Introduction What’s already known?

1. Marked differences in the density, composition and microanatomical distribution of infiltrating immune cells in cutaneous squamous cell carcinoma and the peritumoral tissue of immunosuppressed organ transplant recipients compared to immunocompetent patients S.B. Strobel1, K. Safferling2,3, B. Lahrmann2,3, J.H. Hoffmann1, A.H. Enk1, E.N. Hadaschik1,4, N. Grabe2,3 and A.S. Lonsdorf1 1 Department of Dermatology, Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany 2 Hamamatsu Tissue Imaging and Analysis Center, BIOQUANT, Heidelberg, Germany 3 Department of Medical Oncology, National Center for Tumor Diseases Heidelberg, Ruprecht-Karls-University of Heidelberg, Heidelberg, Germany 4 Department of Dermatology, University of Essen, Essen, Germany British Journal of Dermatology. DOI: 10.111/bjd.16477

2. Introduction What’s already known? • Long-term immunosuppression is a major risk factor for skin carcinogenesis • Cutaneous squamous cell carcinoma (SCC) arising in immunosuppressed organ transplant recipients (OTR) show a considerably increased incidence and more aggressive disease progression compared to immunocompetent patients (IC) • The inflammatory tumor microenvironment, in particular the composition and localization of infiltrating immune cells, may alter tumor behavior and affect the clinical course of several types of cancer, including SCC

3. Objective To determine the density and phenotype of immune cells infiltrating SCC and surrounding skin in OTR, and to characterize the microanatomical distribution patterns in comparison to IC

4. Methods Patient cohorts 20 renal transplant patients (OTR) with an SCC were carefully matched with 18 immunocompetent patients (IC) based on clinical and histopathological features such as gender, age at SCC diagnosis (± 5 years), tumor location and tumor grading. The basic clinical and tumor-related characteristics of the analyzed cohorts are shown.

5. Methods A Immunohistochemical staining (IHC) and regional semi-automated analysis of whole tissue section Analyses of immune cell infiltrates within SCC and at defined regions of interest (ROI) of tumor- surrounding skin in formalin-fixed paraffin-embedded (FFPE) tissue by high-resolution semi-automated microscopy on complete tissue sections stained for CD4, CD8, CD20 and CD68. ROI: tumor area (blue), IM500 (red), IM1500 (orange) and tissue (green) Figure A: original magnification upper panels 200x, lower panels 800x. Figure B: original magnification left panels 20x, right panels 200x. B

6. Results Overall immune cell densities in the different regions of SCC and peritumoral tissue of OTR and IC • Significant reduction of the overall immune cell density in all ROI in OTR compared to IC • Tumors display the overall lowest density of infiltrating leucocytes in both groups compared to the surrounding, non-tumoral tissue sites • Significant reduction of the immune cell density at the invasive margin (IM500) of SCC arising in OTR compared to IC (p= 0.0003) • Also surrounding non-tumoral skin of IC contained significantly more immune cells compared to OTR (p= 0.0071)

7. Density and microanatomical distribution of different immune cell subsets in SCC and peritumoral tissue of OTR and IC • The most abundant tumour-infiltrating leucocytes (TIL) in both groups are CD4+ • Detection of the overall lowest numbers of CD8+ T cells in SCC of OTR compared to SCC from IC as well as compared to all other analyzed ROI of the respective cohort • Significant reduction of the CD20+ B cell density at all ROI in OTR specimens compared to IC (p= <0.0001) • No significant difference in the density and distribution of CD68+ mononuclear histiocytes between the two groups

8. Results

9. Discussion • Density of skin infiltrating immune cells in tissue from OTR is overall reduced compared to IC, particularly at the tumor borders • Previous studies suggested unchanged proportions of CD3+ T cells and CD4+ infiltrating leucocytes in the intra- and peritumoral infiltrate of OTR and IC. We found that the peritumoral tissue surrounding SCC of immunosuppressed OTR, harbors distinct changes in the local microenvironment compared to respective tissue sites in IC • In this study, CD4+ infiltrates at the directly invasive margin and tumor vicinity were significantly reduced in tissue from OTR compared to IC, while the density of CD4+ infiltrates within the tumor and in the peripheral skin did not differ significantly between the groups.

10. Discussion • The density of CD8+ T cells within the SCCs and surrounding skin of OTR was significantly diminished • The significantly reduced CD20+ B cells in tissue from OTR, analogous to significantly reduced CD8+ T cells detected at corresponding intra- and peritumoral tissue sites, may suggest a field effect of neoplastic progression and contribution to the aggressive clinical course of SCC in OTR • Immunosuppressants may influence the ability of immune cells to accumulate in the skin and position themselves at the site of a growing SCC in order to detect and defend against cancer • Additional studies must be done to learn more about the functional relevance of the particular immune cell subsets for the control of skin cancer

11. ConclusionsWhat does this study add? • Long-term iatrogenic immunosuppression markedly disturbs the composition and microanatomical distribution of intra- and peritumoral immune infiltrates of SCC • The overall reduced density of infiltrating adaptive immune cells, particularly reduced CD4+ infiltrates at the invasive margin of SCC and reduced tumor-infiltrating CD8+ T cells, may contribute to the aggressiveness of SCC arising in OTR • The cutaneous CD20+ B cell compartment at the site of a growing SCC is significantly affected by systemic immunosuppression

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