Tumor PD-L1 expression is correlated with increased TILs and poor prognosis in penile squamous cell carcinoma

Abstract

Despite its rare incidence worldwide, penile squamous cell carcinoma (PeSCC) still presents with significant morbidity and mortality due to the limited treatment options for advanced patients, especially those in developing countries. The program death-1 (PD-1)/PD-1 ligand (PD-L1) axis has been demonstrated to play an important role in tumor immune escape, and immunotherapies targeting this pathway have shown great success in certain cancer types. Here, we analyzed the expression pattern of PD-L1 in tumor cells and tumor-infiltrating lymphocytes (TILs) in PeSCC with a multi-center cohort. We found that the majority of PeSCCs (53.4%) were PD-L1-positive and that high PD-L1 expression in tumor cells was associated with a poor prognosis. Notably, PD-L1 expression in tumor cells was significantly associated with the extent of TILs and CD8⁺ TILs. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) showed that PD-L1 was positively correlated with interferon-gamma (IFNγ) and CD8⁺ gene expression. Moreover, we defined the constitutive and inducible surface expression of PD-L1 in newly established primary PeSCC cell lines. Interestingly, two PeSCC cell lines had high intrinsic PD-L1 expression. Another cell line showed low PD-L1 expression, but the PD-L1 expression could be induced by IFNγ stimulation. Overall, our data showed that high PD-L1 expression in penile tumor cells indicated a poor prognosis. The upregulation of PD-L1 in PeSCC included both extrinsic and intrinsic mechanisms. These findings indicated that the PD-1/PD-L1 axis might be a potential therapeutic target for patients with penile squamous cell carcinoma.

Keywords: IFNγ; PD-L1; immunohistochemistry; penile cancer; penile cancer cell line; tumor-infiltrating lymphocytes.

Figure 1.

IHC analysis of PD-L1 expression on the tumor cell membranes or TILs. (A) An isotype control for PD-L1 staining in human placenta tissue (200×). (B) A positive control of PD-L1 staining in human placenta tissue (200×). (C) Positive membrane staining of PD-L1 on penile tumor cell membrane (100×). (D) 200× magnification of the boxed area shown in (C). (E) Positive staining of PD-L1 on TILs (100×). (F) 200× magnification of the boxed area shown in (E).

Figure 2.

Correlation between PD-L1 expression and IFNγ or CD8⁺ gene expression in PeSCC. The expressions levels of PD-L1, IFNγ and CD8⁺ were evaluated by qRT-PCR in 24 primary PeSCC tissues. GAPDH was used as an internal control. Correlation analysis was performed for PD-L1 and IFNγ (A) as well as for PD-L1 and CD8⁺ (B), r: Spearman's correlation coefficient.

Figure 3.

Intrinsic and extrinsic expression of PD-L1 in penile cancer cell lines. (A) Western blot of PD-L1 in three different penile cancer cell lines (Penl1, Penl2 and 149RCa) and the normal human keratinocyte cell line, HaCaT. (B) qPCR analysis of PD-L1 mRNA in penile cancer cell lines relative to HaCaT. (C) Flow cytometric analysis of the cell surface PD-L1 in Penl1 and Penl2. PD-L1, black line; isotype control, gray zone. (D) Induction of PD-L1 expression by different IFNγ doses in Penl2. All experiments were performed in triplicate. Representative data are shown.

Figure 4.

PD-L1 expression on tumor cells and CD8⁺ TILs is associated with shorter CSS. Kaplan–Meier curves for the analysis of tumor PD-L1 expression (A), TILs PD-L1 expression (B), extent of TILs (C) and CD8⁺ TILs (D). p-values were calculated by log-rank test.