Patterns of PD-L1 expression and CD8 T cell infiltration in gastric adenocarcinomas and associated immune stroma

Abstract

Objective: Recent data supports a significant role for immune checkpoint inhibitors in the treatment of solid tumours. Here, we evaluate gastric and gastro-oesophageal junction (G/GEJ) adenocarcinomas for their expression of programmed death-ligand 1 (PD-L1), infiltration by CD8+ T cells and the relationship of both factors to patient survival.

Design: Thirty-four resections of primary invasive G/GEJ were stained by immunohistochemistry for PD-L1 and CD8 and by DNA in situ hybridisation for Epstein-Barr virus (EBV). CD8+ T cell densities both within tumours and at the tumour-stromal interface were analysed using whole slide digital imaging. Patient survival was evaluated according to PD-L1 status and CD8 density.

Results: 12% of resections showed tumour cell membranous PD-L1 expression and 44% showed expression within the immune stroma. Two cases (6%) were EBV positive, with one showing membranous PD-L1 positivity. Increasing CD8+ densities both within tumours and immune stroma was associated with increasing percentage of tumour (p=0.027) and stromal (p=0.005) PD-L1 expression. Both tumour and immune stromal PD-L1 expression and high intratumoral or stromal CD8+ T cell density (>500/mm²) were associated with worse progression-free survival (PFS) and overall survival (OS).

Conclusions: PD-L1 is expressed on both tumour cells and in the immune stroma across all stages and histologies of G/GEJ. Surprisingly, we demonstrate that increasing CD8 infiltration is correlated with impaired PFS and OS. Patients with higher CD8+ T cell densities also have higher PD-L1 expression, indicating an adaptive immune resistance mechanism may be occurring. Further characterisation of the G/GEJ immune microenvironment may highlight targets for immune-based therapy.

Keywords: CANCER IMMUNOBIOLOGY; GASTRIC CANCER; IMMUNE RESPONSE; IMMUNOTHERAPY.

Figure 1

Expression of PD-L1 by gastric adenocarcinomas. H&E (A, B, E and F) and programmed death-ligand 1 (PD-L1) (C, D, G and H) immunohistochemical staining in representative examples of PD-L1^pos (panel 1) and PD-L1^neg (panel 2) intestinal-type gastric adenocarcinoma. PD-L1 staining is observed on both tumour cells and infiltrating immune cells. Panel 1 (×100 original magnification) and panel 2 (×400 original magnification).

Figure 2

Epstein–Barr virus (EBV) status in programmed death-ligand 1 (PD-L1^pos) gastric adenocarcinomas. H&E (A and D) and PD-L1 (B and E) staining and in situ hybridisation (ISH) for EBV (C and F) in two PD-L1^pos gastric adenocarcinomas. Evidence of EBV infection by ISH in shown in C, while the tumour in F is negative for EBV. Shown at ×100 original magnification.

Figure 3

Expression of programmed death-ligand 1 (PD-L1) by immune stroma associated with gastric adenocarcinomas. H&E (A–C) and PD-L1 staining (D–F) in two gastric adenocarcinomas with PD-L1^pos (A, B, D and E) or PD-L1^neg immune stroma (C and F). PD-L1 expression by immune stroma is seen in a peritumoral ‘interface’ pattern. Shown at ×100 (A, C, D and F) and ×400 (B and E) original magnification.

Figure 4

Programmed death-ligand 1 (PD-L1) expression by tumour cells and immune stroma increases with increasing CD8 density in each location. CD8 density within gastric and gastro-oesophageal junction was determined by digital image analysis and densities were divided by quartiles into low (<147/mm²), mid (147–500/mm²) and high (>500/mm²). Correlation between CD8 density and PD-L1 expression by location were determined using the exact version of the Cochran–Armitage trend test. PD-L1 expression by both tumour cells (p=0.0027) and immune stroma (p=0.005) increased with increasing CD8 density in each location.

Figure 5

Tumour programmed death-ligand 1 (PD-L1) expression in gastric and gastro-oesophageal adenocarcinomas is correlated with worse progression-free survival (PFS) and overall survival (OS). PFS and OS associations with PD-L1 expression by the tumour (A) and in the immune stroma (B). Probabilities of overall and event-free survival were estimated using the Kaplan–Meier method and compared using the log-rank statistic or the Cox proportional hazards regression model.

Figure 6

Increasing intratumoral and stromal CD8+ T cell density in gastric and gastro-oesophageal adenocarcinomas is correlated with worse progression-free survival (PFS) and overall survival (OS). PFS and OS associations with CD8+ T cell density within tumours (A) and in the immune stroma (B). Probabilities of overall and event-free survival were estimated using the Kaplan–Meier method and compared using the log-rank statistic or the Cox proportional hazards regression model. The upper quartile was used as the breakpoint for stroma CD8 and the approximate lower and upper quartiles were used for tumour CD8. The reported HRs for intratumoral CD8 density reflect the survival comparison between tumours with high density (>500 CD8 T cells/mm²) and tumours with low density (<147 CD8 T cells/mm²).