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Background Regulatory FoxP3+ lymphocytes function as suppressors of T-cell activity. The clinical impact of high FoxP3+ cell density in cancers is not fully understood, as some studies have linked high FoxP3+ cell density to good prognosis and others to poor prognosis in tumor cohorts with associated clinical data. While some data suggest that these variable data are due to biological differences between tumor entities, it is also possible that methodological differences have caused these discrepancies. This study was undertaken to analyze the density of FoxP3+ cells in various different cancer types by employing standardized methods. Materials and Methods Tissue microarrays and large sections made from >20,000 prostate, breast, colorectal, ovarian, pancreatic, bladder and stomach cancers were analyzed together with various normal and inflamed tissues by conventional brightfield FoxP3 immunohistochemistry. Samples were also analyzed by fluorescent multiplex immunohistochemistry to assess the fraction of Ki67+ FoxP3+ cells. Results Our results indeed suggested a variable role of FoxP3+ cells in different tumor types. High FoxP3+ density was linked to high Gleason grade (p=0.0003) and early biochemical recurrence (p Conclusions Our data demonstrate an inverse prognostic impact of the FoxP3+ cell density in prostate and breast cancers. The increased proliferation rate of immune-regulatory FoxP3+ cells in some bladder cancer is interesting in the light of the variable response of these tumors to immune checkpoint inhibitors. Disclosure Information T. Mandelkow: None. E. Bady: None. N.C. Blessin: None. C. Hube-Magg: None. R. Simon: None. G. Sauter: None. C. Fraune: None. M. Lennartz: None. K. Moller: None. S.A. Weidemann: None. A.M. Luebke: None. D. Hoflmayer: None. F. Buscheck: None.

Background CD112R is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies, but little is known about its molecular epidemiology in healthy and diseased tissues. Materials and Methods To study the prevalence and expression level of CD112R+ immune cells, we analyzed more than 200 samples of normal lymphatic, inflamed and cancerous tissues in a microenvironment tissue microarray format (4 mm tissue spot diameter) and large sections using fluorescent multiplex immunohistochemistry. Results CD112R expression was detected at variable intensity levels in 47% of CD8+ cytotoxic lymphocytes, 49% of CD4+ T helper cells, 30% of FOXP3+ regulatory T helper cells and in 25% of CD56+ natural killer cells, but no expression was seen in CD11c+ dendritic cells and CD68+ macrophages. All analyzed compartments across normal and diseased tissues showed a small subset (CD8: 9±18%, CD4: 5±15%, FOXP3: 2±5%) of immune cells with supramaximal CD112R expression. The highest fraction of cells with supramaximal CD112R expression was found in the subset of CD8+ cytotoxic T cells in the Peyer’s patches of ileum (62%), the intergranuloma area of lymph node sarcoidosis (27%) and in ovarian cancer (37%). In cancerous tissues, the density and the fraction cytotoxic T cells with supramaximal CD112R expression was highly variable and ranged from 5% in bladder cancer to 3% in lung cancer and 36% in ovarian cancer. A high variability of the number of cells with supramaximal CD112R expression was also seen within every tumor entity. Conclusions In summary, our analysis shows that CD112R expression is abundant in various subsets of immune cells but identifies a small fraction of cells with exceedingly high CD112R levels. The widespread occurrence of CD112R+ cytotoxic T cells in the cancer microenvironment may suggest considerable opportunities for checkpoint inhibitors targeting CD112R. Disclosure Information N.C. Blessin: None. T. Mandelkow: None. E. Bady: None. C. Hube-Magg: None. R. Simon: None. G. Sauter: None. C. Fraune: None. M. Lennartz: None. K. Moller: None. D. Hoflmayer: None. S.A. Weidemann: None.

Background Expansion of CD8+ cytotoxic T lymphocytes is a prerequisite for anti-cancer immune activity. In the era of immune checkpoint therapy, profound knowledge of the dynamics of CD8+ has regained considerable interest. However, systematically acquired data on CD8+ proliferation in large sets of normal and diseased tissues are sparse. Materials and Methods Here, we applied multiplex fluorescence immunohistochemistry to conventional large sections and tissue microarrays in order to quantitate Ki67+CD8+ cells in >20 different compartments of normal lymphoid tissues, 7 types of inflammatory diseases and 785 cancers. Results In most normal lymphoid tissues (tonsil, lymph node, thymus, Peyer’s patches, spleen, colon, appendix) the percentage of Ki67+CD8+ cells typically did not exceed 3%. The percentage of Ki67+CD8+ cells was markedly higher (45%) in the immune-active cortex of the thymus, however. In inflammatory conditions (including Hashimoto thyroiditis, Lichen sclerosus of the penis, sarcoidosis, sialadenitis, IgG4 pancreatitis, Crohn’s disease and eczema), the percentage of Ki67+CD8+ cells was much more variable and often sharply higher than in normal tissues. It ranged from 0.5% in one patient with sialadenitis to 19% in the intraepithelial compartment of Crohn’s disease. In 765 colorectal cancers, the fraction of Ki67 positive CD8+ cytotoxic T cells ranged from 0 to 100% (mean: 20.6%). A high fraction of Ki67+CD8+ cells was significantly associated with microsatellite instability (p Conclusions In summary, our data show a variable increase of the fraction of proliferating CD8+ T cells in cancers and in inflammatory diseases as compared to healthy secondary lymphoid organs. The striking link with microsatellite instability and unfavorable tumor features suggest a potential clinical utility of assessing Ki67+CD8+ in colorectal cancer. Disclosure Information K. Moller: None. M. Lennartz: None. R. Abu-Hashem: None. N.C. Blessin: None. T. Mandelkow: None. E. Bady: None. C. Hube-Magg: None. R. Simon: None. G. Sauter: None. C. Fraune: None. T.S. Clauditz: None. F. Buscheck: None. A.M. Luebke: None.