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Multiplex fluorescence IHC (mfIHC) approaches were yet either limited to six markers or limited to a small tissue size that hampers translational studies on large tissue microarray cohorts. Here we have developed a BLEACH&STAIN mfIHC method that enabled the simultaneous analysis of 15 biomarkers (PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, and CD31) in 3,098 tumor samples from 44 different carcinoma entities within one week. To facilitate automated immune checkpoint quantification on tumor and immune cells and study its spatial interplay an artificial intelligence–based framework incorporating 17 different deep-learning systems was established. Unsupervised clustering showed that the three PD-L1 phenotypes (PD-L1+ tumor and immune cells, PD-L1+ immune cells, PD-L1−) were either inflamed or noninflamed. In inflamed PD-L1+patients, spatial analysis revealed that an elevated level of intratumoral M2 macrophages as well as CD11c+ dendritic cell (DC) infiltration (P < 0.001 each) was associated with a high CD3+ CD4± CD8± FOXP3± T-cell exclusion and a high PD-1 expression on T cells (P < 0.001 each). In breast cancer, the PD-L1 fluorescence intensity on tumor cells showed a significantly higher predictive performance for overall survival (OS; AUC, 0.72, P < 0.001) compared with the commonly used percentage of PD-L1+ tumor cells (AUC, 0.54). In conclusion, our deep-learning–based BLEACH&STAIN framework facilitates rapid and comprehensive assessment of more than 60 spatially orchestrated immune cell subpopulations and its prognostic relevance. Implications: The development of an easy-to-use high-throughput 15+1 multiplex fluorescence approach facilitates the in-depth understanding of the immune tumor microenvironment (TME) and enables to study the prognostic relevance of more than 130 immune cell subpopulations.

BACKGROUND: Programmed death ligand 1 (PD-L1) is the target of immune checkpoint inhibitor therapies in a growing number of tumor types, but a unanimous picture on PD-L1 expression across cancer types is lacking. MATERIALS AND METHODS: We analyzed immunohistochemical PD-L1 expression in 11,838 samples from 118 human tumor types and its relationship with tumor infiltrating CD8 positive lymphocytes. RESULTS: At a cut-off level of 10% positive tumor cells, PD-L1 positivity was seen in 85 of 118 (72%) tumor types, including thymoma (100% positive), Hodgkin’s lymphoma (93%), anaplastic thyroid carcinoma (76%), Kaposi sarcoma (71%), sarcomatoid urothelial carcinoma (71%), and squamous cell carcinoma of the penis (67%), cervix (65%), floor of the mouth (61%), the lung (53%), and pharynx (50%). In immune cells, PD-L1 positivity was detectable in 103 (87%) tumor types, including tumors of haematopoetic and lymphoid tissues (75% to 100%), Warthin tumors of the parotid glands (95%) and Merkel cell carcinoma (82%). 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 individual tumor types, including serous carcinoma of the ovary, invasive breast carcinoma of no special type, intestinal gastric adenocarcinoma, and liposarcoma (p< 0.0001 each). CONCLUSIONS: PD-L1 expression in tumor and inflammatory cells is found in a wide range of human tumor types. Higher rates of tumor infiltrating CD8 positive lymphocytes in PD-L1 positive than in PD-L1 negative cancers suggest that the antitumor immune response may trigger tumoral PD-L1 expression.

CD8+ cytotoxic T-lymphocytes are essential components of the anti-tumor immunity. To better understand the expansion of CD8+ T-cells we used multiplex fluorescence immunohistochemistry to study Ki67+CD8+ cells in normal lymphoid tissues, selected inflammatory diseases and cancers in 41 large sections/ microenvironment tissue microarrays (TMAs) as well as 765 samples in a conventional TMA format. The evaluation of more than 20 different compartments of normal lymphoid tissues revealed that the percentage of proliferating (ki67+) CD8+ cells did commonly not exceed 3%. In inflammations, the percentage of Ki67+CD8+ cells was more variable and higher compared to normal tissues. In cancers, the percentage of Ki67+CD8+ cells was higher in the tumor center than at the invasive margin. In the tumor center of 765 colorectal cancers, the density of Ki67+CD8+ cells and the percentage of proliferating CD8+ cytotoxic T-cells was significantly associated with microsatellite instability (p

Secreted frizzled-related protein 4 (SFRP4) controls WNT signaling and is thought to play a role for tumor aggressiveness. Here, we analyzed a tissue microarray containing 11,152 prostate cancers with pathological, clinical and molecular data by immunohistochemistry. SFRP4 expression was higher in cancer than in non-neoplastic acinar cells. SFRP4 staining was seen in 64.9% of tumors and classified as weak in 33.2%, moderate in 23.9% and strong in 7.8% of cancers. SFRP4 overexpression was linked to advanced tumor stage, high classical/quantitative Gleason grade (p

Background: Mesothelin is a glycosylphosphatidylinositol-anchored cell surface protein of widely unknown function that may play a role in tumor growth control. Because mesothelin is overexpressed in a variety of human cancer types including ovarian cancers, it represents an attractive target for novel therapies employing adaptive T-cell transfer in these tumors. However, tumor heterogeneity is a challenge for targeted therapies. Methods: To study the extent of heterogeneity of mesothelin overexpression in primary ovarian cancers and their peritoneal and lymph node metastases, a tissue microarray (TMA) was constructed from multiple sites of 220 ovarian cancers and analyzed by immunohistochemistry. One tissue core each was taken from up to 18 different tumor blocks per cancer, resulting in a total of 2,460 tissue spots from 423 tumor sites (188 primary cancers, 162 peritoneal carcinosis and 73 lymph node metastases). Results: Positive mesothelin expression was found in 2,041 of the 2,342 (87%) arrayed tissue spots and in 372 of the 392 (95%) tumor sites that were interpretable for mesothelin immunohistochemistry. Intratumoral heterogeneity was found in 23% of 168 primary cancer sites interpretable for mesothelin and decreased to 12% in 154 peritoneal carcinosis and to 6% in 71 lymph node metastases (p Conclusions: Our data demonstrate that mesothelin expression is frequent and highly homogeneous in ovarian cancer and prompt for future anti-mesothelin therapy studies in this tumor type. Citation Format: Sören Weidemann, Natalia Gorbokon, Maximilian Lennartz, Claudia Hube-Magg, Christoph Fraune, Christian Bernreuther, Till Clauditz, Frank Jacobsen, Kristina Jansen, Barbara Schmalfeldt, Linn Wölber, Peter Paluchowski, Enikö Berkes, Uwe Heilenkötter, Guido Sauter, Ria Uhlig, Waldemar Wilczak, Stefan Steurer, Ronald Simon, Till Krech, Andreas Marx, Eike Burandt. High homogeneity of mesothelin expressionin primary and metastatic ovarian cancer [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 3303.

Estrogen receptor alpha (ER) is a nuclear transcription factor with a pivotal role in the function of the reproductive organs and of other organ systems. It constitutes a well-established therapeutic target for various small molecules. ER immunohistochemistry is used in surgical pathology for the distinction of tumors of the breast and gynecological organs. To comprehensively determine ER expression in normal and neoplastic tissues, a tissue microarray containing 18,560 samples from 149 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. ER positivity was found in 55 of 149 tumor categories, and 27 of these tumor categories contained at least one case with strong ER staining. ER positivity was most seen in various subtypes of breast cancer (78.9-95.7%) and in several different tumor categories of the ovary and the endometrium (8.5-75.9%). Other commonly ER positive tumor entities included leiomyoma (65.9%), neuroendocrine tumor of the appendix (43.8%), leiomyosarcoma (22.9%), Sertoli Leydig cell tumor of the ovary (1 of 3 positive), rhabdomyosarcoma (16.7%), angiomyolipoma (10%), colorectal neuroendocrine carcinoma (9.1%), myoepithelial carcinoma of the salivary gland (8.3%), granulosa cell tumor of the ovary (8.3%), epithelial-myoepithelial carcinoma of the salivary gland (7.7%), squamous cell carcinomas of various sites of origin (0.8-6.7%), testicular teratoma (5.3%), Brenner tumor of the ovary (5.1%), Ewing sarcoma (5%), adenocarcinoma NOS of the salivary gland (4.8%), papillary thyroid carcinoma (3.3%), sarcoma, not otherwise specified (NOS) (2.8%), paraganglioma (2.6%), non-invasive papillary urothelial carcinoma, pTa G3 (2.4%), pulmonary adenocarcinoma (2.1%), neuroendocrine tumor of the ileum (2.1%), and adenocarcinoma of the prostate, Gleason 5+5 (1.2%). Among 1,411 evaluable invasive breast carcinomas of no special type (NST), absent or low ER immunostaining was significantly linked to high BRE grade (p Citation Format: Florian Viehweger, David Dum, Anne Menz, Ria Uhlig, Andrea Hinsch, Doris Hoeflmayer, Christoph Fraune, Christian Bernreuther, Patrick Lebok, Soeren Weidemann, Guido Sauter, Maximilian Lennartz, Frank Jacobsen, Till Sebastian Clauditz, Till Krech, Andreas H Marx, Ronald Simon, Stefan Steurer, Sarah Minner, Eike Burandt, Natalia Gorbokon. Patterns of estrogen receptor expression across 18,500 tumor samples obtained from 149 cancer types [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 2164.

Background: Automated prognosis marker assessment in prostate and breast cancer using immunohistochemistry is currently hampered by the lack of a reliable differentiation between benign and malignant glands. To evaluate the patient’s risk in routine clinical practice in prostate cancer prognosis parameters such as the Gleason grading, that are accompanied by a high interobserver variability are used. In breast cancer multi-gene panels are used that are influenced by fluctuating tumor purity. A reproducible prognostic evaluation is lacking in both tumor entities. Design: To enable automated prognosis marker quantification, we have developed and validated a framework for automated prostate and breast cancer detection that comprises three different artificial intelligence analysis steps and an algorithm for cell-distance analysis of multiplex fluorescence immunohistochemistry. Pan-cytokeratin (panCK) antibodies were used to detect epithelial cells and antibodies directed against Myosin and p63 were used to identify basal cells. Results: The optimal distance between Myosin+ and p63+ basal cells and benign panCK+ cells was identified as 25 µm in breast and 23 µm in prostate cancer and used to exclude benign glands from the analysis combined with several deep learning-based algorithms. Our framework discriminated normal glands from malignant glands with an AUC of 0.96 in breast and 0.98 % in prostate cancer. The approach for automated prostate and breast cancer detection, by excluding benign gland from the analysis, improved the predictive performance of prognosis markers significantly (p Conclusion: Automated prostate and breast cancer identification enables fully automated prognosis marker assessment in routine clinical practice using deep learning and multiplex fluorescence immunohistochemistry. BLEACH&STAIN as well as other multiplex fluorescence immunohistochemistry approaches that enable the simultaneous analysis of 20+ biomarkers can be used to established prognosis panels that can be now assessed in an automated manner. Citation Format: Tim Mandelkow, Gisa Mehring, Elena Bady, Maximilian Lennartz, Christoph Fraune, Frank Jacobsen, Natalia Gorbokon, Till Krech, Eike Burandt, Anne Menz, Ria Uhlig, Till S. Clauditz, Guido Sauter, Markus Graefen, Sarah Minner, Niclas C. Blessin. The combination of artificial intelligence and BLEACH&STAIN multiplex fluorescence immunohistochemistry facilitates automated prostate and breast cancer detection. [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 5438.

Background: A combination of different immune-checkpoint-inhibitors (ICIs) have shown remarkable success in several tumor entities. However, the likelihood of positive response to ICIs is poor in most tumor entities and recent evidence suggests that the quantity and the expression level of immune checkpoints such as TIM3, CTLA-4, PD-1 of tumor infiltrating lymphocytes (TILs) influences the likelihood of response to immune checkpoint inhibitors. Design: To assess the density and spatial interplay of 42 immune checkpoint expressing leukocyte subpopulations in 6031 tumor samples from 49 carcinoma (sub)entities two different types of tissue microarrays (0.6 mm and 4 mm in diameter) were stained with 21 antibodies 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: We found that the mean overall fraction of TIM3, PD-1, PD-L1 and CTLA-4 expression on M1, M2 macrophages, cD11c+ dendritic cells, CD8+ cytotoxic T-cells, CD4+ T-helper cells, FOXP3+ Tregs and CD20+ B-cells, ranged from 10% (sum of the fractions of immune checkpoint expression) in small cell carcinomas of the prostate up to 123 % in squamous cell carcinoma of the lung. Tumor types approved for checkpoint inhibitor therapy, including adenocarcinomas of the lung (121 %), small cell carcinoma of the lung (74%), malignant melanoma (59 %), and clear cell renal cell cancer (49%) were all ranking among the upper half of our list of mean fractions of overall immune checkpoint expression. Spatial analysis and the shift in immune cell compositions revealed that in most carcinoma (sub)entities, the overall immune cell density and fraction of immune checkpoint expression of individual patients occasionally exceeded the average immune cell density and fraction of tumors for which checkpoint inhibitors have been approved. Conclusion: These data support the concept that among most carcinoma entities at least some individual patients may benefit from treatment with immune checkpoint inhibitors. Citation Format: Zhihao Huang, Nicolaus F. Debatin, Elena Bady, Jan H. Mueller, Tim Mandelkow, Magalie C. Lurati, Ronald Simon, David Dum, Guido Sauter, Franziska Büscheck, Doris Hoeflmayer, Sören Weidemann, Claudia Hube-Magg, Till Clauditz, Maximilian Lennartz, Eike Burandt, Niclas C. Blessin. Prevalence and spatial interplay of mononuclear phagocyte and lymphocyte subpopulations in 49 carcinoma entities in respect to its TIM3, PD-1, PD-L1 and CTLA-4 expression 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 6649.

Background: Kallikrein-related peptidase 7 (KLK7) is a chymotrypsin-like serine protease which is essential for the desquamation of corneocytes and thus plays a pivotal role in maintaining skin homeostasis. In cancer, KLK7 overexpression was suggested to represent a route for metastasis through cleavage of cell junction and extracellular matrix proteins of cancer cells. Methods: To comprehensively determine KLK7 protein expression in normal and neoplastic tissues, a tissue microarray containing 13,447 samples from 147 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. Results: KLK7 positivity was found in 64 of 147 tumor categories, including 17 tumor categories with at least one strongly positive case. The highest rate of KLK7 positivity was found in squamous cell carcinomas from various sites of origin (positive in 18.1%-63.8%), ovarian and endometrium cancers (4.8%-56.2%), salivary gland tumors (4.8%-13.7%), bilio-pancreatic adenocarcinomas (20.0%-40.4%), and adenocarcinomas of the upper gastrointestinal tract (3.3%-12.5%). KLK7 positivity was linked to nodal metastasis (p=0.0005), blood vessel infiltration (p=0.0037), and lymph vessel infiltration (p Conclusions: These data provide a comprehensive overview on KLK7 expression in normal and neoplastic human tissues. The prognostic relevance of KLK7 expression and the possible role of KLK7 as a drug target need to be further investigated. Citation Format: Simon Kind, Carolina Castillo, Ria Uhlig, Natalia Gorbokon, Maximilian Lennartz, Sebastian Dwertmann Rico, Viktor Reiswich, Florian Viehweger, Martina Kluth, Claudia Hube-Magg, Christian Bernreuther, Franziska Büscheck, Till Clauditz, Christroph Fraune, Andrea Hinsch, Till Krech, Patrick Lebok, Stefan Steurer, Eike Burandt, Sarah Minner, Andreas Marx, Ronald Simon, Waldemar Wilczak, Guido Sauter, Anne Menz, Frank Jacobsen. KLK7 expression in human tumors: A tissue microarray study on 13,447 tumors [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 3302.

Progesterone receptor (PR) is a member of the nuclear/steroid hormone receptor family of ligand-dependent transcription factors. It plays an important role in reproduction and mammary gland development and has various tissue-specific effects in nonreproductive organs. In diagnostic pathology, positive PR immunostaining is used to support a diagnosis of breast or gynecologic origin in a tumor. In this study, the expression of PR was analyzed by immunohistochemistry in 18,176 (interpretable: 16,445) samples from 147 different tumor types and subtypes in a tissue microarray format. PR immunostaining was detected in 57.4% of breast tumors, 28.6% of other gynecological tumors, and 1.8% of nongynecological and nonmammary tumors. Among the group of nongynecological and nonmammary tumors, particularly high rates of PR positivity were seen in neuroendocrine tumors (54.3%) and neuroendocrine carcinomas (35.7%) of the pancreas. A comparison with clinico-pathological parameters showed that reduced PR immunostaining was significantly associated with adverse histopathological and clinical features in breast carcinoma, endometrioid endometrial carcinoma, and pancreatic neuroendocrine tumors. In summary, our analysis of 147 different tumor types for PR immunostaining provides a ranking list of tumor entities according to their prevalence of PR positivity, helps to better understand the diagnostic utility of PR, and highlights the distinct PR positivity among neuroendocrine neoplasms of pancreatic origin.

Mesothelin (MSLN) represents an attractive molecule for targeted cancer therapies. To identify tumors that might benefit from such therapies, tissue microarrays including 15,050 tumors from 122 different tumor types and 76 healthy organs were analyzed for MSLN expression by immunohistochemistry. Sixty-six (54%) tumor types showed at least occasional weak staining, including 50 (41%) tumor types with at least one strongly positive sample. Highest prevalence of MSLN positivity had ovarian carcinomas (serous 97%, clear cell 83%, endometrioid 77%, mucinous 71%, carcinosarcoma 65%), pancreatic adenocarcinoma (ductal 75%, ampullary 81%), endometrial carcinomas (clear cell 71%, serous 57%, carcinosarcoma 50%, endometrioid 45%), malignant mesothelioma (69%), and adenocarcinoma of the lung (55%). MSLN was rare in cancers of the breast (7% of 1138), kidney (7% of 807), thyroid gland (1% of 638), soft tissues (0.3% of 931), and prostate (0 of 481). High expression was linked to advanced pathological tumor (pT) stage (p &lt, 0.0001) and metastasis (p &lt, 0.0001) in 1619 colorectal adenocarcinomas, but unrelated to parameters of malignancy in 1072 breast-, 386 ovarian-, 174 lung-, 757 kidney-, 171 endometrial-, 373 gastric-, and 925 bladder carcinomas. In summary, numerous important cancer types with high-level MSLN expression might benefit from future anti-MSLN therapies, but MSLN’s prognostic relevance appears to be limited.

Background PD-L1 expression predicts response to immune checkpoint inhibitors in renal cell carcinomas (RCC), but has also been suggested to be linked to poor patient outcome. Methods We analyzed PD-L1 in > 1400 RCC in a tissue microarray format by immunohistochemistry. Results were compared with histological tumor type, parameters of cancer aggressiveness, and intratumoral CD8+ cytotoxic cells. Result At a cut-off level of 5% PD-L1 positive tumor cells, PD-L1 positivity was seen in 6.3% of 633 clear cell RCC (ccRCC), 18.2% of 165 papillary RCC, 18.8% of 64 chromophobe RCC, and 41.7% of 103 oncocytomas. In ccRCC, PD-L1 positivity was significantly linked to high ISUP (p p p p = 0.0042), short recurrence-free (p p = 0.0002). Intratumoral CD8+ lymphocytes were more frequent in PD-L1 positive (1055 ± 109) than in PD-L1 negative ccRCC (407 ± 28; p + cells (p p p = 0.0027), and Thoenes grade (p p = 0.0280). Conclusions These data suggest that PD-L1 expression in highly immunogenic RCCs facilitates immune evasion and contributes to cancer aggressiveness.

The Ki67 labeling index (Ki67-LI) is a strong prognostic marker in prostate cancer. Its analysis requires cumbersome manual quantification of Ki67 immunostaining in at least 200 tumor cells. To enable automated Ki67-LI assessment in routine clinical practice, we have developed and validated a framework for automated Ki67-LI quantification, which comprises three different artificial intelligence analysis steps and an algorithm for cell-distance analysis of multiplex fluorescence immunohistochemistry staining. The prognostic impact of the Ki67-LI was tested on a tissue microarray (TMA) containing one sample each of 12475 prostate cancers. A “heterogeneity TMA” containing 3 to 6 samples from different tumor areas was used to model Ki67 analysis of multiple different biopsies. The Ki67-LI provided strong and independent prognostic information in 11845 successfully analyzed prostate cancers (p Citation Format: Niclas C. Blessin, Tim Mandelkow, Elena Bady, Ronald Simon, Claudia Hube-Magg, Maximilian Lennartz, Guido Sauter, Markus Graefen, Stefan Steurer. Automated Ki67-LI assessment in prostate cancer using artificial intelligence in multiplex fluorescence immunohistochemistry [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 483.

Focal T-lymphocyte aggregates occur commonly in colorectal cancer, but their biological significance is unknown. To study focal aggregates of T-lymphocytes, a deep learning-based framework for automated identification of T-cell accumulations (T-cell-nests) was developed using CD8, PD-1, CD112R, and Ki67 multiplex fluorescence immunohistochemistry. To evaluate the clinical significance of these parameters, a cohort of 523 colorectal cancers with clinical-follow up data was analyzed. The spatial analysis of locally enriched CD8+ T-cell densities and cell-to-cell contacts identified T-cell-nests in the tumor microenvironment of colorectal cancer. CD112R and PD-1 expression on CD8+ T-cells located in T-cell-nests was found elevated as compared to CD8+ T-cells in all other tumor compartments (p Citation Format: Maximilian Lennartz, Cheng Yang, Tim Mandelkow, Elena Bady, Ronald Simon, Claudia Hube-Magg, Guido Sauter, Niclas C. Blessin. Non-redundant upregulation of CD112R (PVRIG) and PD-1 on cytotoxic T-lymphocytes located in T-cell-nests of colorectal cancer [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 4148.

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 assessment of prognostic markers in routine clinical practice of breast cancer is currently performed using multi gene RNA panels. However, the unknown proportion of normal breast tissue in relation to malignant breast tissue can reduce the predictive value of such tests. Immunohistochemistry holds the potential for a better assessment of tumors because tumor cells can be separately analyzed. To enable automated prognosis marker detection (i.e. HER2, GATA3, progesterone receptor [PR], estrogen receptor [ER], androgen receptor [AR], TOP2A, Ki-67, TROP2, Mammaglobin), we have developed and validated a framework for automated breast cancer identification, which comprises three different artificial intelligence analysis steps and an algorithm for cell-distance analysis of 12+1 marker BLEACH&STAIN multiplex fluorescence immunohistochemistry staining, in 2004 breast cancers. The optimal distance between Myosin+ basal cells and benign panCK+ benign cells was identified as 31µm and used to exclude benign glands from the analysis combined with and deep learning-based algorithm for benign gland detection. Our deep learning-based framework discriminated normal glands from malignant glands with an area under receiver-operating characteristic curves (AUC) of 0.96 (95% confidence interval [CI], 0.92 to 0.99). The accuracy of the approach was also validated by several well-characterized biological findings, such as the identification of 13% HER2+, 73% PR+/ER+, and 14% triple negative cases in the study cohort as well as a significant higher Fraction of TOP2A in the HER2+ cases as compared to the HER2- cases (p Citation Format: Tim Mandelkow, Elena Bady, Magalie C. Lurati, Claudia Hube-Magg, Maximilian Lennartz, Guido Sauter, Niclas C. Blessin, Ronald Simon. An artificial intelligence-based framework for BLEACH&STAIN mfIHC facilitates automated prognosis marker assessment in breast cancer [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 1934.

An increasing number of therapy regimens using a combination of different immune checkpoint inhibitors (ICIs) have shown remarkable results in several different tumor entities. However, the likelihood of a positive response rate to combined ICIs is poor in most tumor entities and depends on several parameters including the tumor microenvironment. Particularly little is known about the spatial orchestration and spatial interplay between different immune checkpoint expressing cells. Given that the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is expressed on both immune cells as well as tumor cells and that several phase I/II studies are currently evaluating anti-TIM3 drugs, the interplay between these immune checkpoints in human cancers is of topical interest. To study the spatial orchestration and interplay between TIM3, CTLA-4, PD-1, and PD-L1 expression on T-cell subsets, macrophage subsets, CD11c+ dendritic cells, CD20+B-cells in relation to panCK+ malignant cells, CD31+ vessels and other structural tumor compartments, a multiplex fluorescence immunohistochemistry approach was used to stain 18 different antibodies on a set of tissue microarrays containing samples from more than 3000 carcinoma samples. In addition, a deep learning-based framework for cell type identification was developed and validated in this study. TIM3, PD-1, PD-L1, and CTLA-4 expression was measured on tumor cells (panCK+), cytotoxic T-cells (CD3+CD8+), T-helper cells (CD3+CD4+), regulatory T-cells (CD3+CD4+FOXP3+), subsets of macrophages (CD68+CD163+/CD68+iNOS+) and dendritic cells (CD11c+). Interestingly, TIM3 as well as CTLA-4 expression on CD3+CD8+ cytotoxic T-cells and CD3+CD4+FOXP3+ regulatory T-cells showed a spatially more diverse expression pattern - particularly inverse expression profile - compared to PD-1 expression on all analyzed T-cells subsets that was consistently accompanied by PD-L1 expression on immune and tumor cells (p Citation Format: Nicolaus F. Debatin, Elena Bady, Tim Mandelkow, Magalie C. Lurati, Ronald Simon, Claudia Hube-Magg, Maximilian Lennartz, Guido Sauter, Niclas C. Blessin. Interplay between TIM3+ immune cells and other immune checkpoints in more than 40 different human carcinoma entities using 18+1 BLEACH&STAIN mfIHC [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 1935.

TIGIT is an inhibitory immune checkpoint receptor and a putative target for novel immune therapies. Here, we analysed two different types of tissue microarrays of healthy lymphatic and various inflamed tissues, colorectal and lung cancers, as well as >1700 tumour samples from 86 different tumour entities for TIGIT and/or PD-1 by bright field and/or multiplex fluorescence immunohistochemistry. TIGIT was detected in CD8+ cytotoxic T cells, CD4+ T helper cells, FOXP3+ regulatory T cells, and NK cells, but not in CD11c+ dendritic cells, CD68+ macrophages, and CD20+ B lymphocytes. TIGIT expression paralleled that of PD-1. More than 70% of TIGIT+ cells were PD-1+, and more than 90% of the PD-1+ cells were TIGIT+. Expression varied between different tissue compartments. TIGIT expression in tonsil gradually increased from the interfollicular area over the marginal/mantle zone to the germinal centre in all T cell subtypes. In inflammatory diseases, the strongest expression of TIGIT/PD-1 was found in Hashimoto thyroiditis. TIGIT+ lymphocytes were seen in all 86 different tumour entities with considerable high variability of TIGIT positivity within and between different cancer entities. Particularly, high densities of TIGIT+ lymphocytes were, for example, seen in squamous cell cancers of various origins. In summary, the variable expression levels of TIGIT and PD-1 in cell types and tissue compartments illustrate the high complexity of immune microenvironments. The high frequency of TIGIT (and PD-1) expressing lymphocytes in cancers highlights considerable opportunities for cotargeting with checkpoint inhibitors.

Introduction: CTLA-4 is an inhibitory immune checkpoint receptor and a negative regulator of anti-tumor T-cell function. This study aimed at a comparative analysis of CTLA-4+ cells between different tumor entities. Methods: To quantify CTLA-4+ cells, 4,582 tumor samples from 90 different tumor entities as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Two different antibody clones (MSVA-152R and CAL49) were validated and quantified using a deep learning framework for automated exclusion of unspecific immunostaining. Results: Comparing both CTLA-4 antibodies revealed a clone dependent cytoplasmic cross-reactivity in adrenal cortical adenoma (63%) for MSVA-152R and in pheochromocytoma (67%) as well as hepatocellular carcinoma (36%) for CAL49. After automated exclusion of non-specific staining reaction (3.6%), a strong correlation was observed for the densities of CTLA-4+ lymphocytes obtained by both antibodies (r=0.87; p Conclusion: Marked differences exist in the number of CTLA-4+ lymphocytes between tumors. Analyzing two independent antibodies by a deep learning framework identifies clone-specific cross-reactivity and facilitates automated quantification of target proteins such as CTLA-4. Citation Format: David Dum, Tjark L. C. Henke, Tim Mandelkow, Elena Bady, Jonas B. Raedler, Ronald Simon, Guido Sauter, Maximilian Lennartz, Waldemar Wilczak, Eike Burandt, Stefan Steurer, Niclas C. Blessin. Semi-automated validation and quantification of CTLA-4 in 90 different Tumor entities using multiple antibodies and artificial intelligence [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P069.

Introduction: The quantification of PD-L1 (programmed cell death ligand 1) has been used to predict patient's survival, to characterize the tumor immune microenvironment, and to predict response to immune checkpoint therapies. However, a framework to assess the PD-L1 status with a high interobserver reproducibility on tumor cells and different types of immune cells has yet to be established. Methods: To study the impact of PD-L1 expression on the tumor immune microenvironment and patient outcome, a framework for fully automated PD-L1 quantification on tumor cells and immune cells was established and validated. Automated PD-L1 quantification was facilitated by incorporating three different deep learning steps for the analysis of more than 80 different neoplasms from more than 10'000 tumor specimens using a bleach & stain 15-marker multiplex fluorescence immunohistochemistry panel (i.e., PD-L1, PD-1, CTLA-4, panCK, CD68, CD163, CD11c, iNOS, CD3, CD8, CD4, FOXP3, CD20, Ki67, CD31). Clinicopathological parameter were available for more than 30 tumor entities and overall survival data were available for 1517 breast cancer specimens. Results: Comparing the automated deep-learning based PD-L1 quantification with conventional brightfield PD-L1 data revealed a high concordance in tumor cells (p Conclusion: In conclusion, multiplex immunofluorescence PD-L1 assessment provides cutoff-free/continuous PD-L1 data which are superior to the conventional percentage of PD-L1+ tumor cells and of high prognostic relevance. The combined analysis of spatial PD-L1/PD-1 data and more than 20 different immune cell subtypes of the immune tumor microenvironment revealed distinct PD-L1 immune phenotypes. Citation Format: Niclas C. Blessin, Elena Bady, Tim Mandelkow, Cheng Yang, Jonas B. Raedler, Ronald Simon, Christoph Fraune, Maximilian Lennartz, Sarah Minner, Eike Burandt, Doris Höflmayer, Guido Sauter, Katharina Möller, Sören A. Weidemann. Automated cell type specific PD-L1 quantification by artificial intelligence using high throughput bleach & stain 15-marker multiplex fluorescence immunohistochemistry in human cancers [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P068.

Checkpoint kinase 2 (CHK2) is a serine-threonine kinase with a role in DNA repair, cell cycle arrest or apoptosis in response to DNA damage. Both reduced and increased CHK2 expression has been described in different tumour types with impact on patient prognosis. To evaluate prevalence and significance of altered CHK2 expression in prostate cancer, a tissue microarray containing 17,747 tumours was analysed by immunohistochemistry. Nuclear CHK2 immunostaining was absent or weak in benign prostate epithelium but often more prominent in cancers. CHK2 immunostaining was considered weak in 38.8%, moderate in 33.6% and strong in 11.2% of prostate cancers. High CHK2 expression was strongly associated with TMPRSS2:ERG fusions (p0.0001). Subgroup analysis of ERG positive and negative cancers revealed that high CHK2 staining was significantly linked to advanced tumour stage, high Gleason score, positive nodal status, positive surgical margin, high preoperative PSA (p0.0001 each) and early prostate-specific antigen (PSA) recurrence (p=0.0001) in the subset of ERG negative cancers, while most of these associations were absent in ERG positive cancers. In ERG negative cancers, high CHK2 expression was an independent predictor of patient prognosis, even if parameters were included that were only available postoperatively. High CHK2 expression was also linked to presence of chromosomal deletions, high level of androgen receptor expression, positive p53 immunostaining, and high Ki-67 labelling index. These provide further in vivo evidence for previously described functional interactions. In summary, high CHK2 expression is linked to adverse tumour features and independently predicts early biochemical recurrence in ERG negative prostate cancer. CHK2 measurement, either alone or in combination, might be of clinical utility in this prostate cancer subgroup.

A characteristic feature of testicular seminoma is the abundance of immune cells in the tumor microenvironment, raising the possibility that immune checkpoint inhibitors may serve as a therapeutic option in these types of tumors. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory immune checkpoint receptor in analogy to PD-1, and drugs targeting TIGIT are currently being investigated in clinical trials. Little is known about the expression of these proteins in testicular seminomas. Therefore the present study performed immunohistochemical analysis to determine the relative abundance of TIGIT and PD-1 in relation to the total CD3+ immune cell infiltration in a tissue microarray (TMA) constructed from 78 seminoma patients. The fraction of TIGIT+ and PD-1+ lymphocytes was highly variable in individual cancers and ranged from 2.3 to 69.4% (mean: 32.2±14.7%) for TIGIT and from 0.8 to 56.5% (mean: 21.6±13.2%) for PD-1. The same high degree of variability was also identified for the ratio of PD-1 to TIGIT positive cells, which varied from a dominance of TIGIT (PD-1: TIGIT ratio=0.02) in 74% of patients, to a predominance of PD-1 (PD-1: TIGIT ratio=12.5) in 23% of patients. In summary, the immune checkpoint receptors TIGIT and PD-1 are abundantly expressed in human seminomas. Once available, anti-TIGIT antibodies, possibly in combination with anti-PD-1 drugs, may be a reasonable therapeutic strategy for this type of cancer.

The amount of tumor infiltrating lymphocytes (TILs) is an established prognostic feature in the cancer microenvironment of colorectal and breast cancers, but data are less clear for most other solid cancer types. To study the prognostic impact of TILs in vulvar squamous cell carcinomas, a cohort of 530 cancers was analyzed by immunohistochemistry for CD3 and CD8 positive cells (CD3+, CD8+). Automated digital image analysis was used to quantitate the densities of CD3+, and CD8+ TILs in the invasive margin and the tumor center. The average immune cell density was significantly higher in the invasive margin (CD3+: 1772±1105, CD8+: 769±644 cells/mm2) as compared to the center of the tumor (CD3+: 518±570, CD8+: 301±445 cells/mm2, p≤0.0001 each). Comparison with clinic-pathological data revealed a significant prognostic impact of CD3+ but not of CD8+ cell density and a stronger link with tumor phenotype and outcome for TILs at the invasive tumor margin as compared to the tumor center. Elevated densities of CD3+ cells within the invasive margin were significantly associated with a favorable pT stage (p=0.0012) and a prolonged overall (p=0.0025) and progression free survival (p=0.0231). A high CD3+ cell density in the tumor center was linked to overall (p=0.0457) and progression free survival (p=0.0266). Unsupervised clustering of CD3+/CD8+ cell densities identified 3 major subsets corresponding to the immune desert (sparse TILs at invasive margin and tumor center), immune excluded (high TIL numbers at invasive margin but low TIL counts in the tumor center), and immune inflamed phenotypes (high TIL numbers at invasive margins and tumor center). Survival analysis revealed a particularly poor prognosis for the immune desert phenotype for overall (p=0.0071) and progression free survival (p=0.0027). In summary, our data suggest a clinical utility of TIL quantification in vulva squamous cell cancer. Citation Format: Maximilian Lennartz, Niclas Christian Blessin, Ann-Christin Rolschewski, Ronald Simon, Guido Sauter, Ria Uhlig, Linn Wölber, Sven Mahner, Nikolaus de Gregorio, Rüdiger Klapdor, Matthias Kalder, Stefan Steurer, Waldemar Wilczak, Eike Burandt, Simon Kind. Prognostic impact of tumor infiltrating lymphocytes in the tumor microenvironment of vulvar squamous cell carcinoma [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 2750.

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.

Purpose: Aberrant DNA content has been discussed as a potential prognostic feature in prostate cancer. Experimental Design: We analyzed the clinical significance of DNA ploidy in combination with prognostic relevant deletions of PTEN and 6q15 in 3,845 prostate cancers. Result: The DNA status was diploid in 67.8%, tetraploid in 25.6%, and aneuploid in 6.8% of tumors, and deletions of PTEN and 6q15 occurred in 17.8% and 20.3% of tumors. Abnormal DNA content and deletions were linked to high Gleason score, advanced tumor stage, and positive nodal stage (P < 0.0001 each). The risk of PSA recurrence increased from diploid to tetraploid and from tetraploid to aneuploid DNA status (P < 0.0001 each). However, 40% of patients with Gleason score ≥4+4 and 55% of patients with PSA recurrence had diploid cancers. This fraction decreased to 21% (Gleason ≥4+4) and 29% (PSA recurrence) if PTEN and/or 6q deletion data were added to ploidy data to identify cancers with an aberrant DNA status. The significance of combining both deletions and ploidy was further demonstrated in a combined recurrence analysis. Presence of deletions increased the risk of PSA recurrence in diploid (P < 0.0001), tetraploid (P < 0.0001), and aneuploid cancers (P = 0.0049), and the combination of ploidy data and deletions provided clinically relevant information beyond the CAPRA-S nomogram. Multivariate modeling including preoperatively and postoperatively available parameters identified the “combined DNA status” as a strong independent predictor of poor patient outcome. Conclusions: The combinatorial DNA content analysis involving general (ploidy) and specific events (deletions) has the potential for clinical utility in prostate cancer. Clin Cancer Res; 22(11); 2802–11. ©2016 AACR.

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.

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. 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. 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 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. Citation Format: Tim Mandelkow, Elena Bady, Niclas Blessin, Claudia Hube-Magg, Ronald Simon, Guido Sauter, Christoph Fraune, Maximilian Lennartz, Katharin Möller, Sören Weidemann, Andreas Luebke, Doris Höflmayer. Prevalence and prognostic role of FoxP3regulatory T lymphocytes in cancer: A tissue microarray study on >20,000 cancers [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 3854.

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.

Hodgkin’s lymphoma (HL) is characterized by a high background of inflammatory cells which play an important role for the pathogenesis of the disease. T cell immunoreceptor with Ig and ITIM domains (TIGIT) is an inhibitory immune checkpoint receptor and a putative target for novel immunotherapies. To study patterns of TIGIT expression in the T cell background surrounding malignant cells including Hodgkin cells, Reed-Sternberg cells and histiocytic cells, a microenvironment (ME) tissue microarray (TMA) was constructed from tissue punches measuring 2 mm in diameter obtained from formalin-fixed tissue samples of Hodgkin’s lymphoma lymph nodes (n = 40) and normal human tonsil (n = 2). The ME-TMA was stained by brightfield and fluorescence multiplex immunohistochemistry (IHC) to evaluate expression levels of TIGIT and PD-1 as well as standard lymphocyte markers (CD3, CD8, CD4, FOXP3) in the lymphocytic background. All analyzed cases of HL contained 9–99% (median: 86%) of TIGIT+ lymphoid cells. In general, TIGIT localized to the same cells as PD-1. Strikingly, expression levels of TIGIT and PD-1 were highly variable among the analyzed samples. Highest levels of TIGIT and PD-1 were found in one sample of nodular lymphocytic-predominant HL (NLPHL). In conclusion, TIGIT expression is highly variable between patients with Hodgkin’s lymphoma. Our results encourage further studies evaluating the role of TIGIT as a target for immunotherapies in Hodgkin’s lymphoma. Electronic supplementary material The online version of this article (10.1186/s12885-018-5111-1) contains supplementary material, which is available to authorized users.

Since several decades, prostate specific antigen (PSA) is regarded as a specific marker for healthy and neoplastic prostate tissues, and PSA level changes in blood and tissue are discussed as prognostic markers of prostate cancer. Using a custom-made mouse monoclonal anti-PSA antibody, we studied expression of PSA in a tissue microarray (TMAs) comprising 17,456 prostate cancers with long-term follow-up data. PSA staining was typically present in the cytoplasm and pronounced at the apical cell pole. PSA expression was often reduced (15%) or even completely lost (1%). Reduction of PSA expression was significantly linked to adverse features of prostate cancer, including advanced stage, high classical and quantitative Gleason grade, presence of lymph node metastasis, a positive surgical margin, increased cell proliferation, and reduced time to recurrence (p Citation Format: Ronald Simon, Martina Kluth, Kristine Fischer, Sarah Bonk, Claudia Hube-Magg, Doris Höflmayer, Maximilian Lennartz, Florian Lutz, Guido Sauter, Franziska Büscheck, Ria Uhlig, David Dum, Andreas M. Luebke, Corinna Wittmer, Frank Jacobsen, Eike Burandt, Stefan Steurer, Waldemar Wilczak, Sarah Minner. Prostate specific antigen (PSA) as a diagnostic and prognostic marker of prostate cancer [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 430.

We have recently shown that quantification of Gleason 4 patterns (Q-Gleason) provides strong prognostic information beyond traditional Gleason grade groups (3+3, 3+4, 4+3, 8, 9-10) in prostate cancer, and that the prognostic power of Q-Gleason scoring can be further improved if Gleason 5 patterns are integrated to build the Integrative Quantitative Gleason (IQ-Gleason) score, a continuous value ranging from 0 – 117.5. The current study was undertaken, to determine whether (and how) factoring in molecular markers could further improve the predictive power. We selected deletions of PTEN and 6q (by FISH analysis) as well as DNA ploidy information obtained by flow cytometry as examples for established molecular prognostic markers that provided strong and independent prognostic information in our study cohort of more than 17,000 prostate cancers. A “molecular score” was established for these markers including “penalty” points for presence of deletions/aneuploidy and “bonus” points for lack of deletions/diploid DNA status. Penalty and bonus points were separately determined for each parameter based on differences in the recurrence rate between cancers with and without deletions or aneuploidy. The “penalty” points were then added to the IQ-Gleason score points while the “bonus” points were subtracted from the IQ-Gleason in order to obtain the MiQ-Gleason score. The MiQ-Gleason provided strong prognostic information in univariate analysis (p Citation Format: Ronald Simon, Martina Kluth, Maximilian Lennartz, Claudia Hube-Magg, Sarah Minner, Maria Christina Tsourlakis, Stefan Steurer, Markus Graefen, Hartwig Huland, Lars Budäus, Guido Sauter, Thorsten Schlomm. Improving Gleason scoring in prostate cancer: Development and clinical value of the Molecular Integrative Quantitative Gleason (MIQ-Gleason) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3741. doi:10.1158/1538-7445.AM2017-3741

5027 Background: Aberrant DNA ploidy has been long discussed as a potential prognostic feature in prostate cancer. Methods: In this project, we analyzed the clinical significance of DNA ploidy in c...