Programmed Death Ligand 1 Is a Negative Prognostic Marker in Recurrent Isocitrate Dehydrogenase-Wildtype Glioblastoma

Abstract

Background: Checkpoint inhibition has demonstrated clinical efficacy in a variety of solid tumors. Reports of programmed death ligand 1 (PD-L1) expression in glioblastoma are highly variable (ranging from 6% to 88%) and its role as a prognostic marker has yielded conflicting results.

Objective: To validate the prevalence and prognostic role of PD-L1 expression in a large cohort of diffuse gliomas according to the 2016 revised WHO classification.

Methods: Using tissue microarrays, we compared 5 PD-L1 monoclonal antibodies (n = 56) and validated expression (n = 183) using quantitative immunohistochemistry (IHC) and RNA in situ hybridization (RISH). Expression data from The Cancer Genome Atlas (TCGA) and published studies were compared with clinical outcome. Multiplexed immunophenotyping was used to identify PD-L1+ cell populations in post-treatment glioblastoma.

Results: Using a 5% cut-off, PD-L1 expression was significantly associated with a poor prognosis in both histologically defined (n = 125, log-rank P < .001) and recurrent isocitrate dehydrogenase (IDH)-wildtype glioblastoma (n = 60, log-rank P = .015). PD-L1 remained a significant negative prognosticator in Cox regression analysis (hazard ratio: 1.96, P = .021). Analysis of TCGA data confirmed decreased overall survival in recurrent non-glioma CpG island methylator phenotype (G-CIMP) glioblastoma (n = 12, log-rank P = .023), but not in glioblastoma as a group (n = 444, log-rank P = .135). PD-L1 RISH showed a significant correlation with IHC (P < .0001). PD-L1 was observed in the proliferating perivascular stem cell and immune niche of post-treatment glioblastoma.

Conclusion: A 5% PD-L1 expression cut-off identified a subset of glioblastoma that is associated with a worse clinical outcome. This association remained significant within the newly defined IDH-wildtype classification. These findings could have implications for patient stratification in future clinical trials of PD-1/PD-L1 blockade.

Keywords: Glioblastoma; Immunohistochemistry; Immunotherapy; In situ hybridization; PD-1; PD-L1; RNA.

FIGURE 1.

Comparison of 5 monoclonal antibodies to PD-L1 in glioblastoma. Both distribution of staining (staining heterogeneity) and cellular localization were compared. CAL10, SP142, and SP263 showed concordant intensity and extent of staining. All clones showed cell surface (membranous) staining. Cytoplasmic immunoreactivity (arrowhead) was more evident with CAL10, SP263, and E1L3N. Clone 28-8 was comparatively weaker in intensity. Scale bar = 50 μm.

FIGURE 2.

PD-L1 IHC across 2016 WHO histomolecular subtypes and RISH correlation. A, Representative images of IDH-wildtype (GBIDHWT) and IDH-mutant (GBIDHM) glioblastoma stained with anti-PD-L1 (SP263). B, Stacked bar chart showing PD-L1 IHC distributed across WHO 2016 diagnoses in the NIH cohort (n = 183). C, Overall, PD-L1 staining intensity (not quantified in the current study) was associated with an increase in PD-L1 RISH signals. D, Across the entire cohort (n = 183), quantitative PD-L1 IHC and mRNA showed a significant positive correlation. Scale bar = 100 μm.

FIGURE 3.

Kaplan–Meier survival estimates comparing high and low PD-L1 expression in glioblastoma. A, Differences in survival curves from tumors meeting the 2007 WHO histologic criteria for glioblastoma (5% PD-L1 cut-off). B, Differences in survival curves from recurrent IDH-wildtype glioblastoma reclassified according to the 2016 WHO nomenclature (5% PD-L1 cut-off). C, TCGA survival curves, classified only as glioblastoma, stratified by median PD-L1 mRNA expression. D, Survival curves from TCGA patients after filtering for recurrent, non–G-CIMP (IDH-wildtype) tumors (median PD-L1 mRNA cut-off).

FIGURE 4.

Multiplex immunofluorescence of the tumor microenvironment in post-treatment glioblastoma with inflammatory response to checkpoint inhibition. A, Immunolabeling with the nuclear stem cell marker SOX-2 (green), proliferation marker PCNA (yellow), and PD-L1 (red; SP142). PD-L1/SOX-2/PCNA+ cells are depicted in the merged image. B, Representative images of perivascular immune cells co-expression of IBA1 (aqua), CD68 (purple), and PD-L1 (red). C, Representative images of T-cells (CD3, yellow) showing co-expression of both CD4 helper T-cells (green) and CD8 cytotoxic T cells (orange) with PD-L1 (red). Nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI; blue). Scale bar = 50 μm.