The PD-1, PD-L1 expression and CD3+ T cell infiltration in relation to outcome in advanced gastric signet-ring cell carcinoma, representing a potential biomarker for immunotherapy

Abstract

Recent data supports a potentially significant role for immune checkpoint inhibitors in the treatment of gastric cancer. However, there are few data on the clinical implications of immunotherapy markers in gastric signet-ring cell carcinoma (SRCC). We evaluated the expression of programmed cell death protein-1 (PD-1), programmed cell death ligand 1(PD-L1), infiltration by CD3+ T cell, microsatellite instability (MSI), and Epstein-Barr Virus (EBV), and the relationship of each factor to survival in 89 advanced SRCC patients. All patients received 5-FU-based first-line chemotherapy. PD-L1 and PD-1 were expressed in 40.4% and 18.0% of the patients, respectively. There was a significant correlation between PD-L1 and PD-1 expression (r=0.363, p<0.001). There was loss of at least 1 of the 4 DNA mismatch repair (DNA-MMR) gene proteins in 32.6% of samples. Only 1 case out of 89 was EBV positive, with concurrent PD-L1 positivity, a high degree of CD3+ T cell infiltration and MSI. Increased CD3+ T cells numbers was associated with increased PD-1 expression (r=0.256, p=0.012) and MSI status (r=0.208, p=0.049). High CD3+ T cell infiltration was related to better OS (23.7 months, 95% CI: 19.0-38.0 vs 15.8 months, 95% CI: 13.0-22.0, p=0.033), but was not an independent prognostic factor for survival after multivariate analysis (HR=0.68, 95% CI: 0.42-1.10, p=0.116). CD3+ T cell was more infiltrated in PD-1 positive, tumors with MSI and were associated with better OS, indicating an adaptive immune resistance may be occurring. Further research into the cancer immunotherapy markers of SRCC immune microenvironment may highlight targets for immunotherapy.

Keywords: CD3+ T cell; gastric signet-ring cell carcinoma; microsatellite instability; programmed cell death ligand 1; programmed cell death protein 1.

Figure 1. Immunohistochemical staining for programmed death-1 ligand-1 (PD-L1) and programmed death-1 (PD-1) in gastric signet-ring cell carcinoma (SRCC) tissue

(A) Representative immunohistochemical staining for PD-1 cells infiltrating gastric cancer tissue. (B) Representative immunohistochemical staining for PD-L1 in tumor cells. (C) Representative immunohistochemical staining for PD-L1 in immune cells. Original magnification ×400.

Figure 2. Immunohistochemical staining for CD3+ TILs infiltration in SRCC tissue

(A) Rare CD3+ TILs infiltrated in SRCC tissue. (B) Representative immunohistochemical staining for low-CD3 group. (C) Representative immunohistochemical staining for high-CD3 group. Original magnification ×400.

Figure 3. CD3+ TILs increased with the expression of PD-L1, PD-1 and MSI status

(A) The average CD3+ TILs number with or without PD-L1/PD-1 expression and in MSI or MSS status were determined using independent-sample t-tests. CD3+ TILs were more infiltrated in PD-L1-positive (p=0.007), PD-1-positive (p=0.023), and tumor with MSI (p=0.049). (B) Correlation between CD3 number and PD-L1, PD-1 expression, and MSI status were determined using the Chi-square exact test. PD-L1 expression (p=0.101), PD-1 expression (p=0.072) and MSI status (p=0.049) increased with increasing CD3 number in SRCC tissue.

Figure 4. Chromogenic in-situ hybridization for Epstein-Barr Virus-encoded RNA (EBER) in SRCC tissue

(A) EBER-positive highlights carcinoma cells. (B) EBER-negative. Original magnification ×400.

Figure 5. Immunohistochemical staining for MLH1, MSH2, MSH6 and PMS2 in SRCC tissue

(A) Representative immunohistochemical staining for MLH1 in tumor cells. (B) Missing immunohistochemical staining of MLH1 in tumor cells. Original magnification ×400.

Figure 6

Kaplan–Meier estimates of overall survival in SRCC patients according to the expression of (A) PD-L1 and (B) PD-1, (C) CD3+ T cell infiltration, and (D) MSI status.