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Abstract
Background Penile squamous cell carcinoma (PSCC) is rare in the US accounting for 0.7% of the total cancer incidence. Around 50% of PSCC cases are associated with HPV infection and between 40–60% have high PD-L1 expression. The identification of the immune landscape in PSCC using the spatial proximity between tumor cells and immune phenotypes has not been described yet.
Methods We performed multiplex immunofluorescence (mIF) on 54 formalin-fixed, paraffin-embedded tissue sections of PSCC. The staining was performed with the opal-7 kit (Akoya/PerkinElmer) in the Leica Bond RX autostainer using 7 markers against, Cytokeratins, CD3, CD8, CD68, PD-1, and PD-L1. The slides were scanned in the multispectral microscopy Vectra Polaris (Akoya/PerkinElmer), and 5 regions of interest (ROI) per case were selected and analyzed using the digital image analysis software InForm 2.2.4. The X and Y coordinate from each cell phenotype was obtained from each ROI. Nearest neighbor median distance and the infiltration analysis using the G-function metric of the different tumor associated immune cells (TAICs) within a distance of 20, 40 and 60 microns from the tumor cells were obtained through the R studio software and correlated with available clinical information.
Results Using the nearest neighbor cell analysis we identified the distance of ≤87.33 microns as a close median distance from tumor cells to the multiple TAICs. In our cohort of PSCC tumors, the closest TAIC phenotype to tumor cells was the CD3+ T cells with a median distance of 20.13 microns, followed by macrophages CD68 (median distance, 21.19 microns) and cytotoxic T cells (CD3+, CD8+) with a median distance of 36.09 microns, compared with the others TAICs located farther than 87.33 microns (table 1, figure 1). Interestingly, cytotoxic T cells (median distance, 59.5 micros), T-cells (median distance, 65.6 microns) and macrophages expressing PD-L1 (median distance, 61.2 microns) are located closer to tumor cells expressing PD-L1 than from tumor cells not expressing PD-L1 (median distance, 104.08, 116.05 and 118.74 microns, respectively). Unexpectedly, HPV negative patients had closer cytotoxic T cells CD3+CD8+ median distance, 30.88 microns) and cytotoxic T cells antigen experienced CD3+CD8+PD-1+ (median distance, 50.09 microns), compared to HPV positive patients (median distance of 36.09 and 59.83 microns, respectively). Additionally, the G-function AUC metric from tumor cells to macrophages CD68 showed high interaction at 40 microns in HPV cases when compared with others distances and not HPV cases (P=0.049, figure 2).
Median distance from tumor cells to different tumor associated immune cells (TAIC)
Heat map of median distances (μm) from tumor to tumor associated immune cells (TAIC) with HPV status
G function of tumor cells to CD68+ macrophages vs HPV status
Conclusions Although, higher densities of cytotoxic T cells were observed relatively closer from tumor cells than others TAIC subsets, we didn’t find strong interaction with this subset in using the G-function AUC metric. The PD-1/PD-L1 axis was also found in close proximity suggesting that there are more likely to interact with tumor cells generating a strongly immunosuppressive microenvironment. In addition, while CD68+ macrophages were found to be closely associated with tumor cells, PD-L1+ macrophages were found to have the closest interaction with tumor cells. The potential of these cell phenotypes to generate a strongly immunosuppressive microenvironment need to be explored in additional cases.
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