Prognostic value of tumor-infiltrating lymphocytes and PD-L1 expression in esophageal squamous cell carcinoma

Abstract

Background: Tumor cells (TC) participate in tumor progression by altering the immune responses in the tumor microenvironment. However, the clinical relevance and prognostic effect of PD-L1 expression and tumor-infiltrating lymphocytes (TILs) in esophageal squamous cell carcinoma (ESCC) are unknown. The purpose of this study was to investigate the interactions and clinical significance of PD-L1 expression and TILs in ESCC.

Methods: Tissue specimens were collected from 126 patients with ESCC who underwent curative esophagectomy. Immunohistochemical analysis and multiplex immunofluorescence for CD4, CD8, CD25, FOXP3, and PD-L1 in the tumor were used to identify multiple tumor-infiltrating immune cells (TIIC), Tregs, and TC.

Results: PD-L1 was expressed in tumor cells (PD-L1 TC). PD-L1 TIIC and PD-L1 TC affected the biological behavior of TC. The positive expression rate of PD-L1 TC and CD8⁺ TILs was 27.8% (35/126) and 31.7% (40/126), respectively. Kaplan-Meier analysis showed that overall survival (OS) was significantly associated with decreased CD8⁺ TILs and PD-L1 TC-positive expression, which promote ESCC progression and metastasis.

Conclusion: Tumor depth, CD8, and PD-L1 TC were independent prognostic factors in ESCC, and a predictive nomogram with these three risk factors improved the accuracy of predicting OS in patients with ESCC after surgical resection. The conjoint analysis of multiple immune-related factors is beneficial for stratifying patient survival risk.

Keywords: PD‐L1; esophageal squamous cell carcinoma; regulatory T cells; tumor microenvironment; tumor‐infiltrating lymphocytes.

FIGURE 1

Tumor‐infiltrating lymphocytes, PD‐L1 expression and their associations with survival of patients with esophageal squamous cell carcinoma. (A) Representative immunohistochemistry images illustrating the infiltration of CD8⁺ TILs, FOXP3⁺ TILs, PD‐L1 TC, and PD‐L1 TIIC. (B) Kaplan–Meier analysis was used to determine the overall survival (OS) according to the prognostic effect of tumor depth (p = 0.0003), (C) CD8 (p = 0.0028), (D) PD‐L1 TC (p = 0.0402), and (E) PD‐L1 TIIC (p = 0.7978). (F) Kaplan–Meier curve for OS according to CD8/PD‐L1 TC status. (G) Representative immunofluorescence images of CD8 (green) and DAPI (blue) staining. (H) Immunofluorescence staining of CD4 (green), CD25 (red), and FOXP3 (blue) to label CD4⁺CD25⁺FOXP3⁺ Tregs, CD4⁺CD25⁺FOXP3⁻ T cells, CD4⁺CD25⁻FOXP3⁺ T cells, and CD4⁺CD25⁻FOXP3⁻ T cells, respectively. (I) Kaplan–Meier analysis was used to determine OS according to the prognostic effect of CD4⁺CD25⁺FOXP3⁺ Tregs (p = 0.0349) and (J) CD8⁺ TILs/CD4⁺CD25⁺FOXP3⁺ Tregs ratio (p = 0.0233). CD, cluster of differentiation; OS, overall survival; PD‐L1, programmed death ligand 1; TC, tumor cell; TIIC, tumor‐infiltrating immune cell; TILs, tumor‐infiltrating lymphocytes; Treg, regulatory T cells.

FIGURE 2

Prognostic nomogram to predict the overall survival (OS) of patients with esophageal squamous cell carcinoma. (A) A predictive nomogram for overall survival was constructed by combining proven independent prognostic factors with UICC tumor depth (T1 + 2, T3 + 4), CD8 expression (positive, negative), and PD‐L1 TC (positive, negative) expression. (B) The calibration plot for the nomogram to predict 3‐year survival and observed survival. (C) Time‐dependent receiver operating characteristic curves for nomogram, tumor depth, and CD8 and PD‐L1 TC expression to evaluate the 3‐year overall survival probability. p‐values were determined using the log‐rank test.