Pre-existing immunity and hrHPV status determine immunotherapy response in advanced penile cancer: biomarkers and overall survival in the PERICLES trial

Abstract Penile squamous cell carcinoma (PeSCC) is a rare malignancy with limited treatment options in advanced stages. The phase II PERICLES trial (NCT03686332) investigated atezolizumab with or without radiotherapy in advanced penile cancer. Here, we report mature overall survival (OS) and updated progression-free survival (PFS) outcomes and an in-depth biomarker analysis. Patients were treated with atezolizumab monotherapy (n=12) or atezolizumab in combination with radiotherapy (n=20, 33 fractions of 1.5-1.8 Gy) for locoregional disease control. Comprehensive analysis of baseline tumor tissue (n=28/31) was performed using bulk RNA sequencing, immunohistochemistry and multiplex immunofluorescence. Additionally, spatial transcriptomics (n=9) was conducted to further explore functionality and (co-)localization of immune cell subsets. With extended follow-up (median: 38 months), landmark 2-year OS and PFS in the full study cohort was 18.8% (95% CI, 9.1-38.6) and 12.5% (95% CI, 5.0-31.3), respectively. High-risk human papillomavirus (hrHPV)–positive tumors were enriched for IFN-γ and IFN-α signatures, potentially explaining the improved PFS and OS observed in this subgroup. Both stromal CD68+ myeloid cell density and intratumoral CD8+PD1+ T-cell density were associated with non-progression at 1 year. Increased intratumoral CD8+PD1+ T-cell density was also associated with improved PFS and OS, with consistent findings in the hrHPV–positive subgroup. Spatial transcriptomics identified these CD8+ T cells as tissue-resident cytotoxic effector cells co-expressing checkpoint molecules (LAG3 and TIGIT), chemokines (CCL4 and CXCL13), and cytotoxic molecules (PRF1 and GZMB). Interrogation of the penile cancer immune microenvironment highlights CD8+PD1+ T-cells as candidate biomarkers in relation to clinical benefit from immune checkpoint blockade in advanced PeSCC.