Immunophenotypes Based on the Tumor Immune Microenvironment Allow for Unsupervised Penile Cancer Patient Stratification

Abstract

The tumor immune microenvironment (TIME) plays an important role in penile squamous cell carcinoma (peSCC) pathogenesis. Here, the immunophenotype of the TIME in peSCC was determined by integrating the expression patterns of immune checkpoints (programmed cell death-1 (PD-1)/programmed cell death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen 4 (CTLA-4), and Siglec-15) and the components of tumor-infiltrating lymphocytes, including CD8⁺ or Granzyme B⁺ T cells, FOXP3⁺ regulatory T cells, and CD68⁺ or CD206⁺ macrophages, in 178 patients. A high density of Granzyme B, FOXP3, CD68, CD206, PD-1, and CTLA-4 was associated with better disease-specific survival (DSS). The patients with diffuse PD-L1 tumor cell expression had worse prognoses than those with marginal or negative PD-L1 expression. Four immunophenotypes were identified by unsupervised clustering analysis, based on certain immune markers, which were associated with DSS and lymph node metastasis (LNM) in peSCC. There was no significant relationship between the immunophenotypes and high-risk human papillomavirus (hrHPV) infection. However, the hrHPV-positive peSCC exhibited a higher density of stromal Granzyme B and intratumoral PD-1 than the hrHPV-negative tumors (p = 0.049 and 0.002, respectively). In conclusion, the immunophenotypes of peSCC were of great value in predicting LNM and prognosis, and may provide support for clinical stratification management and immunotherapy intervention.

Keywords: CTLA-4; HPV; PD-L1; Siglec-15; peSCC; prognosis; tumor immune microenvironment.

Figure 1

The expression of PD-L1 and Siglec-15 in peSCC tissues by immunohistochemistry. Representative immunohistochemistry (IHC) images show marginal (a) and diffuse (b) expression of PD-L1 expression (b). The low (c) and high (d) expressions of Siglec-15 in intratumoral or stromal tumor-infiltrating myeloid cells were also presented. Magnification: 200×.

Figure 2

The expression of immune markers in hrHPV⁻ and hrHPV⁺ samples. (a) The box plots indicate the transformed densities of stromal CD8 (CD8s), intratumoral CD8 (CD8t), GrBt, GrBs,CD68t, CD68s, CD206t, CD206s, FOXP3t, FOXP3s, PD-1t, PD-1s and CTLA-4s. Compared with the hrHPV⁻ patients, the hrHPV⁺ patients expressed a higher density of stromal GrB and intratumoral PD-1. * p < 0.05, ** p < 0.01, independent t-test. (b) Spineplot diagrams show the expression patterns of PD-L1 and Siglec-15 in the intratumoral and stromal regions. There was no difference between the expression of PD-L1 and Siglec-15 between hrHPV⁻ and hrHPV⁺ tumors.

Figure 3

The correlation of patient prognosis and expression of immune markers in peSCC. (a) The densities of immune markers were treated as continuous variables. Forest plot shows the hazard ratios (HR) for disease-specific survival (DSS). Values shown in bold are statistically significant. (b–e) Kaplan–Meier survival analyses (log-rank tests) were conducted, according to PD-L1 expression in tumor (b), PD-L1 expression in stroma (c), Siglec-15 expression in tumor (d), and Siglec-15 expression in stroma (e).

Figure 4

The immunophenotypes of the tumor immune microenvironment (TIME) in peSCC. (a) Correlation matrix plots by Spearman correlation analyses show pairwise positively stronger correlation of the expression of tested markers. * p < 0.05, ** p < 0.01, *** p < 0.001. (b) Cluster analyses using correlation distance and ward.D were conducted based on the expression of immune markers. The heatmap shows the high expression (red) or low expression (blue) of each immune marker. Annotations of the samples on the top of the heatmap indicate histopathological features, including hrHPV status, histological subtypes, grade, T stage, N stage, survival status, and the four clusters identified by the cutree method. (c) Box plots show the expression of immune markers, according to identified four clusters. The transformed densities of CD8, GrB, FOXP3, PD-1, CD68, CD206, and CTLA-4 in the intratumoral and stromal regions were indicated by box plots. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, Kruskal-Wallis test. (d–f) Kaplan–Meier survival curves for disease-specific survival (DSS) show patients in clusters A and C had better prognosis than clusters B and D in all cases (d), hrHPV⁻ cases (e) and hrHPV⁺ cases(f). (g) Patients in high-risk group (clusters A and C) were likely to experience tumor progression, compared with those in low-risk group (clusters B and D).

Figure 5

The association of immunophenotypes and disease stages of patients with peSCC. Spineplot diagrams show the percentages of patients at different T (a) and N (b) stages. Results indicate that patients in cluster A were frequently accompanied with advanced T and N stages.

Figure 6

Immunohistochemical quantitative analyses of CD8 (a–d) and PD-1 (e–h) expression in intratumoral and stromal regions. Analyses were performed using inForm 2.1 Image Analysis Software (Mantra Software/PerkinElmer). Images of IHC staining, as well as the simulative tissue segmentation (tumor: red, stroma: green) (b,f), positive cell segmentation (c,g) and merged images (d,h), were shown. Magnification: 200×.