Spatial analysis of the immune microenvironment and identification of phenotypes of prognostic significance in penile squamous cell carcinoma (PSCC).
Authors: Gullapalli Keerthi , Miller Justin , Fazili Adnan Nazir , Johnson Jeffrey S. , Guske Christopher , Whiting Junmin , Kim Youngchul , Miyagi Hiroko , Zacharias Niki Marie , Johns Andrew , Segura Carlos Moran , Nguyen Jonathan , Rao Priya , Spiess Philippe E. , Pettaway Curtis Alvin , Chahoud Jad 
Published: February 2025

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Background:
Advanced penile cancer is a rare but aggressive malignancy. Over 70% of patients with bulky metastases relapse or have primary refractory disease with current treatments. Immunotherapy represents a promising modality, but response rates remain low. While prior studies explored single immune factors within the tumor microenvironment (TME), understanding complex interactions between T cells and macrophages and in relation to the tumor cells is crucial to best characterize the TME.
Methods:
We performed multiplex immunofluorescence (mIF) on 59 PSCC tissues obtained from MD Anderson to analyze expression of 12 key immune cell markers (CD3, CD8, CD68, CD86, CD206, CD163, ARG, CSF1R, MHC-II, PD-1, PD-L1). Spatial analysis and co-clustering of cell phenotypes was performed using Ripley’s K. Overall (OS), recurrence free (RFS), and cancer specific survival (CSS) curves were determined using Kaplan Meier method and tested using logrank and cox regression models.
Results:
The median age was 60 (IQR 24-86) and majority were HPV negative (64%). High densities and clustering of cytotoxic CD8+ T cells and CD68+ tumor associated macrophages (TAMs) were each associated with improved OS (126 vs 61 months, p= 0.03; 140 vs 61 months, p= 0.04, respectively]. Conversely, high densities of antigen-experienced CD8+PD-1+ cytotoxic T cells and antigen-experienced CD68+PD-1+ macrophages were associated with worse OS [HR 1.44 (1-2.05), p= 0.04 and HR 1.48 (1.04-2.12), p=0.03 respectively], PFS [HR 1.45 (1.03-2.05), p= 0.03 and HR 1.53 (1.06-2.19), p= 0.02 respectively ] and CSS [HR 1.52 (1.03-2.24), p= 0.03 and HR 1.63 (1.12-2.36), p= 0.01 respectively]. Clustering of CD86+ M1 macrophages was associated with improved OS (104.5 vs 29.1 months, p=0.02), while clustering of CD163+ and CD206+ M2 macrophages correlated with decreased OS (67.8 vs 199.7 months, p=0.03; 100.6 vs NA months, p=0.01 respectively). Bivariate analysis revealed improved OS associated with co-clustering of CD8+ T cells with TAMs (104 vs 42 months, p=0.03). Similarly, co-clustering of CD8+T cells with CK+ tumor cells was associated with improved OS (140 vs 61 months; p=0.02). While co-clustering of CD86+ M1 macrophages to cytokeratin (CK+) tumor cells correlated with improved OS (126 vs 42 months, p=0.03), co-clustering of CD 206+ M2 macrophages to CK+ tumor cells associated with poor RFS (48 vs NA months, p=0.03). Co-clustering of antigen experienced T cells with TAMs was also associated with decreased RFS (29 vs NA months, p =0.03).
Conclusions:
Using spatial analysis, we showed that interaction between T cells and macrophages impact clinical outcomes in PSCC. High densities and proximity of CD8+ T cells and M1 macrophages, and low levels of antigen experienced T cells and macrophages were associated with improved survival in PSCC.