Patterns and prognostic values of programmed cell death-ligand 1 expression and CD8 + T-cell infiltration in small cell carcinoma of the esophagus: a retrospective analysis of 34 years of National Cancer Center data in China

Abstract

Background: Small cell carcinoma of the esophagus (SCCE) is an extremely rare and highly aggressive neuroendocrine malignancy with a strikingly poor prognosis. Given the great clinical successes of checkpoint immunotherapies, we explored the expression profile and clinical significance of programmed cell death-ligand 1 (PD-L1) and CD8 + T cell in SCCE for the first time.

Materials and methods: Tumor-infiltrating immune cells (TIICs) and tumor cells in postoperative, whole tumor sections from 147 SCCE patients were stained for PD-LI expression. We also evaluated each patient's Combined Positive Score (CPS). Multiplex immunofluorescence staining (CD3, CD20, CD68, and PD-L1) was introduced to clarify the location of PD-L1. CD8 density was analyzed by digital imaging and analysis of entire slides. Clinical outcomes were tested for correlations with both PD-L1 expression and CD8 density.

Results: No patients had PD-L1 expressed in their tumor cells. PD-L1 + expression in TIICs was detected in 65 patients (44.2%) and 42 (28.6%) exhibited CPS positivity. Multiplex immunofluorescence staining demonstrated that most of the PD-L1 was expressed on the CD68 + monocytes/macrophages. PD-L1 expression in the TIICs and CPS was found to be correlated with paraffin block age, tumor length, macroscopic type, T stage, and increased overall survival (OS). Expression of PD-L1 in TIICs showed significantly prolonged relapse-free survival (RFS). Increasing CD8 densities were associated with increased PD-L1 expression ( Ptrend <0.0001). Multivariate regression confirmed that PD-L1 in TIICs and CD8 states were independent predictors of OS, and CD8 status were found to be independently predictive of RFS. A stratification based on PD-L1 and CD8 status was also significantly associated with both OS and RFS.

Conclusion: Expression of PD-L1 was only detected in TIICs from approximately half of the patients with SCCEs. In SCCEs, PD-L1 and CD8 status are novel prognostic biomarkers and may inform the implementation of risk-related therapeutic strategies. SCCEs with higher CD8 infiltration also had higher expression of PD-L1, suggesting the development of resistance against adaptive immunity. These findings support the assertion that PD-L1/programmed cell death 1 inhibitors should be investigated in this rare malignancy.

Figure 1

Expression of programmed cell death-ligand 1 (PD-L1) in small cell carcinoma of the esophagus. (A) Tumor cells (TCs) in small cell carcinoma of the esophagus did not show any PD-L1 staining. (B) One percent of the tumor-infiltrating immune cells (TIICs) showed PD-L1 positive. (C) Fifty percent of the TIICs showed PD-L1 positive. Hematoxylin–eosin staining (upper panel, ×100, scale bars, 100 μm) and PD-L1 staining (middle panel, ×100, scale bars, 100 μm; lower panel, ×400, scale bars, 20 μm) of small cell carcinoma of the esophagus.

Figure 2

PD-L1 was mainly located on the CD68⁺ tumor-infiltrating immune cells. Representative multiple immunofluorescence images of CD68/PD-L1 (A), CD3/PD-L1 (B), CD20/PD-L1 (C), and CD3/CD20/CD68/PD-L1 (D) from tissue microarrays. Left panel, ×100, scale bars, 100 μm; right panel, ×400, scale bars, 20 μm. PD-L1, programmed cell death-ligand 1.

Figure 3

CD8⁺ T-cell infiltration and the association with PD-L1 patterns in SCCE. (A) CD8⁺ T-cell infiltration in the intratumoral compartment (left panel, ×200) and the peritumoral compartment (right panel, ×200). Scale bars, 50 μm. The distributions of CD8⁺ T-cell density in SCCEs with or without expression of PD-L1 in TIICs (B) or by CPS (C). (D) PD-L1 expression in TIICs or by CPS increases with increasing CD8 density. (E) The landscape of distributions of CD8 densities and PD-L1 status in SCCE. ****P<0.0001. CPS, Combined Positive Score; PD-L1, programmed cell death-ligand 1; SCCE, small cell carcinoma of the esophagus; TIIC, tumor-infiltrating immune cell.

Figure 4

Correlations of PD-L1 and CD8 status and survival in small cell carcinoma of the esophagus. Relapse-free survival and overall survival in patients with small cell carcinoma of the esophagus according to PD-L1 expression in TIICs (A and B), PD-L1 expression by CPS (C and D), and CD8 status (E and F). CPS, Combined Positive Score; PD-L1, programmed cell death-ligand 1; TIIC, tumor-infiltrating immune cell.

Figure 5

Survival analysis of the four different immunophenotypes in small cell carcinoma of the esophagus. (A) Representative multiple immunofluorescence images of the four different types (left panel, ×100, scale bars, 100 μm; right panel, ×400, scale bars, 20 μm). The Kaplan–Meier curves for relapse-free survival (B) and overall survival (C) in patients with small cell carcinoma of the esophagus according to programmed cell death-ligand 1 (PD-L1) expression and CD8 status.