Prognostic effect of different PD-L1 expression patterns in squamous cell carcinoma and adenocarcinoma of the cervix

Abstract

Programmed death-ligand 1 (PD-L1) is expressed in various immune cells and tumor cells, and is able to bind to PD-1 on T lymphocytes, thereby inhibiting their function. At present, the PD-1/PD-L1 axis is a major immunotherapeutic target for checkpoint inhibition in various cancer types, but information on the clinical significance of PD-L1 expression in cervical cancer is largely lacking. Here, we studied PD-L1 expression in paraffin-embedded samples from two cohorts of patients with cervical cancer: primary tumor samples from cohort I (squamous cell carcinoma, n=156 and adenocarcinoma, n=49) and primary and paired metastatic tumor samples from cohort II (squamous cell carcinoma, n=96 and adenocarcinoma, n=31). Squamous cell carcinomas were more frequently positive for PD-L1 and also contained more PD-L1-positive tumor-associated macrophages as compared with adenocarcinomas (both P<0.001). PD-L1-positive tumor-associated macrophages were found to express CD163 and/or CD14 by triple fluorescent immunohistochemistry, demonstrating an M2-like phenotype. Interestingly, disease-free survival (P=0.022) and disease-specific survival (P=0.046) were significantly poorer in squamous cell carcinoma patients with diffuse PD-L1 expression as compared with patients with marginal PD-L1 expression (i.e., on the interface between tumor and stroma) in primary tumors. Disease-specific survival was significantly worse in adenocarcinoma patients with PD-L1-positive tumor-associated macrophages compared with adenocarcinoma patients without PD-L1-positive tumor-associated macrophages (P=0.014). No differences in PD-L1 expression between primary tumors and paired metastatic lymph nodes were detected. However, PD-L1-positive immune cells were found in greater abundance around the metastatic tumors as compared with the paired primary tumors (P=0.001 for squamous cell carcinoma and P=0.041 for adenocarcinoma). These findings point to a key role of PD-L1 in immune escape of cervical cancer, and provide a rationale for therapeutic targeting of the PD-1/PD-L1 pathway.

Figures

Figure 1

Programmed death-ligand 1 (PD-L1) expression patterns in cervical cancer. Different patterns for PD-L1 expression (in brown) were detected in primary squamous cell carcinoma and adenocarcinoma. (a) Diffuse PD-L1 expression by primary squamous cell carcinoma cells. (b) Marginal PD-L1 expression by primary squamous cell carcinoma cells. (c) PD-L1-negative primary squamous cell carcinoma. (d) Primary squamous cell carcinoma with PD-L1-positive tumor-associated macrophages (examples indicated by black arrows). (e) PD-L1-negative primary adenocarcinoma. (f) PD-L1-negative primary adenocarcinoma with PD-L1-positive tumor-associated macrophages (examples indicated by black arrows). Scale bar is 100 μm.

Figure 2

Identification of programmed death-ligand 1 (PD-L1)-positive tumor-associated macrophages. A representative triple immunofluorescence staining shows monochromatic (a) PD-L1, (b) CD14, (c) CD163 images, and (d) colocalized PD-L1 (in blue), CD14 (in red), CD163 (in green), and DAPI (4',6-diamidino-2-phenylindole) (in gray) in cervical cancer patients with PD-L1-positive tumor-associated macrophages in primary tumors. NB: Varying intensity of CD163 staining can be observed. Scale bar is 30 μm.

Figure 3

Survival analysis for PD-L1 positivity. Kaplan–Meier 5-year survival curves show disease-free survival (DFS) (a) and disease-specific survival (DSS) (b) for patients with diffuse PD-L1 expression by tumor cells, for patients with PD-L1-negative (PD-L1−) tumors, and for patients with marginal PD-L1 expression in squamous cell carcinoma. Kaplan–Meier 5-year survival curve shows disease-specific survival (DSS) for patients with (c) squamous cell carcinoma and (d) adenocarcinoma with PD-L1-positive tumor-associated macrophages (TAM(+)) and for patients without PD-L1-positive tumor-associated macrophages (TAM(−)). P-values were calculated using the log-rank test.

Figure 4

PD-L1 expression in metastatic lymph nodes. PD-L1 (in brown) expression in (a) metastatic squamous cell carcinoma with a PD-L1-positive cordon indicated by the black arrow and high numbers of PD-L1-positive peritumoral immune cells, and (b) metastatic adenocarcinoma lymph node samples. PD-L1 positivity in primary squamous cell carcinoma (SCC) and adenocarcinoma (AC) and metastatic lymph nodes (c) with PD-L1-positive tumor cells, (d) with diffuse PD-L1 expression, (e) with the presence of PD-L1-positive tumor-associated macrophages (TAM), (f) with the presence of peritumoral PD-L1-positive immune cells, and (g) with the presence of a PD-L1-positive cordon. **P=0.001 and *P=0.041 calculated with McNemar test. Scale bar is 50 μm.