Change in PD-L1 expression after acquiring resistance to gefitinib in EGFR-mutant non-small cell lung cancer

Abstract

Introduction: Therapies targeting the programmed death-1 (PD-1) and its ligand (PD-L1) has been successful in a subset of patients with non-small cell lung cancer (NSCLC). PD-L1 expression in tumor tissues has been suggested as a predictive and prognostic marker. We examined the change of PD-L1 expression after gefitinib treatment in patients with EGFR- mutant NSCLC. Materials and Methods: We established gefitinib-resistant NSCLC cell lines and compared with parental PC-9 cells for regulation of PD-L1 expression. We also collected paired tumor tissues before and after gefitinib from eighteen NSCLC patients. PD-L1 expression on tumor and immune cells was defined by H-score of immunohistochemical stain (range: 0 to 300). The correlations between change of PD-L1 expression and clinicopathologic characteristics were analyzed. Results: The PD-L1 expression level was higher in gefitinib-resistant PC-9 cells (PC9GR1 and PC9GR2) than in parental PC-9 cells. Expression of PD-L1 in gefitinib-resistant PC-9 cells was up-regulated by interferon-γ and down-regulated by MEK inhibitor, selumetinib. In addition, MET and mesenchymal markers were increased in association with PD-L1 protein expression in gefitinib-resistant PC-9 cells. PD-L1 expression on tumor cells showed an increase in the median H-score from 25 to 40 (P = 0.067). Among them, seven patients (38.9%) showed a marked increase in median H-score (80 to 180, group A) and the other eleven patients (61.1%) showed no change in median H-score (0 to 0, group B). In group A and B, the median progression free survival for gefitinib was 13 versus 12 months (P=0.594), and the median overall survival after gefitinib resistance was not reached versus 16 months (P=0.068), respectively. MET positivity by immunohistochemistry in post-gefitinib biopsies was significantly associated with group A (P = 0.028). PD-L1 expressing immune cells was observed in 3 (16.7%) of pre-gefitinib tumor tissues and 7 (38.9%) of post-gefitinib tumor tissues. PD-L1 expression by immune cells showed statistically significant correlation with pre-gefitinib FOXP3+ TILs (ρ = 0.584, P = 0.014) and with post-gefitinib CD3+ TILs (ρ = 0.547, P = 0.028), CD8+ TILs (ρ = 0.650, P = 0.005), and PD-1+ TILs (ρ = 0.590, P = 0.016), respectively. Patients who had acquired mutation T790M after gefitinib was likely to have higher TILs infiltration score in tumor microenvironments. Conclusions: The level of PD-L1 expression was changed not only in tumor cells but also in tumor infiltrating immune cells after acquiring resistance to gefitinib. Patients who had increased tumoral PD-L1 expression after gefitinib showed longer overall survival than those who did not, which may be associated with increased MET expression or activation of intracellular MEK signaling pathway. Tumor tissues that did not showed increase in tumoral PD-L1 expression after gefitinib had secondary T790M mutation or increased TILs. Re-evaluation of the level of PD-L1 expression is needed after acquiring resistance to gefitinib in tumor and also in tumor infiltrating immune cells. Thus, we can understand the mechanism of drug resistance and improve the outcomes of treatments in NSCLC.