Abstract 2488: The penile cancer tumor microenvironment and immunotherapy response: Results from the PERICLES trial
Authors: Rafael Tynisha S. , Gil-Jimenez Alberto , de Vries Hielke M. , Seignette Iris M. , Bekers Elise , Lopez-Yurda Marta , Peters Dennis , Hooijberg Erik , Broeks Annegien , Brouwer Oscar R. , Schaake Eva , Vis Daniel J. , de Gruijl Tanja D. , Wessels Lodewyk F.A. , van der Heijden Michiel S. 
Published: March 2024

Abstract
Introduction: The phase II PERICLES trial investigated the efficacy of atezolizumab (anti-PD-L1) for advanced penile cancer, with or without radiotherapy. As previously published, durable responses (progression-free at 12 months) were observed in 4 out of 32 enrolled patients. Here, we present the final overall survival (OS) results and an analysis of potential biomarkers for immunotherapy response.
Methods: Preplanned analysis projected that at 27/32 events, the study would have 80% power (2-sided α=0.05) to assess whether the OS for the entire cohort significantly differed from the hypothesized historical median for stage IV patients. Baseline tumor tissue samples were subjected to bulk-RNA sequencing (n=31), fluorescent (n=28) and chromogenic (n=30) multiplex immunohistochemistry. We quantified the densities of CD3+CD8+ cytotoxic T-cells, CD3+CD8−FoxP3− T-helper cells, CD3+CD8−FoxP3+ regulatory T-cells, CD68+ macrophages and CD20+ B-cells in both the tumor and stromal compartments and assessed spatial relationships. We also quantified CD8+PD1+ (potentially enriched for tumor reactivity) and CD8+PD1− T-cells. Differential expression and gene set enrichment analysis (GSEA) were performed using the hallmarks gene sets.
Results: The median OS for the entire study cohort was 11.3 months (95% CI, 5.5-16.7), which is an improvement compared to the hypothesized historical median of 7.8 months for stage IV patients (one sample log-rank p=0.033). Biomarker analyses showed an increased density of stromal macrophages (p&lt;0.001) and intratumoral cytotoxic T-cells (p=0.035) in durable responders compared to non-responders. Additionally, we observed an increased density of intratumoral CD8+PD1+ in durable responders (p&lt;0.001). In contrast, intratumoral CD8+PD1− densities were comparable between durable and non-responders (p=0.057). Patients with a high density of intratumoral CD8+PD1+ also had an improved progression-free survival (PFS) (log-rank, p&lt;0.001) and OS (log-rank, p=0.002). Analysis of the spatial relationships in the tumor microenvironment (TME) showed that durable responders had larger distances between cytotoxic T-cells and T-helper cells (p=0.034). GSEA revealed significant (FDR≤5%) up-regulation of IFN-α/γ responses in durable responders. In contrast, non-responders showed up-regulation of epithelial-mesenchymal transition, KRAS signaling and angiogenesis, suggesting potential barriers to an anti-tumor response.
Conclusion: In the final clinical analysis, OS was improved with atezolizumab compared to the historical population of stage IV patients. CD8+PD1+ T-cells, a common proxy for tumor-reactive T-cells, and spatial relationships in the TME might serve as biomarkers of durable response to immunotherapy in advanced penile cancer.
Citation Format: Tynisha S. Rafael, Alberto Gil-Jimenez, Hielke M. de Vries, Iris M. Seignette, Elise Bekers, Marta Lopez-Yurda, Dennis Peters, Erik Hooijberg, Annegien Broeks, Oscar R. Brouwer, Eva Schaake, Daniel J. Vis, Tanja D. de Gruijl, Lodewyk F.A. Wessels, Michiel S. van der Heijden. The penile cancer tumor microenvironment and immunotherapy response: Results from the PERICLES trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2488.