Programmed death ligand 1 expression and tumor-infiltrating lymphocytes in glioblastoma

Abstract

Background: Immune checkpoint inhibitors targeting programmed cell death 1 (PD1) or its ligand (PD-L1) showed activity in several cancer types.

Methods: We performed immunohistochemistry for CD3, CD8, CD20, HLA-DR, phosphatase and tensin homolog (PTEN), PD-1, and PD-L1 and pyrosequencing for assessment of the O6-methylguanine-methyltransferase (MGMT) promoter methylation status in 135 glioblastoma specimens (117 initial resection, 18 first local recurrence). PD-L1 gene expression was analyzed in 446 cases from The Cancer Genome Atlas.

Results: Diffuse/fibrillary PD-L1 expression of variable extent, with or without interspersed epithelioid tumor cells with membranous PD-L1 expression, was observed in 103 of 117 (88.0%) newly diagnosed and 13 of 18 (72.2%) recurrent glioblastoma specimens. Sparse-to-moderate density of tumor-infiltrating lymphocytes (TILs) was found in 85 of 117 (72.6%) specimens (CD3+ 78/117, 66.7%; CD8+ 52/117, 44.4%; CD20+ 27/117, 23.1%; PD1+ 34/117, 29.1%). PD1+ TIL density correlated positively with CD3+ (P < .001), CD8+ (P < .001), CD20+ TIL density (P < .001), and PTEN expression (P = .035). Enrichment of specimens with low PD-L1 gene expression levels was observed in the proneural and G-CIMP glioblastoma subtypes and in specimens with high PD-L1 gene expression in the mesenchymal subtype (P = 5.966e-10). No significant differences in PD-L1 expression or TIL density between initial and recurrent glioblastoma specimens or correlation of PD-L1 expression or TIL density with patient age or outcome were evident.

Conclusion: TILs and PD-L1 expression are detectable in the majority of glioblastoma samples but are not related to outcome. Because the target is present, a clinical study with specific immune checkpoint inhibitors seems to be warranted in glioblastoma.

Keywords: glioblastoma; immune checkpoint; programmed death 1; programmed death ligand 1.

Fig. 1.

Low-magnification overview (anti-PD-L1 immunostaining, original magnification ×2) showing prominent and patchy PD-L1 expression (arrow 1: positive area, arrow 2: negative area) in tumor tissue of a glioblastoma and lack of PD-L1 expression in the adjacent brain parenchyma (asterisk).

Fig. 2.

Diffuse/fibrillary and membraneous PD-L1 expression in glioblastoma. (A–D) (adjacent sections of the same tissue specimen): In glioblastoma areas with diffuse/fibrillary histomorphology (A: hematoxylin and eosin (H&E) staining, original magnification ×200; Aa: H&E staining, original magnification ×800) and expression of glial fibrillary acidic protein (GFAP; B: anti-GFAP, original magnification ×200) we found prominent diffuse/fibrillary PD-L1 expression (C and D: anti-PD-L1; C: original magnification ×200, D: original magnification ×400). (E–H) (adjacent sections of the same tissue specimen): In glioblastoma areas with epithelioid tumor cells (E: H&E staining, original magnification ×200; Ee: H&E staining, original magnification ×800) and expression of glial fibrillary acidic protein (GFAP; F: anti-GFAP, original magnification ×200) we found prominent membranous PD-L1 expression and some faint diffuse or granular cytoplasmic staining (G and H: anti-PD-L1; C: original magnification ×200, D: original magnification ×400).

Fig. 3.

(A) Dense CD3+ TIL infiltration (magnification ×200). (B) PD1+ TILs infiltrating glioblastoma (magnification ×400). (C) Perivascular infiltration with CD3+ TILs (magnification ×200). (D) CD3+ TILs at the infiltration zone (magnification ×200).

Fig. 4.

A–G: Kaplan-Meier curves in the Vienna retrospective cohort. (A) Overall survival according to age. (B) Overall survival according to Karnofsky performance status. (C) Overall survival according to extent of resection. (D) Overall survival according to MGMT methylation status. (E) Overall survival according to membranous PD-L1 expression. (F) Overall survival according to diffuse/fibrillary PD-L1 expression. (G) Overall survival according to PD-L1 expression in neuron. (H) Kaplan-Meier curve in the TCGA cohort showing overall survival according to PD-L1 gene expression. All P values are according to the log-rank test.