Differential regulation of PD-L1 expression by immune and tumor cells in NSCLC and the response to treatment with atezolizumab (anti-PD-L1)

Abstract

Programmed death-ligand 1 (PD-L1) expression on tumor cells (TCs) by immunohistochemistry is rapidly gaining importance as a diagnostic for the selection or stratification of patients with non-small cell lung cancer (NSCLC) most likely to respond to single-agent checkpoint inhibitors. However, at least two distinct patterns of PD-L1 expression have been observed with potential biological and clinical relevance in NSCLC: expression on TC or on tumor-infiltrating immune cells (ICs). We investigated the molecular and cellular characteristics associated with PD-L1 expression in these distinct cell compartments in 4,549 cases of NSCLC. PD-L1 expression on IC was more prevalent and likely reflected IFN-γ-induced adaptive regulation accompanied by increased tumor-infiltrating lymphocytes and effector T cells. High PD-L1 expression on TC, however, reflected an epigenetic dysregulation of the PD-L1 gene and was associated with a distinct histology described by poor immune infiltration, sclerotic/desmoplastic stroma, and mesenchymal molecular features. Importantly, durable clinical responses to atezolizumab (anti-PD-L1) were observed in patients with tumors expressing high PD-L1 levels on either TC alone [40% objective response rate (ORR)] or IC alone (22% ORR). Thus, PD-L1 expression on TC or IC can independently attenuate anticancer immunity and emphasizes the functional importance of IC in regulating the antitumor T cell response.

Keywords: PD-1; PD-L1; atezolizumab; cancer immunotherapy; checkpoints.

Fig. 1.

Prevalence and patterns of PD-L1 expression on TC and IC. (A) Prevalence of PD-L1 in tumor biopsy specimens from patients prescreened and/or enrolled for the atezolizumab clinical trials FIR, BIRCH, and POPLAR. PD-L1 expression on TC and IC was determined by IHC and scored as described (Materials and Methods). ^aTC2/3 or IC2/3 excluding TC3 or IC3; ^bTC1/2/3 or IC1/2/3 excluding TC2/3 or IC2/3. (B) Association of PD-L1 protein expression with PD-L1 mRNA in clinical specimens. Pretreatment tumor specimens from patients enrolled in the POPLAR trial binned into mutually exclusive subgroups based on PD-L1 IHC status on TC and IC. PD-L1 gene expression: *P < 0.05 vs. TC0 and IC0 subgroups determined by Wilcoxon rank sum test; **P < 0.001 vs. TC0 and IC0 subgroups determined by Wilcoxon rank sum test. (C) The Venn diagram (Left) illustrates the percentage overlap of PD-L1 on TC and IC relative to NSCLC tumor specimens from atezolizumab trials. Representative images (Right) of PD-L1 (brown) on TC and IC by IHC.

Fig. 2.

Immune-rich TME of IC3 tumors. (A and B) CD8+ T cell infiltration and PD-L1 expression in IC3 vs. TC3 tumors. (A) Representative images with H&E and IHC staining (brown) are shown. (Scale bar: 50 µm.) (B) Median CD8+ T cell infiltration. NS, not significant. (C–F) Immune infiltration across distinct locations within IC3 vs. TC3 tumors. Representative images (H&E and PD-L1 IHC staining) are shown. (G and H) High expression of T_eff markers in IC3 tumors. Violin plot shows T_eff gene expression; plus signs indicate median. P values were determined using Wilcoxon rank sum test. *P < 0.001; **P < 0.0001.

Fig. 3.

TC3 tumors were characterized by a sclerotic/desmoplastic TME, mesenchymal markers expression, and epigenetic regulation of the PD-L1 promoter. (A and B) TC3 and IC3 tumors scored for sclerosis and desmoplasia as described (Materials and Methods). (C) Representative images with H&E staining of TC3 and IC0 as well as IC3 and TC0 tumors. Sclerotic/desmoplastic characteristics of TC3 tumors are indicated with arrows. (D and E) High expression of EMT markers in TC3 tumors collected from lung. Plus signs in violin plot indicate median. P values were determined using the Wilcoxon rank sum test. (F and G) Correlation between PD-L1 promoter methylation and PD-L1 expression in human NSCLC cell lines and tumors. P values are based on permutation tests in a multiple regression between log-transformed methylation and PD-L1 TC and IC scores.

Fig. 4.

TC3 tumors and clinical response to atezolizumab. (A) Clinical outcomes from atezolizumab treatment in PD-L1 TC and IC tumor subgroups. NE, not estimable; NR, not reached. (B) Changes in sum of longest diameters (SLD) over time in TC0 and IC0, TC3 and IC0, IC3 and TC0, and TC3 and IC3 patients responding to atezolizumab treatment. Thin lines represent individual patients. Thick lines show trends for each subset. (C) Waterfall plot showing best percentage change from baseline in SLD. Lanes represent individual patients. (D and E) Mechanism of response to atezolizumab in TC3 tumors. CD8 and PD-L1 expression (IHC) and gene expression analysis of paired pretreatment (archival and baseline biopsies) and on-treatment biopsy from a tumor that subsequently responded to treatment with atezolizumab. For the archival sample, tumor biopsy from a lymph node was collected 10 mo before the first dose of atezolizumab. For the baseline sample, lung tumor was collected 4 d before the first dose of atezolizumab. For the on-treatment samples, biopsy from a lymph node was collected ∼5 mo after the first dose of atezolizumab. (F) Changes in SLD over time in the same patient. The arrow indicates time when the on-treatment biopsy was collected. SI Appendix has the patient narrative.