Alterations in tumor immune microenvironment and progressive immune exhaustion with advancing penile squamous cell carcinoma using multiplex immunofluorescence and image analysis.
Authors: Gullapalli Keerthi , Ionescu Filip , Roman Souza Gabriel , Paravathaneni Mahati , Eschrich Steven A. , Kim Youngchul , Whiting Junmin , Grass G. Daniel , Jain Rohit K. , Segura Carlos Moran , Nguyen Jonathan , Yu Alice , Zemp Logan , Spiess Philippe E. , Chahoud Jad 
Published: January 2024

9 Background: Penile squamous cell carcinoma (PSCC) is a rare and aggressive malignancy with limited treatment options in the advanced or recurrent settings. Immunotherapy can yield clinical responses in PSCC, but less than 20% of patients benefit. Herein, using multiplex immunofluorescence (MIF), we analyzed the composition of the tumor immune microenvironment as it varies with human papillomavirus (HPV) status and across advancing disease stages with an aim to further our understanding of how immune composition may impact clinical outcome, potential role in disease progression and response to immune therapies. Methods: Formalin-fixed and paraffin-embedded tissue samples from men with PSCC treated at Moffitt cancer center (Tampa, FL) were immunostained for CD3, CD4, CD8, CD68, PD-1, PD-L1, CD163 and CD 206 using OPAL TM 7 kit (AKOYA Biosciences) for MIF. Densities of various cell phenotypes (expressed as cells/mm2) were quantified using an automated quantitative image analysis system (InForm). Immune cell phenotype densities were stratified by HPV status and by tumor stage. We will apply the nearest neighbor cell analysis to identify the spatial orientation of immune cells in the tumor microenvironment. Results: 57 PSCC patients (median age, 60 years [IQR (interquartile range) 31-92]; 60% HPV negative) had MIF analysis. More than half (31/57) had lymph nodes involved (6 N1, 25 N2-3). The immune composition did not differ significantly with HPV status. When investigated by individual clinical stage, we observed an increase in median density of total (CD3+), helper (CD3+CD4+), cytotoxic (CD3+CD8+) and CD3+PD-L1+ T cells from N0 to N1 stage (pN0: CD3+ 44.23, CD3+CD4+ 14.77, CD3+CD8+ 16.15 cells/mm2; pN1: CD3+ 188.36, CD3+CD4+ 71.69, CD3+CD8+ 40.22 cells/mm2) followed by a decrease in N2-N3 stage (CD3+CD4+ 18.45 and CD3+CD8+ 28.22 cells/mm2).The median density of total macrophages increased with stage (pN0: 306.38; pN1: 502.47; pN2-3: 648.27 cells/mm2). While the activated M1 macrophages increased from N0 to N1 and decreased in the more advanced N2-3 stage, M2 macrophages steadily increased across stages and became the dominant type in N2-3. Conclusions: This study describes the interplay of T cells and macrophages across disease stages using MIF. We observed an initial T cell and myeloid immune response in the early locoregional stage, followed by the emergence of immune exhaustion, marked by a decline in the density of cytotoxic T cells, rising PD-L1 expression, and the progressive replacement of anti-tumor M1 macrophages with pro-tumorigenic M2 macrophages across stages. Our findings can inform treatments utilizing immune manipulation. Geospatial analysis exploring the proximity relationships of different immune cell types to each other and to the tumor is ongoing and will be presented at the meeting.