Abstract Introduction/Objective Although most prostate cancers behave in an indolent manner, a small proportion is highly aggressive. Both primary and advanced prostate cancer is widely known as a non-inflamed cancer that is characterized by a paucity of immune infiltration. Methods/Case Report To assess the spatial interplay of more than 30 TIM3, CTLA-4, PD-1/-L1 expressing leukocyte subpopulations in 453 prostate cancers, tissue microarrays were stained with 21 antibodies using our BLEACH&STAIN multiplex fluorescence immunohistochemistry approach and analyzed using a deep learning-based image analysis framework. Results (if a Case Study enter NA) The immune cell density of CD8+ cytotoxic T-cells, CD4+ T-helper cells, FOXP3+ regulatory T-cells, M1/ M2 macrophages, as well as CD11c+ dendritic cells increased consistently along with the Gleason grade in primary prostate cancer (p≤ 0.034 each). In recurrent prostate cancers under therapy, the density of FOXP3+regulatory T-cells and M1 macrophages further increased, while the density of CD8+ cytotoxic T-cells, CD4+ T-helper cells, as well as CD11c+ dendritic cells decreased (p≤0.017 each). Although the immune checkpoint expression of TIM3 on T-cell subsets, macrophages and dendritic cells was upregulated in advanced/ recurrent tumors, the expression level of PD-1 was downregulated in all analyzed T-cell subsets. Conclusion Although prostate cancer is a generally considered a low inflamed tumor, the degree of tumor infiltration by various immune cell subtypes increases markedly along with tumor progression and in recurrent tumors under therapy. Taken together, these data suggest that the evaluation of the spatial distribution of immune cell types along with their immune checkpoint expression can provide relevant clinical information in prostate cancer.

e15181 Background: Carcinoembryonic antigen (CEA) is a cell surface glycoprotein which is overexpressed in various cancers but reported prevalence data vary considerably for many tumor types. Because of its overexpression in various cancer types and the limited expression in normal tissue CEA has become a potential drug target with several potential drug candidates currently being evaluated. CEA is also shed into the blood stream, so that CEA measurement in the serum is used as a tool for early detection and recurrence monitoring of cancer. Methods: To comprehensively determine CEA expression in normal and neoplastic tissues, a tissue microarray containing 15,413 samples from 120 different tumor types and subtypes as well as 76 different normal tissue types were analyzed by immunohistochemistry. Results: CEA was detectable in 65 (54.2%) of 120 tumor categories including 49 (40.8%) tumor types with at least one strongly positive case. CEA positivity was most common in colorectal adenomas (100%) and carcinomas (98.7%), other gastrointestinal adenocarcinomas (61.1%-80.3%), medullary carcinomas of the thyroid (96.3%), pulmonary adenocarcinoma (73.7%), mucinous carcinomas of the ovary (79.8%) and the breast (43.2%), squamous cell carcinomas of various sites (30.2%-69.1%), and small cell carcinomas of the lung (64.3%), the urinary bladder (38.9%), and the prostate (50.0%) as well as non-invasive papillary urothelial carcinoma pTa G3 (33,6%), pTa G2 high-grade (25,0%) and pTa G2 low-grade (5,7%). High CEA expression was linked to high grade tumors (p < 0.0001) and invasive growth (p < 0.0001) in urinary bladder cancer as well as estrogen receptor positivity (p = 0.0005) and HER2 positivity (p = 0,0158) in invasive breast cancer of no special type. In 1.250 colorectal adenocarcinomas, reduced CEA expression was associated with mismatch repair deficiency (p < 0.0001) but not with pT and pN stage. CEA expression level was unrelated to clinico-pathological tumor parameters in adenocarcinomas of the pancreas and the stomach, endometroid endometrium carcinoma as well as in serous and endometroid carcinomas of the ovaries. Conclusions: In summary, CEA is abundantly expressed in a broad range of epithelial neoplasms. Our data thus identify various tumor entities where CEA positive cancers might best benefit from CEA serum monitoring and anti-CEA therapies.

Abstract Introduction/Objective Quantity and the spatial relationship of individual immune cell (sub)types can provide prognostic information in muscle-invasive bladder cancer. Methods/Case Report To study the prognostic role of different immune cell subpopulations in muscle-invasive bladder cancer, we stained 521 muscle-invasive urothelial bladder carcinomas with 21 antibodies using BLEACH&STAIN multiplex fluorescence immunohistochemistry (mfIHC) in a tissue microarray format. A framework of neuronal networks was trained and used for image analysis. Results (if a Case Study enter NA) The identification of more than 300 different immune cell subpopulations and the characterization of their spatial relationship resulted in numerous spatial interaction patterns. 40 immune parameters showed prognostic significance in univariate analyses of which 15 were independent from pT, pN, and histologic grade. The strongest association to clinical outcome was identified for intraepithelial CD8+cytotoxic T-cells (tAUC: 0.70) compared to all other 15 independent prognosis parameters (AUC: ≤0.68, p=0.039). Deeper spatial analysis revealed an increased expression levels of TIM3, CTLA-4 and PD-1 on CD8+ T cells that were located in the intraepithelial compartment. Furthermore, the cell-to-cell interaction profile of intraepithelial CD8+ T-cells was dominated by intraepithelial dendritic cells as well as intraepithelial M1-macrophages. Conclusion The intraepithelial CD8+T-cells that showed the highest expression level of TIM-3, PD-1 and CTLA-4 and were strongly interacting with M1-macrophages as well as dendritic cells and represented the strongest prognostic parameter in muscle-invasive bladder cancer. This can be explained by the fact that tumor cell destruction by CD8+ cytotoxic T-lymphocytes through direct cell-to-cell-contacts represents the “terminal-end-route” of anti-tumor immunity. The strongest prognostic immune-factor – intraepithelial CD8+T-cells – can be measured straightaway in routine pathology as the CD8-labelling index using conventional bright field immunohistochemistry.

Carcinoembryonic antigen (CEA; CEACAM5) is a cell surface glycoprotein which constitutes an attractive therapeutic target and serum CEA is used for cancer monitoring. CEA is overexpressed in various cancers, but the reported prevalence data vary considerably for many tumor types. To comprehensively determine CEA expression in normal and neoplastic tissues, a tissue microarray containing 15,413 samples from 120 different tumor types and subtypes as well as 76 different normal tissue types were analyzed by immunohistochemistry. CEA positivity occurred in 65 of 120 tumor categories including 49 entities with at least one strongly positive case. CEA positivity was most common in colorectal carcinomas (98.7%), other gastrointestinal adenocarcinomas (61.1%-80.3%), medullary carcinomas of the thyroid (96.3%), pulmonary adenocarcinoma (73.7%), mucinous carcinomas of the ovary (79.8%) and the breast (43.2%), squamous cell carcinomas of various sites (30.2%-69.1%), and small cell carcinomas of the lung (64.3%), the urinary bladder (38.9%), and the prostate (50.0%). High CEA expression was linked to high grade (p<0.0001) and invasive growth (p<0.0001) in urothelial carcinoma. Reduced CEA expression was associated with mismatch repair deficiency (p<0.0001) but not with pT and pN stage in colorectal cancer. In summary, CEA is abundantly expressed in a broad range of epithelial neoplasms. Our data thus identify various tumor entities where CEA positive cancers might best benefit from CEA serum monitoring and anti-CEA therapies.

Deletion of CDKN2A, encoded for the tumor suppressor protein p16, represents a common cause for reduced or absent of p16 expression in tumor cells. In this study, we analyzed the impact of CDKN2A deletion on tumor aggressiveness, patient prognosis, and p16 expression in patients with urothelial bladder carcinomas. CDKN2A copy number status was analyzed by fluorescence in-situ hybridization (FISH) on more than 2,500 urothelial bladder carcinomas in a tissue microarray format. Data of CDKN2A deletion were compared with p16 expression data measured by immunohistochemistry in a previous study. The results were compared with parameters of tumor phenotype and patient outcome. CDKN2A deletions occurred in 28% of 1,880 analyzable carcinomas, including 9% heterozygous and 19% homozygous CDKN2A deletions. The CDKN2A deletion rate, especially for homozygous deletions, increased from pTa G2 low (14%), to pTa G2 high (35%) but slightly decreased in pTa G3 (20%) carcinomas (p<0.0001). In muscle-invasive urothelial carcinomas, especially homozygous CDKN2A deletions were associated with advanced pT stage (p<0.0001), but unrelated to patient prognosis. However, in the subset of pT4 urothelial bladder carcinomas, a tendency for shorter overall survival for patients with CDKN2A deleted tumors was found (p=0.0828). CDKN2A deletions were significantly associated with p16 immunostaining (p<0.0001). Cancers without p16 immunostaining harbored in 62% of all cases a CDKN2A deletion and 97% of 343 homozygous CDKN2A deleted cancers were p16 negative. Cancers with CDKN2A deletion and negative p16 immunostaining were associated with advanced stage (p<0.0001) but unrelated to patient prognosis in muscle-invasive urothelial carcinomas. CDKN2A deletions were most commonly seen in p16-negative carcinomas, supporting their role for p16 inactivation in urothelial bladder carcinomas. The association of CDKN2A deletion – especially homozygous deletion – with advanced stage may reflect increasing genomic instability going along with stage progression.

Prognostic markers in routine clinical practice of breast cancer are often assessed using RNA based multi-gene panels that are depending on a fluctuating tumor purity. Multiplex fluorescence immunohistochemistry (mfIHC) holds the potential for improved risk assessment. To enable automated prognosis marker quantification, we have developed and validated a framework for automated breast cancer detection involving three different artificial intelligence analysis steps and an algorithm for cell-distance analysis using BLEACH&STAIN 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. The optimal distance between Myosin+ and p63+ basal cells and benign panCK+ cells was identified as 25 μm in breast cancer and used – combined with deep learning-based algorithms – to exclude benign glands from the analysis. Our framework discriminated normal glands from malignant glands with an accuracy of 98.4% (95% confidence interval [CI]: 97.4 – 99.3). The approach for automated breast cancer detection improved the predictive performance of several prognosis markers significantly (each p<0.05) and a comparison with manually assessed data using conventional brightfield immunohistochemistry showed a high concordance for a multitude of different prognosis marker such as PR, ER, GATA3, HER2, and PD-L1 (each p<0.0001). The combined assessment of up to 5 markers in a prognosis score showed strong prognostic relevance (p<0.001) and was an independent risk factor in multivariate analysis (p=0.005). Thus, the data from this study show that automated breast cancer detection in combination with artificial intelligence-based analysis of multiplex fluorescence immunohistochemistry enables a rapid and reliable analysis of multiple prognostic parameters. The major advantage of this method is the analysis of malignant cells exclusively that cannot be achieved using RNA-based panel analysis.

Abstract Introduction/Objective Gene amplifications of the proto-oncogen MYC are common events in carcinomas. In this study, we analyzed the impact of MYC amplifications on tumor aggressiveness and patient prognosis in urothelial bladder carcinomas. Methods/Case Report MYC copy number status was analyzed on more than 2,500 urothelial bladder carcinomas in a tissue microarray format by using dual-labeling fluorescence in-situ hybridization (FISH) with probes for centromere 8 and MYC (8q24). The results were compared with tumor phenotype and patient outcome. Results (if a Case Study enter NA) MYC amplification (ratio MYC/centromere 8 ≥2) occurred in 10% of 2,052 analyzable urothelial bladder carcinomas. Of these, 141 (6.8%) had a low level MYC copy number gain (MYC/centromere 8 ratio ≥2 and ≤ 3) and 68 (3.3%) had high-level MYC amplifications (MYC/centromere 8 ratio >3) . The rate of MYC gain and MYC amplification increased from pTa G2 low (0.6%), to pTa G2 high (7.7%), and pTa G3 (8.2%) carcinomas, and were highest in muscle-invasive pT2-4 (13.2%) carcinomas (p<0.0001). In muscle-invasive urothelial carcinomas, the MYC amplification rate increased from pT2 (13.1%), to pT3 (14.7%), and pT4 (19.8%) but these differences failed to reach statistical significance (p=0.0902). MYC amplification was also unrelated to patient survival in 526 patients with pT2-4 urothelial carcinomas who had undergone cystectomy (p=0.5526). Conclusion MYC amplification is a common event in urothelial bladder carcinoma - especially in case of pTa G3 and muscle-invasive carcinoma. The association of MYC amplification with advanced stage may reflect increasing genomic instability going along with tumor progression.

Abstract Introduction/Objective Prostein (P501S), also termed solute carrier family 45 member 3 (SLC45A3) is an androgen regulated protein which is preferentially expressed in prostate epithelial cells. Because of its frequent expression in prostate cancer, prostein was suggested a diagnostic prostate cancer marker. Methods/Case Report In order to comprehensively assess the diagnostic utility of prostein immunohistochemistry, a tissue microarray containing 19,202 samples from 152 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. Results (if a Case Study enter NA) Prostein immunostaining was typically cytoplasmic, granular and perinuclear and predominated in prostate cancer. Prostein positivity was seen in 96.7% of 419 prostate cancers including 78.3% with a strong staining. In 12,233 extra-prostatic tumors, prostein positivity was observed in 9.8% of cases but only 0.4% had a strong staining. Extra-prostatic prostein positive tumors were 50 different tumor categories, 12 of which included at least one strongly positive case. Extra-prostatic tumors with highest rates of prostein positivity included salivary gland tumors (7.6%-44.4%), neuroendocrine neoplasms (15.8%-44.4%), adenocarcinomas of the gastrointestinal tract (7.3%-14.8%), biliopancreatic adenocarcinomas (3.6%-38.7%), hepatocellular carcinomas (8.1%), and adenocarcinomas of other organs (up to 21%). Conclusion In summary, our data provide a comprehensive overview on prostein expression in human cancers. Prostein is a highly sensitive prostate cancer marker occurring in >96% of prostate cancers. Because prostein can also be expressed in various other tumor entities, labeling of a tumor mass as a prostate cancer should not be based on prostein positivity alone.

Prognostic markers in routine clinical practice of breast cancer are currently assessed using multi-gene panels. However, the fluctuating tumor purity can reduce the predictive value of such tests. Immunohistochemistry (IHC) holds the potential for a better risk assessment. To enable automated prognosis marker detection (i.e. HER2, GATA3, PR, ER, and AR, TOP2A, Ki-67, TROP2), 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 11+1 marker BLEACH&STAIN multiplex fluorescence immunohistochemistry (mfIHC) staining in 2′004 breast cancers. The optimal distance between Myosin+ basal cells and benign panCK+ cells was identified as 25 μm 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. The accuracy of the approach was also validated by well-characterized biological findings, such as the identification of 13% HER2+, 73% PR+/ER+, and 14 triple negative cases. Furthermore, the automated assessment of GATA3, PR, ER, TOP2A-LI, Ki-67-LI and TROP2 was significantly liked to the tumor grade (p<0.001 each). A high expression level of HER2, GATA3, PR, and ER was associated with a prolonged overall survival (p≥0.002 each). A deep learning-based framework for automated breast cancer identification using BLEACH&STAIN mfIHC facilitates automated prognosis marker quantification in breast cancer.

Abstract Introduction/Objective Introduction: Pancreatic acinar cell carcinoma (PACC) is a rare tumor of the pancreas with an intermediate prognosis as compared to pancreatic neuroendocrine tumors (PNE) and pancreatic ductal adenocarcinoma (PDAC) from which it may be difficult to distinguish by morphology alone. Methods/Case Report Methods: To study was the efficiency of immunohistochemical markers, 18 PACCs, 531 PDACs, 64 PNEs, 117 extra pancreatic neuroendocrine neoplasms (EPNN), 826 colorectal carcinomas (CRC) and 252 gastric carcinomas (GC) were analyzed with antibodies for CPA1 (MSVA-601M), bcl10 (Santa Cruz sc5273), and chymotrypsin (Biorad 2100-0657) in a tissue microarray format. Results (if a Case Study enter NA) Results: CPA1 was positive in 18 of 18 (100%) of PACCs, 0 of 49 (0%) of PNEs, 0 of 88 (0%) of EPNNs, 10 of 404 (2.5%) of CRCs, and 0 of 178 (0%) of GCs. Chymotrypsin was positive in 16 (87,5%) PACCs, 1 (2%) PNEs, 2 (2.3%) EPNNs, 10 (2.5%) CRCs, and 1 (0.6%) GCs. Bcl10 was positive in 18 (100%) PACCs, 2 (4.1%) PNEs, 5 (1%) EPNNs, 109 (27%) CRCs, and 18 (10%) GCs. These data resulted in a sensitivity and specificity of 100%/99.2% for CPA1, 100%/88.4% for bcl10, and 94.4%/98.6% for chymotrypsin. Conclusion CPA1 and chymotrypsin are both highly specific and sensitive for ACC while bcl10 is sensitive but has markedly lower specificity. Because all “false positive” cases identified by CPA1 were CRCs that only showed a positive staining in goblet cells and an identical staining pattern was observed in all these cases for chymotrypsin and bcl10, a pancreatic origin of the mucus in these goblet cells is concluded.

5599 Background: Tumor infiltrating lymphocytes (TILs) in the cancer microenvironment are of prognostic value in many solid tumors. However, only little is known about TILs infiltration and its predictive value in vulvar cancer. Methods: Immunohistochemistry and automated digital image analysis was applied to measure the densities of CD3+ (DAKO, Santa Clara, US; #IR503) and CD8+ (DAKO, Santa Clara, US; #IR623) TILs at the invasive margin (IM) and in the center of 530 vulvar carcinomas. Results: At the IM the mean immune cell density was significantly higher compared to the center of the tumor (CD3: 1772±1105, CD8: 769±644 cells/mm2 vs. CD3: 518±570, CD8: 301±445 cells/mm2, p≤0.0001). An elevated density of CD3+ T-cell at the IM was significantly associated with low tumor stage (p = 0.0012). The 2-years OS and PFS rate was significantly different between the group with a high (OS: 82%, PFS: 65%), moderate (OS: 76%, PFS: 55%), or low CD3+ T-cell density at the IM (OS: 64%, p = 0.008, PFS: 44%, p = 0.02). The prognostic impact of CD3+ cells in the center of the tumor was weaker compared to the IM (OS p = 0.046, PPS p = 0.031) and lacking for CD8+ T-cell densities at any location (p≥0.14 each). Unsupervised clustering of CD3+ and CD8+ T-cell densities identified three major subgroups corresponding to the immune desert (137 patients), immune excluded (220 patients) and immune inflamed phenotypes (133 patients). Survival analysis revealed a particular poor prognosis for the immune desert phenotype for OS (0.0071) and PFS (0.0027). Conclusions: This study demonstrates a prognostical relevance of the immunphenotype and the distribution of CD3+ T-cells in vulvar cancer. Their value for therapeutic decision making has to be determined in the future.

Background 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. Materials and 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 unspecific staining pattern 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<0.0001). The mean density of CTLA-4+ cells was 674±1482 cells/mm2 and ranged from 71±175 cells/mm2 in leiomyoma to 5916±3826 cells/mm2 in Hodgkin's lymphoma. Within epithelial tumors, the density of CTLA-4+ lymphocytes were higher in squamous cell (421±467 cells/mm2) and urothelial carcinomas (419±347 cells/mm2) than in adenocarcinomas (269±375 cells/mm2) and renal cell neoplasms (256±269 cells/mm2). A high CTLA-4+ cell density was linked to low pT category (p<0.0001), absent lymph node metastases (p=0.0354), and PD-L1 expression in tumor cells or inflammatory cells (p<0.0001 each). A high CTLA-4/CD3-ratio was linked to absent lymph node metastases (p=0.0295) and to PD-L1 positivity on immune cells (p<0.0026). Conclusions Marked differences exist in the number of CTLA-4+ lymphocytes between tumors. Analyzing two independent antibodies by a deep learning framework can facilitate automated quantification of immunohistochemically analyzed target proteins such as CTLA-4. Disclosure Information D. Dum: None. T.L.C. Henke: None. T. Mandelkow: None. E. Bady: None. R. Simon: None. G. Sauter: None. S. Steuerer: None. W. Wilczak: None. E. Burandt: None. J. Raedler: None. M. Lennartz: None. N.C. Blessin: None.

Background 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. Materials and 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<0.0001) as well as immune cells (p<0.0001) and an accuracy of the automated PD-L1 quantification ranging from 90% to 95.2%. Across all tumor entities, the PD-L1 expression level was significantly higher in distinct macrophage/dendritic cell (DC) subsets (identified by CD68, CD163, CD11c, iNOS; p<000.1) and in macrophages/DCs located in the Stroma (p<0.0001) as compared to intratumoral macrophages/DC subsets. Across all different tumor entities, the PD-L1 expression was highly variable and distinct PD-L1 driven immune phenotypes were identified based on the PD-L1 intensity on both tumor and immune cells, the distance between non-exhausted T-cell subsets (i.e. PD-1 and CTLA-4 expression on CD3+CD8+ cytotoxic T-cells, CD3+CD4+ T-helper cells, CD3+CD4+FOXP3+ regulatory T-cells) and tumor cells as well as macrophage/(DC) subtypes. In breast cancer, the PD-L1 fluorescence intensity on tumor cells showed a significantly higher predictive performance for overall survival with an area under receiver operating curves (AUC) of 0.72 (p<0.0001) than the percentage of PD-L1+ tumor cells (AUC: 0.54). In PD-L1 positive as well as negative breast cancers a close spatial relationship between T- cell subsets (CD3+CD4±CD8±FOXP3±PD-1±CTLA-4±) and Macrophage/DC subsets (CD68±CD163±CD11c±iNOS) was found prognostic relevant (p<0.0001). Conclusions 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. Disclosure Information N.C. Blessin: None. E. Bady: None. T. Mandelkow: None. C. Yang: None. J. Raedler: None. R. Simon: None. C. Fraune: None. M. Lennartz: None. S. Minner: None. E. Burandt: None. D. Höflmayer: None. G. Sauter: None. S.A. Weidemann: None.

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<0.0001) in 16923 prostate cancers, but to low tumor stage (p=0.027) and prolonged survival (p=0.0029) in 1341 breast cancers, and to low tumor stage (p<0.0001) in 744 colorectal cancers. No significant associations were found to tumor phenotype in 549 ovarian, 574 pancreatic, 549 bladder and 346 stomach cancers. Multiplex fluorescence IHC analysis of FoxP3 and Ki67 revealed comparable fractions of proliferating FoxP3+ cells in healthy tissues (average 12.3%, range 5.8–18.5%) and inflammatory conditions (average 7.6%, range 2.6–17.2%). Interestingly, the rate of Ki67+FoxP3+ cells was markedly higher in 36 bladder cancers (average 14.2%, range 0–49.3%) suggesting active expansion of FoxP3+ cells in cancer. 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. Möller: None. S.A. Weidemann: None. A.M. Luebke: None. D. Höflmayer: None. F. Büscheck: 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<0.0001), low pT stage (p<0.0001) and absence of nodal metastases (p=0.0005). 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. Möller: 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. Büscheck: None. A.M. Luebke: 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 \u003e20 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.

Abstract 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<0.0001) in 16923 prostate cancers, but to low tumor stage (p=0.027) and prolonged survival (p=0.0029) in 1341 breast cancers, and to low tumor stage (p<0.0001) in 744 colorectal cancers. No significant associations were found to tumor phenotype in 549 ovarian, 574 pancreatic, 549 bladder and 346 stomach cancers. Multiplex fluorescence IHC analysis of FoxP3 and Ki67 revealed comparable fractions of proliferating FoxP3+ cells in healthy tissues (average 12.3%, range 5.8-18.5%) and inflammatory conditions (average 7.6%, range 2.6-17.2%). Interestingly, the rate of Ki67+FoxP3+ cells was markedly higher in 36 bladder cancers (average 14.2%, range 0-49.3%) suggesting active expansion of FOXP3+ cells in cancer. 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.

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.