Programmed death ligand-1 expression in non-small cell lung cancer

Abstract

Recent strategies targeting the interaction of the programmed cell death ligand-1 (PD-L1, B7-H1, CD274) with its receptor, PD-1, resulted in promising activity in early phase clinical trials. In this study, we used various antibodies and in situ mRNA hybridization to measure PD-L1 in non-small cell lung cancer (NSCLC) using a quantitative fluorescence (QIF) approach to determine the frequency of expression and prognostic value in two independent populations. A control tissue microarray (TMA) was constructed using PD-L1-transfected cells, normal human placenta and known PD-L1-positive NSCLC cases. Only one of four antibodies against PD-L1 (5H1) validated for specificity on this TMA. In situ PD-L1 mRNA using the RNAscope method was similarly validated. Two cohorts of NSCLC cases in TMAs including 340 cases from hospitals in Greece and 204 cases from Yale University were assessed. Tumors showed PD-L1 protein expression in 36% (Greek) and 25% (Yale) of the cases. PD-L1 expression was significantly associated with tumor-infiltrating lymphocytes in both cohorts. Patients with PD-L1 (both protein and mRNA) expression above the detection threshold showed statistically significant better outcome in both series (log-rank P=0.036 and P=0.027). Multivariate analysis showed that PD-L1 expression was significantly associated with better outcome independent of histology. Measurement of PD-L1 requires specific conditions and some commercial antibodies show lack of specificity. Expression of PD-L1 protein or mRNA is associated with better outcome. Further studies are required to determine the value of this marker in prognosis and prediction of response to treatments targeting this pathway.

Figures

Figure 1.. PD-L1 protein and mRNA detection using chromogenic immunohistochemistry and quantitative fluorescence in cell lines and human tissues.

(a) Representative microphotographs showing PD-L1 protein expression using diaminobenzidine (brown chromogen, upper panels), immunofluorescence (red channel, middle panels) and fluorescent in situ PD-L1 mRNA (red channel, bottom panels) in human placenta, PD-L1 expressing Mel624 cells and parental Mel624 cells, × 400 magnification. Bar=60 μm. (b) PD-L1 protein and mRNA fluorescence measurements in several areas of FFPE preparations containing parental Mel624 (Mel624 (−)), PD-L1 Mel624 transfectants (Mel624 (+)) or human placenta. Each dot shows the fluorescence score within the mask in an independent field of view and scores are expressed as arbitrary units (AU). ***P

Figure 2.. Distribution of PD-L1 protein and mRNA scores in the lung cohorts and assay reproducibility.

(a, b) Distribution of PD-L1 protein scores in cases from the Greek (a) and Yale (b) lung cohorts. The dashed line indicates the cutoff for positive/negative designation as described in the Materials and methods section. Inset shows reproducibility of PD-L1 protein measurements in two different cores (core 1 and core 2) obtained from the same case and paraffin block. R2 represents the linear regression coefficient and y represents the slope, (c, d) Distribution of PD-L1 mRNA scores in cases from the Greek (c) and Yale (d) lung cohorts. The dashed line indicates the cutoff for positive/negative designation as per negative control DapB scores. Inset shows relationship between PD-L1 protein and mRNA in both cohorts. R represents the Spearman correlation coefficient.

Figure 3.. PD-L1 positivity and increased TILs is associated with better outcome in lung carcinomas.

(a, b) Kaplan-Meier graphical analysis of 5-year survival in patients with lung carcinomas in the Greek cohort (a) and the Yale University cohort (b) according to PD-L1 protein status. In both cohorts PD-L1-positive patients (black line) showed longer survival compared with negative cases (gray lines, log-rank P=0.0276 for Greek cohort and P=0.0369 for the Yale University cohort). (c, d) 5-Year survival in patients with lung carcinomas in the Greek cohort (c) and the Yale University cohort (d) according to PD-L1 mRNA status. PD-L1 mRNA-positive patients (black line) showed longer survival compared with negative cases (gray lines, log-rank P=0.012 for Greek cohort and P=0.003 for the Yale University cohort). (e, f) 5-Year survival in patients with lung carcinomas in the Greek cohort (e) and the Yale University cohort (f) according to the lymphocytic infiltrate. Cases with high lymphocytic infiltrate (black line, scores 2 and 3) showed longer survival compared with cases with low TILs (gray lines, scores 0 and 1) in both cohorts (log-rank P=0.0158 and 0.009, respectively). The number of cases in each group is indicated within the chart.