Quantitative Assessment of the Heterogeneity of PD-L1 Expression in Non-Small-Cell Lung Cancer

Abstract

Importance: Early-phase trials with monoclonal antibodies targeting PD-1 (programmed cell death protein 1) and PD-L1 (programmed cell death 1 ligand 1) have demonstrated durable clinical responses in patients with non-small-cell lung cancer (NSCLC). However, current assays for the prognostic and/or predictive role of tumor PD-L1 expression are not standardized with respect to either quantity or distribution of expression.

Objective: To demonstrate PD-L1 protein distribution in NSCLC tumors using both conventional immunohistochemistry (IHC) and quantitative immunofluorescence (QIF) and compare results obtained using 2 different PD-L1 antibodies.

Design, setting, and participants: PD-L1 was measured using E1L3N and SP142, 2 rabbit monoclonal antibodies, in 49 NSCLC whole-tissue sections and a corresponding tissue microarray with the same 49 cases. Non-small-cell lung cancer biopsy specimens from 2011 to 2012 were collected retrospectively from the Yale Thoracic Oncology Program Tissue Bank. Human melanoma Mel 624 cells stably transfected with PD-L1 as well as Mel 624 parental cells, and human term placenta whole tissue sections were used as controls and for antibody validation. PD-L1 protein expression in tumor and stroma was assessed using chromogenic IHC and the AQUA (Automated Quantitative Analysis) method of QIF. Tumor-infiltrating lymphocytes (TILs) were scored in hematoxylin-eosin slides using current consensus guidelines. The association between PD-L1 protein expression, TILs, and clinicopathological features were determined.

Main outcomes and measures: PD-L1 expression discordance or heterogeneity using the diaminobenzidine chromogen and QIF was the main outcome measure selected prior to performing the study.

Results: Using chromogenic IHC, both antibodies showed fair to poor concordance. The PD-L1 antibodies showed poor concordance (Cohen κ range, 0.124-0.340) using conventional chromogenic IHC and showed intra-assay heterogeneity (E1L3N coefficient of variation [CV], 6.75%-75.24%; SP142 CV, 12.17%-109.61%) and significant interassay discordance using QIF (26.6%). Quantitative immunofluorescence showed that PD-L1 expression using both PD-L1 antibodies was heterogeneous. Using QIF, the scores obtained with E1L3N and SP142 for each tumor were significantly different according to nonparametric paired test (P < .001). Assessment of 588 serial section fields of view from whole tissue showed discordant expression at a frequency of 25%. Expression of PD-L1 was correlated with high TILs using both E1L3N (P = .007) and SP142 (P = .02).

Conclusions and relevance: Objective determination of PD-L1 protein levels in NSCLC reveals heterogeneity within tumors and prominent interassay variability or discordance. This could be due to different antibody affinities, limited specificity, or distinct target epitopes. Efforts to determine the clinical value of these observations are under way.

Figure 1. PD-L1 protein heterogeneity using DAB

Intra-tumor PD-L1 protein heterogeneity with DAB using E1L3N and SP142. Bars in DAB panels represent 100 µm.

Figure 2. PD-L1 protein heterogeneity using QIF

Intra-tumor PD-L1 protein heterogeneity with QIF in different cases using E1L3N (A) and SP142 (B). H&E with heat maps using QIF of representative cases for each antibody showing PD-L1 protein intra-assay heterogeneity were generated, with intensity of QIF scores ranging from gray (low) to high (red). Black squares represent FOV in which QIF scores were not calculated (lack of tumor, poor quality, etc.). Representative positive and negative FOV from each heat map case are shown using IF. PD-L1 protein represented in the red channel, DAPI (nuclear) in the blue channel, and cytokeratin (tumor) in the green channel.

Figure 3. PD-L1 protein correlation with TILs using QIF

Correlation of two PD-L1 antibodies (A and B) with tumor infiltrating lymphocytes (TILs). QIF scores measured in arbitrary units (AU).

Figure 4. FOV comparison of two PD-L1 antibodies using QIF

A. QIF PD-L1 scores for E1L3N and SP142 (A) sorted by E1L3N mean score for individual cases. B. Comparison of identical FOV for all cases using both PD-L1 antibodies. Mean score represented by black bar. Dotted red line represents visual detection threshold for both antibodies. Scores in arbitrary units (AU).