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Background: The composition and functional state of T-cell subpopulations can highly impact patient’s outcome and response to immune checkpoint therapy. However, only little is known about the spatial interplay of most rare T-cell subpopulations. Design: To assess the density, composition, degree of immune checkpoint expression, and spatial interplay of T-cell subpopulations in 5989 tumor samples from more than 100 tumor entities, two different types of tissue microarrays (0.6 mm and 4 mm in diameter) were stained with antibodies directed against CD3, CD4, CD8, FOXP3, T-bet, GATA3, RORyT, BCL6, FOXP3, CD56, CD45RA, CD45RO, TIM3, PD-1, CTLA-4 Granzym B, and Ki67 using our BLEACH&STAIN multiplex fluorescence immunohistochemistry approach. A deep learning-based framework comprising two different convolutional neuronal networks (U-Net and DeepLabv3+) was used for image analysis. Results: For identification and definition of immune cell subpopulations unsupervised X-shift clustering and 2D/3D t-distributed stochastic neighbor embedding (t-SNE) using the “Rtsne” package (Rtsne (RRID:SCR_016342)) were applied and revealed 102 T-cell subpopulations at certain functional state. Within these subpopulations, the well-characterized expression profiles were visually matched with single T-cell expression profiles and documented as digital images. This process resulted in 12 main T-cell subsets that were further subclassified according to their functional state (proliferation, immune checkpoint expression) and studied according to their spatial orchestration. Interestingly, the vast majority of T-cell subsets were found in all analyzed tumor entities. However, their spatial orchestration, immune checkpoint expression profile was highly variable between different tumor entities. Conclusion: This study provides a comprehensive overview of rare T-cells subpopulations and its spatial orchestration in more than 100 different tumor entities. Citation Format: Zhihao Huang, Elena Bady, Jan H. Müller, Tim Mandelkow, Magalie C. Lurati, Ronald Simon, Christian Bernreuther, Frank Jacobsen, Guido Sauter, Katharina Möller, Andreas Luebke, Andrea Hinsch, Till S. Clauditz, Eike Burandt, Niclas C. Blessin. Spatial T-cell atlas in more than 100 different tumor entities using BLEACH&STAIN. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5144.

Multiplex fluorescence immunohistochemistry (mfIHC) approaches were yet either limited to 6 markers or limited to a small (1.5cmx1.5cm) tissue size that hampers translational studies on large tissue microarray (TMA) cohorts. To be able to assess more marker in a large patient cohort, we have developed a BLEACH&STAIN multiplex fluorescence immunohistochemistry approach that enabled the simultaneous analysis of 15 biomarkers. To study the relationship of PD-L1 expression on multiple different cell types and the relationship with various lymphocyte subtypes, PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, and CD31 were analyzed in 3098 tumor samples from 44 different tumor types. An artificial intelligence-based framework - incorporating three different deep learning systems - for automated marker quantification on tumor as well as immune cells was further established to study the spatial interplay between PD-L1 expression and the composition of tumor infiltrating leucocytes (TILs). Comparing the automated deep learning-based PD-L1 quantification with conventional brightfield PD-L1 data revealed a high concordance in tumor cells (p Citation Format: Elena Bady, Katharina Möller, Tim Mandelkow, Ronald Simon, Maximilian Lennartz, Claudia Hube-Magg, Guido Sauter, Niclas C. Blessin. BLEACH&STAIN 15 marker multiplexed imaging in 3098 human carcinomas revealed six major PD-L1 driven immune phenotypes with distinct spatial orchestration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 597.

The programmed death protein 1 (PD-1) and his ligand PD-L1 are frequently expressed in human cancers and immune checkpoint inhibitor therapies targeting the PD1/PD-L1 pathway are increasingly employed in a growing number of tumor types. However, a unanimous picture on PD-L1 expression in cancer cells and immune cells in various cancer types are so far lacking. In this study we analyzed PD-L1 expression in 5,561 tumor samples by immunohistochemistry in a highly standardized way to define the relative importance of PD-L1 expression and its relationship with tumor infiltrating CD8 positive lymphocytes across 87 human tumor types and subtypes. At a cut-off level of 10% positive tumor cells, PD-L1 positivity was seen in 62 of 87 (71%) tumor types. More than 50% PD-L1 positive cases were found in 7 (8%) tumor types, including thymoma (100%), Hodgkin lymphoma (93%), anaplastic thyroid carcinoma (67%), as well as squamous cell carcinomas of the penis (67%), cervix (65%), oral cavity (61%), and pharynx (50%). PD-L1 immunostaining in tumor cells was absent in 25 (29%) tumor types, including non-Hodgkin lymphomas, seminoma, endometrial malignant mixed Müllerian tumors, and mucinous carcinoma of the ovary. In immune cells, PD-L1 positivity was detectable in 79 (91%) tumor types, with the highest positivity rates in tumors of haemotopoetic and lymphoid tissues (75% to 100%), seminoma (85%), Warthin tumors of the parotid glands (83%), and Merkel cell carcinoma (82%). Immune cell PD-L1 positivity was absent in only 8 (9%) tumor types (e.g. chondosarcoma, pleomorphic adenoma of the salivary gland, and chromophobe renal cell carcinoma). PD-L1 positivity in tumor cells was significantly correlated with the number of intratumoral CD8 positive lymphocytes across all tumor types as well as in the separate analyses of several individual tumor types, such as basal cell carcinoma (p Citation Format: Katharina Möller, Ronald Simon, Martina Kluth, Guido Sauter, Claudia Hube-Magg, Christoph Fraune, Niclas Blessin, Maximilian Lennartz, Doris Höflmayer, Sarah Minner, Eike Burandt, Sören Weidemann, Andreas Luebke. PD-L1 expression in human tumors: a tissue microarray study on 5,561 tissue samples and 87 tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2775.

Mesothelin is a glycoprotein which is normally expressed in only few non-vital tissues. Due to its membranous location and expression in various cancer types, it represents an attractive molecule for target-specific cancer therapies. To examine, what tumor types might benefit most from anti-mesothelin therapies, a set of tissue microarrays containing samples from 13,298 tumors from 122 different tumor types and 608 samples from 76 different normal tissue categories were analyzed for mesothelin expression by immunohistochemistry. Sixty-six (54%) of these 122 tumor types and subtypes showed at least occasional weak staining, including 49 tumor types with at least one strongly positive sample. Tumor types with highest prevalence of mesothelin positivity included ovarian cancers (serous 97%, clear cell 83%, endometroid 77%, mucinous 71%, Müllerian mixed tumors 65%), pancreatic adenocarcinoma (81%), endometrial cancers (clear cell 71%, serous 57%, Müllerian mixed tumors 50%, endometroid 45%), malignant mesothelioma (71%), and lung adenocarcinoma (55%). Mesothelin positivity was particularly rare or absent in urothelial carcinoma (10% positive out of 925), breast cancer (6% of 1,285), renal cell cancer (7% of 1,049), adenocarcinoma of the prostate (0 of 498), thyroid carcinomas (0 of 238), and soft tissue tumors (0 of 920). Significant associations with patient outcome or parameters of malignancy were not observed in 1,072 breast cancers of no special type, 925 urinary bladder cancers, 386 serous ovarian cancers, 266 lung cancers or 373 stomach cancers. Taken together, our systematic and highly standardized analysis identified numerous clinically important cancer types with frequent and high level mesothelin expression. These tumor types might benefit most from future anti-mesothelin therapies. The prognostic relevance of mesothelin appears to be low in the analyzed tumor types. Citation Format: Sören Weidemann, Natalia Gorbokon, Doris Höflmayer, Katharina Möller, David Dum, Andreas Luebke, Martina Kluth, Claudia Hube-Magg, Niclas Blessin, Guido Sauter, Jakob Izbicki, Ronald Simon, Stefan Steurer, Andreas Marx, Till Krech, Kristina Jansen, Maximilian Lennartz. Mesothelin expression in human tumor types: a tissue microarray study on more than 13,000 tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2833.

Immune checkpoint inhibitor (CPI) therapies targeting the PD-1/PD-L1 pathway have shown remarkable results in a rising number of different tumor entities. The likelihood of a positive response rate to CPIs depends - amongst other factors - on the PD-L1 expression level in tumor cells and tumor infiltrating immune cells. PD-L1 expression on different cell types is known to impact both the function and the composition of immune cells in the tumor microenvironment. However, this process is not fully understood. To study the impact of PD-L1 expression on the intratumoral immune environment, a multiplex fluorescence immunohistochemistry approach was used enabling the simultaneous analysis of 15 different antibodies on a set of tissue microarrays containing samples from more than 10000 neoplasms from more than 100 different tumor types and subtypes. Image analysis was performed using a U-net deep learning algorithm for cell detection and the “R” software package for statistical analysis. PD-L1 expression was measured on tumor cells (AE1/AE3+), subsets of macrophages (CD68+CD163+/CD68+iNOS+) and dendritic cells (CD11c+). To address the complexity of the tumor microenvironment, PD-1 expression was measured on B-cells (CD20+) and T-cell subsets, including cytotoxic T-cells (CD3+CD8+), T-helper cells (CD3+CD4+), regulatory T-cells (CD3+CD4+FOXP3+) and their memory (CD45RO+), activated (CD7-) or expanding (ki67+) phenotypes. Although PD-L1 expression varied significantly between cell types, patients and different tumor entities, the number of PD-1+ T-cells was consistently linked to PD-L1 expression. Combined analysis of cell densities, expression patterns, intensity measurements, interaction and distance analysis between immune cells and tumor cells revealed distinct changes in the immune cell infiltration pattern linked to PD-L1 expression. Previously uncharacterized immune cell-composition dynamics in clustered tumor phenotypes, according to the PD-L1 expression, were detected. This included significant associations between PD-L1+ tumor cells and PD-L1+ antigen-presenting cells and various PD-1+ T-cell subsets. In conclusion, deep profiling of 15 biomarkers in 10 000 cancers revealed complex differences in the composition of tumor infiltrating immune cells depending on the PD-L1 expression level of tumor cells and the mononuclear phagocyte system. Citation Format: Niclas Christian Blessin, Cheng Yang, Jonas Raedler, Julia Ebner, Tim Mandelkow, Ronald Simon, Christoph Fraune, Maximilian Lennartz, Andreas M. Luebke, Sarah Minner, Eike Burandt, Doris Höflmayer, Guido Sauter, Katharina Möller, Sören A Weidemann. Deep profiling of the PD-1/PD-L1 pathway in 10000 cancers revealed changes in the immune cell composition between cancer entities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2773.

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. 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. 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 Citation Format: Maximilian Lennartz, Raed Abu-Hashem, Niclas Christian Blessin, Tim Mandelkow, Elena Bady, Ronald Simon, Claudia Hube-Magg, Guido Sauter, Christoph Fraune, Katharina Möller, Till S. Clauditz, Andreas M. Luebke, Franziska Büscheck. Prevalence of proliferating CD8+cells in normal lymphatic tissues, inflammation and cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4979.

Expression of inhibitory immune checkpoint receptors such as CD112R (Poliovirus Receptor-Related Immunoglobulin Domain-Containing Protein, PVRIG) and PD-1 on CD8+ lymphocytes are considered to indicate an “exhausted” stage of T cell activation while Ki67 protein expression in these cells is viewed as sign of activity or expansion. To assess the prognostic impact of the clinical impact of CD112R, PD-1, and Ki67 expression of CD8+ lymphocytes, expression of these proteins was analyzed by fluorescent multiplex immunohistochemistry on a tissue microarray containing 384 colorectal cancers with clinical follow-up data. An elevated fraction of CD8+Ki67+ cells was linked to the absence of nodal metastasis and longer survival in these tumors (p≤0.0039 each). CD112R and PD-1 expression was detected at variable intensity levels and were separately quantitated in both ki67+ (expanding) and Ki67 negative (non-expanding) CD8+ cytotoxic lymphocytes. Elevated PD-1 expression in CD8+ T cells was associated with an absence of nodal metastasis (p=0.004) and longer overall survival in all CD8+ cells (p=0.005). This association was more evident in the subset of expanding (p Citation Format: Ronald Simon, Niclas Christian Blessin, Tim Mandelkow, Elena Bady, Claudia Hube-Magg, Guido Sauter, Maximilian Lennartz, Christoph Fraune, Sören A. Weidemann, Katharina Möller, Franziska Büscheck. Prognostic role of CD112R, PD-1 and Ki67 expression in CD8+cytotoxic T cells in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4970.

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. 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 (4mm tissue spot diameter) and large sections using fluorescent multiplex immunohistochemistry. 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. 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. Citation Format: Niclas Christian Blessin, Tim Mandelkow, Elena Bady, Claudia Hube-Magg, Guido Sauter, Ronald Simon, Christoph Fraune, Maximilian Lennartz, Sören A. Weidemann, Katharina Möller, Doris Höflmayer. Patterns of CD112R expression in normal lymphatic tissues, inflammation and cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3870.

Evidence suggests that the quantity of cytotoxic lymphocytes influences the likelihood for a successful application of immune checkpoint inhibitors. To compare the density of CD8+lymphocytes across various different tumor types, a tissue microarray (TMA) composed of up to 50 tumor samples each from 85 different cancer types and subtypes was analyzed. A total of 2652 cancers and 608 normal tissues were successfully analyzed by CD8 immunohistochemistry followed by automated image analysis of digitized slides. The median number of CD8+lymphocytes ranged from 6 cells/mm2in pleomorphic adenoma up to 1573 cells/mm2in Hodgkin’s lymphoma. CD8 counts were generally lower in normal tissues. Blood vessels of the spleen was the only non-lymphatic tissue staining for CD8.In solid tumors, highest CD8 densities (cells/mm2) were found in seminoma (median: 424), Warthin’s tumor (median: 425), squamous cell cervical cancer (median 468), medullary breast cancer (median: 657) and thymoma (median: 889).Tumor types approved for therapy with checkpoint inhibitors such malignant melanoma (median: 81), muscle invasive urothelial carcinomas (median: 119), small cell lung cancer (median: 120), clear cell kidney cancer (median: 153), squamous cell cancer (median: 189) and adenocarcinoma of the lung (median: 328) as well as Hodgkin’s lymphoma (median:1573) were all ranking among the upper half of our list. Comparably high CD8 densities (cells/mm2) were also found for several rare and aggressive cancer types including Merkel cell carcinoma (median: 70), angiosarcoma (median: 95), anaplastic thyroid cancer (median: 156), anal carcinoma (median: 104), squamous cell carcinoma of the vagina (median: 128) and embryonal carcinoma of the testis (median: 186). The CD8 cell count was highly variable within tumor types. In 73 of 84 analyzed cancer types, the CD8 count at least occasionally exceeded the average CD8 count of tumors for which checkpoint inhibitors have been approved.These data support the concept, that in most tumor types at least some individual cancers may benefit from treatment with immune checkpoint inhibitors. Citation Format: Niclas C. Blessin, Florian Lutz, Patrick Spriesterbach, Wenchao Li, Tim Mandelkow, Vera Nickelsen, Ronald Simon, Claudia Hube-Magg, Florian Viehweger, Maximillian Lennartz, Christoph Fraune, Kristine Fischer, Katharina Möller, Stefan Steurer, Jacob R. Izbicki, Guido Sauter, Sarah Minner, Frank Jacobsen, Andreas M. Luebke, Franziska Büscheck, Doris Höflmayer, Waldemar Wilczak, Eike Burandt, Andrea Hinsch. Distribution of CD8+cytotoxic lymphocytes in human neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4567.