PD-L1 Studies Across Tumor Types, Its Differential Expression and Predictive Value in Patients Treated with Immune Checkpoint Inhibitors

Abstract

Purpose: With recent approval of inhibitors of PD-1 in melanoma, non-small cell lung cancer (NSCLC) and renal cell carcinoma, extensive efforts are under way to develop biomarkers predictive of response. PD-L1 expression has been most widely studied, and is more predictive in NSCLC than renal cell carcinoma or melanoma. We therefore studied differences in expression patterns across tumor types.Experimental Design: We used tissue microarrays with tumors from NSCLC, renal cell carcinoma, or melanoma and a panel of cell lines to study differences between tumor types. Predictive studies were conducted on samples from 65 melanoma patients treated with PD-1 inhibitors alone or with CTLA-4 inhibitors, characterized for outcome. PD-L1 expression was studied by quantitative immunofluorescence using two well-validated antibodies.Results: PD-L1 expression was higher in NSCLC specimens than renal cell carcinoma, and lowest in melanoma (P = 0.001), and this finding was confirmed in a panel of cell lines. In melanoma tumors, PD-L1 was expressed either on tumor cells or immune-infiltrating cells. The association between PD-L1 expression in immune-infiltrating cells and progression-free or overall-survival in melanoma patients treated with ipilimumab and nivolumab was stronger than PD-L1 expression in tumor cells, and remained significant on multivariable analysis.Conclusions: PD-L1 expression in melanoma tumor cells is lower than NSCLC or renal cell carcinoma cells. The higher response rate in melanoma patients treated with PD-1 inhibitors is likely related to PD-L1 in tumor-associated inflammatory cells. Further studies are warranted to validate the predictive role of inflammatory cell PD-L1 expression in melanoma and determine its biological significance. Clin Cancer Res; 23(15); 4270-9. ©2017 AACR.

Figure 1

Examples of strong PD-L1 fluorescent staining. Staining is shown in paraffin embedded pellets of MEL624 transfected to overexpress PD-L1 or parental cells, histospots non-small cell lung cancer (NSCLC), renal cell carcinoma (RCC) and melanoma specimens. Either an anti-S100 antibody or anti-cytokeratin (CK) antibody and fluorophore tyramide were used to distinguish tumor cells (green) from the surrounding stroma and leukocytes. 5H1 antibody (upper panels) or E1L3N antibody (lower panels) conjugated to Cyanine-5-tyramide (red) was used to visualize PD-L1. Overlaid images show PD-L1 expression within the entire histospot comprising both the tumor (orange/yellow) and stroma (red), at 10× magnification.

Figure 2

PD-L1 levels in a panel of tumor-derived metastatic melanoma, non-small cell lung cancer (NSCLC) or renal cell carcinoma (RCC) cell lines. MEL-624 cell lines, overexpressing or not overexpressing PD-L1 were used as controls (boxed). Protein was extracted from NSCLC, RCC and melanoma cell lines and subjected to SDS-PAGE and Western blot analysis to detect PD-L1 with either the 5H1 or E1L3N antibody. The level of PD-L1 normalized to the β-actin level in each sample was measured by ImageJ software.

Figure 3

Association of PD-L1 expression in the tumor or stromal compartments and response to therapy (ipilimumab and nivolumab concurrent therapy). Unpaired t-tests were utilized to compare PD-L1 expression and response to therapy; PD-L1 expression was higher in responders (CR+PR) compared to non-responders (SD+PD).

Figure 4

Kaplan–Meier curves showing the association between PD-L1 expression and progression-free survival (PFS). The median PD-L1 intensity score, measured either in tumor cells or stroma by the 5H1 or E1L3N antibodies, was utilized to dichotomize our cohort into low/high categories. Patients with high PD-L1 levels in the stromal compartment had a longer median PFS compared to low expressers.

Figure 5

Kaplan–Meier curves showing the association between PD-L1 expression and overall survival (OS). PD-L1 scores were dichotomized by the median score in the tumor or stromal compartment. High PD-L1 level in the stromal compartment was significantly associated with prolonged OS.