The Prognostic Value of Immune Factors in the Tumor Microenvironment of Penile Squamous Cell Carcinoma

Abstract

The host's immune system plays a pivotal role in many tumor types, including squamous cell carcinomas (SCCs). We aim to identify immunological prognosticators for lymph node metastases (LNM) and disease-specific survival (DSS) in penile SCC. For this retrospective observational cohort study, penile SCC patients (n = 213) treated in the Netherlands Cancer Institute, were selected if sufficient formalin-fixed, paraffin-embedded tumor material was available. Analysis included previously described high-risk human papilloma virus (hrHPV) status, immunohistochemical scores for classical and non-classical human leukocyte antigen (HLA) class I, programmed death ligand-1 (PD-L1) expression, and novel data on tumor-infiltrating macrophages and cytotoxic an regulatory T-cells. Clinicopathological characteristics and extended follow-up were also included. Regression analyses investigated relationships of the immune parameters with LNM and DSS. In the total cohort, diffuse PD-L1 tumor-cell expression, CD163⁺ macrophage infiltration, non-classical HLA class I upregulation, and low stromal CD8⁺ T-cell infiltration were all associated with LNM. In the multivariable model, only tumor PD-L1 expression remained a significant predictor for LNM (odds ratio (OR) 2.8, p = 0.05). hrHPV negativity and diffuse PD-L1 tumor-cell expression were significantly associated with poor DSS and remained so upon correction for clinical parameters [hazard ratio (HR) 9.7, p < 0.01 and HR 2.8, p = 0.03]. The only immune factor with different expression in HPV⁺ and HPV^- tumors was PD-L1, with higher PD-L1 expression in the latter (p = 0.03). In the HPV^- cohort (n = 158), LNM were associated with diffuse PD-L1 tumor-cell expression, high intratumoral CD163⁺ macrophage infiltration, and low number of stromal CD8⁺ T-cells. The first two parameters were also linked to DSS. In the multivariable regression model, diffuse PD-L1 expression remained significantly unfavorable for DSS (HR 5.0, p < 0.01). These results emphasize the complexity of the tumor microenvironment in penile cancer and point toward several possible immunotherapy targets. Here described immune factors can aid risk-stratification and should be evaluated in clinical immunotherapy studies to ultimately lead to patient tailored treatment.

Keywords: B7-H1; HPV; T-cells; immune escape; microenvironment; penile cancer; programmed death ligand-1; squamous cell carcinoma.

Figure 1

Kaplan–Meier survival plots with log-rank test analysis of high-risk HPV-positive and -negative penile cancer cases.

Figure 2

Tumor-microenvironmental characteristics. Plots of immune parameters in hrHPV⁻ and hrHPV⁺ samples; categorical variables in bar plots, continuous variables in box plots. p-Values of testing comparable distribution in hrHPV groups. Table S2 in Supplementary Material presents the same data. Abbreviations: HLA, human leukocyte antigen; PD-L1, Programmed death ligand-1; TIM, tumor-infiltrating macrophages; hrHPV, high-risk human papilloma virus. Expression of PD-L1 on tumor cells was compared in two ways: negative vs. positive (neg vs. pos) and negative vs. positive at margin vs. diffusely positive (pattern).

Figure 3

Examples of representative stainings for high and low infiltration of CD8⁺ T cells (A,B), FoxP3⁺ regulatory T-cells (C,D), and CD163⁺ macrophages (E,F). Scale bars: 100 µm.

Figure 4

CD163 and CD68 double staining of an hrHPV⁻ (A) and hrHPV⁺ (B) case, indicative of M2 macrophage polarization. Colors: green, CD163; red, CD68; and blue, DAPI. Scale bars: 40 µm.