of PD-1–positive CD8 and PD-L1–positive CD4-positive CD25-positive (PD-1 hi PD-L1 hi ) regulatory T cells (Tregs) have a better response to PD1/PD-L1 blockade. Methods In our study between April 1, 2014, and May 30, 2017, a total of 73 NSCLC peripheral blood samples and fresh tumor specimens were collected for study. Of these, 42 large (10-mm 3 ) fresh tumor specimens were obtained from surgical procedures and checked for expression of immunology biomarkers, including PD-L1, PD-1, CD8, CD4, and CD25, in tumor cells and tumor-infiltrating lymphocytes (TILs) by flow cytometry, immunohistochemistry, and immunofluorescence (IF). Moreover, 31 small biopsy specimens from patients who received immunotherapy (pembrolizumab or nivolumab) were analyzed by immunohistochemistry and IF. The correlation between flow cytometry and IF detected for TILs' density was evaluated by Spearman's rank correlation test; the primary end point was progression-free survival. For the PD-1/PD-L1 blockade assay, the TILs and peripheral blood mononuclear CD8 T cells were cultured (1×10 5 per well) with anti–PD-1 (clone MIH4), anti–PD-L1 (clone MIH1). The cytotoxic activity of TILs in killing NSCLC cells after stimulation by anti–PD-1 and anti–PD-L1 was measured by a conventional 51 Cr release assay. Results We first identified a population of high–PD-L1–expressing CD25-positive CD4-positive T cells (PD-L1 hi Tregs) in the tumor microenvironment. The frequency of PD-L1 hi Tregs was higher in tumor tissue (mean 48.6 ± 14.3% in CD25-positive CD3-positive CD4-positive T cells) than in blood (mean 35.4 ± 10.2% in CD25-positive CD3-positive CD4-positive T cells) and normal tissue (mean 38.6 ± 9.7% in CD25-positive CD3-positive CD4-positive T cells) ( p hi Tregs was positively correlated with PD-1–positive CD8 in Tregs. In addition, the TILs from these patients (PD-1 hi PD-L1 hi ) showed PD-1/PD-L1 pathway dependence and could induce a greater killing effect of TILs by PD-1/PD-L1 blockade treatment. The patients with PD-L1–positive NSCLC with PD-1 hi PD-L1 hi TILs showed a better clinical outcome than those with a low frequency of PD-1 hi CD8 or PD-L1 hi Tregs (median progression-free survival not reached versus 2 months). Conclusions Our findings suggested that the density of PD-L1–positive CD4-positive CD25-positive Tregs in the tumor microenvironment can serve as a diagnostic factor to supplement PD-L1 expression in tumor cells and predict the response to PD-1/PD-L1 blockade immunotherapy in NSCLC.