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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.

Cytokeratin 13 (CK13) is a type I acidic low molecular weight cytokeratin, which is mainly expressed in urothelium and in the squamous epithelium of various sites of origin. Loss of CK13 has been implicated in the development and progression of squamous epithelial neoplasms. To comprehensively determine CK13 expression in normal and neoplastic tissues, a tissue microarray containing 10,439 samples from 131 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. CK13 immunostaining was detectable in 42 (32.1%) of the 131 tumor categories including 24 (18.3%) tumor types with at least one strongly positive case. The highest rate of positive staining was found in various urothelial neoplasms (52.1-92.3%) including Brenner tumor of the ovary (86.8%) and in squamous cell carcinomas from various sites of origin (39.1-77.6%), Warthin tumors of parotid glands (66.7%), adenosquamous carcinomas of the cervix (33.3%), thymomas (16.0%), and endometroid carcinomas of the ovary (15.3%). Twenty other epithelial or germ cell neoplasms showed - a usually weak - CK13 positivity in less than 15% of the cases. In bladder cancer, reduced CK13 expression was linked to high grade and advanced stage (p 0.0001 each). In squamous cell carcinoma of the cervix, reduced CK13 immunostaining was related to high grade (p = 0.0295) and shortened recurrence-free (p = 0.0094) and overall survival (p = 0.0274). In a combined analysis of 1,151 squamous cell carcinomas from 11 different sites of origin, reduced CK13 staining was linked to high grade (p = 0.0050). Our data provide a comprehensive overview on CK13 expression in normal and neoplastic human tissues. CK13 expression predominates in urothelial neoplasms and in squamous cell carcinomas of different organs, and a loss of CK13 expression is associated with aggressive disease in these tumors.

Fatty acid–binding proteins (FABPs) play a pivotal role in the metabolism of fatty acids and are expressed in a tissue-specific manner. FABP1 is most abundantly expressed in the liver where it accounts for about 10% of the total cytosolic protein and is thought to have diagnostic utility. To comprehensively determine FABP1 expression in normal and neoplastic tissues, a tissue microarray containing 17,071 samples from 150 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. Among normal tissues, a strong FABP1 immunostaining was observed in hepatocytes, proximal tubuli of the kidney and epithelium of small intestine, appendix, and the colorectum. FABP1 positivity was found in 24 of 150 tumor categories, including 17 tumor categories with at least 1 strongly positive case. The highest FABP1 positivity rates were seen in colorectal adenomas (86%), in colorectal adenocarcinomas (71.1%), and in hepatocellular carcinomas (65.3%), followed by mucinous carcinoma of the ovary (34.6%), cholangiocarcinoma (21.6%), and various adenocarcinomas from the digestive tract (10–23%). Eleven additional entities had positivity rates between 0.2 and 6.5%. FABP1 staining was not seen in 169 primary adenocarcinomas of the lung. In colorectal cancer, reduced FABP1 expression was linked to poor-grade, right-sided tumor location, microsatellite instability (p p = 0.001), but unrelated to pT and pN status. FABP1 expression has considerably high tumor specificity. As FABP1 expression was virtually absent in adenocarcinomas of the lung, FABP1 immunohistochemistry might be particularly helpful to assist in the identification of metastatic colorectal or gastrointestinal adenocarcinoma to the lung.

Context.— Special AT-rich sequence–binding protein 2 (SATB2) induces local chromatin loops to facilitate transcription. SATB2 immunostaining is commonly used as a marker for colorectal adenocarcinoma and osteosarcoma. Objective.— To extend our knowledge on the diagnostic value of SATB2 analysis in a comprehensive set of human tumors. Design.— Tissue microarrays with 15 012 samples from 120 tumor types and 608 samples of 76 different normal tissues were analyzed. Results.— SATB2 positivity was found in 89 of 120 different tumor types (74%), including 59 of 120 (49%) with at least 1 moderately positive tumor and 38 of 120 tumor types (32%) with at least 1 strongly positive tumor. Expression was frequent in adenomas (44/42–47/44; 94%–96% positive), adenocarcinomas (1747 of 2023; 86%), and various subtypes of neuroendocrine neoplasms (3/7–12/12; 43%–100%) of the colorectum and appendix, Merkel cell carcinoma (25 of 34, 74%), osteosarcomas (15 of 25; 60%), and papillary renal cell carcinoma (RCC) (121 of 235; 52%). Associations to clinicopathologic tumor features were assessed in colorectal and kidney cancers. In colorectal cancer, weak SATB2 expression was linked to high pT (P < .001), nodal metastasis (P < .001), right-sided tumor location (P < .001), microsatellite instability (P < .001), and BRAF mutations (P = .02). In papillary RCC, low SATB2 expression was associated with high pT (P = .02), distant metastasis (P = .04), and reduced tumor-specific survival (P = .04). In clear cell RCC, low SATB2 expression was linked to high pT (P < .001), high Union for International Cancer Control stage (P < .001), high Thoenes grade (P = .02), and reduced recurrence-free survival (P = .02). Conclusions.— Strong SATB2 expression argues for a colorectal origin within adenocarcinomas and neuroendocrine neoplasms. Weak SATB2 expression reflects progression and poor prognosis in colorectal and kidney cancer.

Uroplakin 1B (Upk1b) stabilizes epithelial cells lining the bladder lumen to prevent rupturing during bladder distension. Little is known about Upk1b expression in other normal and malignant tissues. To comprehensively evaluate the potential diagnostic and prognostic utility of Upk1b expression analysis, a tissue microarray containing 14,061 samples from 127 different tumor types and subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. Upk1b immunostaining was found in 61 (48%) different tumor types including 50 (39%) with at least one moderately positive and 39 tumor types (31%) with at least one strongly positive tumor. Highest positivity rates were found in urothelial neoplasms (58-95%), Brenner tumors of the ovary (92%), epithelioid mesothelioma (87%), serous carcinoma of the ovary (58%) and the endometrium (53%) as well as in squamous cell carcinoma of the head and neck (18-37%), lung (39%), and esophagus (26%). In urothelial carcinoma, low Upk1b expression was linked to high grade and invasive tumor growth (P .0001 each) and nodal metastasis (P = .0006). Our data suggest diagnostic applications of Upk1b immunohistochemistry in panels for the distinction of malignant mesothelioma from adenocarcinoma of the lung, urothelial carcinoma from prostatic adenocarcinoma in the bladder, or pancreaticobiliary and gastroesophageal from colorectal adenocarcinoma.

Villin is a protein of the brush border of epithelial cells, which is used as an immunohistochemical marker for colorectal and gastrointestinal neoplasms. However, other tumor entities can also express villin.To comprehensively determine villin expression, tissue microarrays containing 14,398 samples from 118 different tumor types as well as 608 samples of 76 different normal tissues were analyzed by immunohistochemistry.Villin was found in 54 of 118 tumor categories, including 36 tumor categories with strong staining. Villin expression was frequent in colorectal (60-100%), upper gastrointestinal tract (61-100%), pancreatobiliary (25-86%), and renal tumors (≤18%) as well as in mucinous ovarian cancers (67%), yolk sac tumors (76%) and in neuroendocrine neoplasms (22-41%). Reduced villin expression was linked to advanced pT stage, lymph vessel invasion, and microsatellite instability (Our data support a high utility of villin immunohistochemistry for the identification of tumors with gastrointestinal, pancreatobiliary, and yolk sac tumor origin. However, considering that at least a weak villin positivity in some tumor cells occurred in 54 different tumor categories, villin immunohistochemistry should be applied as a part of a marker panel rather than as a stand-alone marker.

CTLA-4 is an inhibitory immune checkpoint receptor and a negative regulator of anti-tumor T-cell function. This study is aimed for a comparative analysis of CTLA-4

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.

Figure S1a: Representative images of the BLEACH&STAIN 15+1 staining. Figure S1b: BLEACH&STAIN 15+1 validation. Figure S2: Deep learning framework for automated cell type identification. Figure S3: Performance of a Deep Learning-based framework for cell type identification compared to manual set thresholds. Figure S4: Comparison between BLEACH&STAIN and manual PD-L1 assessment. Figure S5: Immune phenotype identification. Figure S6: Association between the immune phenotypes and the individual immune cell parameters. Figure S7: Representative image of the 6 major immune phenotypes of Figure 2 Figure S8a: Association between PD-L1 phenotypes and clinico-histopathological parameters. Figure S8b: Distribution of six PD-L1 phenotypes across 44 carcinoma entities. Figure S9: Spatial orchestration of immune cells across the PD-L1 immune phenotypes. Figure S10: Representative image of the spatial orchestration shown in Figure 3 Figure S11: Composition of immune cells across the three PD-L1 phenotypes Figure S12: Prognosis analysis of spatial immune parameters in breast cancer for overall 5-years survival after surgery. Table S1: TMA cohort of the PD-L1 15+1 study. Table S2: List of the used antibodies, antigen retrieval (AR), dilutions, and Opal dyes for multiplex fluorescence immunohistochemistry. Table S3: List of 132 immune cell (sub)population. Table S4: Associations between spatial parameters and PD-L1 phenotypes were independent from the used threshold. Table S5: Immune landscape of 3098 human carcinoma samples.

Carboxypeptidase A1 (CPA1) is a zinc metalloprotease that is produced in pancreatic acinar cells and plays a role in cleaving C-terminal branched-chain and aromatic amino acids from dietary proteins. This study assessed the utility of immunohistochemical CPA1 staining for diagnosing pancreatic acinar cell carcinoma (ACC). A total of 12,274 tumor samples from 132 different tumor types and subtypes as well as 8 samples each of 76 different normal tissue types were interpretable by immunohistochemistry in a tissue microarray format. CPA1 was strongly expressed in acinar cells of all normal pancreas samples but not in any other normal tissues. CPA1 immunostaining was detected in 100% of 11 pancreatic ACCs and 1 mixed acinar endocrine carcinoma, but absent in 449 pancreatic ductal adenocarcinomas, 75 adenocarcinomas of the ampulla Vateri, and 11,739 other evaluable cancers from 128 different tumor entities. A weak to moderate diffuse staining of epithelial and stromal cells of cancer tissues immediately adjacent to non-neoplastic pancreatic acinar cells often occurred and was considered to be caused by the diffusion of the highly abundant CPA1 from normal acinar cells that may have suffered some autolytic cell damage. In conclusion, our data show that CPA1 is a highly sensitive and largely specific marker for normal and neoplastic pancreatic acinar cells. CPA1 immunohistochemistry greatly facilitates the otherwise often difficult diagnosis of pancreatic ACC.

To evaluate cytokeratin 19 (CK19) expression in normal and cancerous tissues, 15,977 samples from 122 tumor types and 608 samples of 76 normal tissue types were analyzed by immunohistochemistry (IHC). In normal tissues, CK19 expression occurred in epithelial cells of most glandular organs but was strictly limited to the basal cell layer of nonkeratinizing squamous epithelium and absent in the skin. CK19 expression in ≥90% of cases was seen in 34% of the tumor entities including the adenocarcinomas of the pancreas (99.4%), colorectum (99.8%), esophagus (98.7%), and stomach (97.7%), as well as breast cancer (90.0%-100%), high-grade serous (99.1%) or endometrioid (97.8%) ovarian cancer, and urothelial carcinoma (92.6%-100%). A low CK19 positivity rate (0.1-10%) was seen in 5 of 122 tumor entities including hepatocellular carcinoma and seminoma. A comparison of tumor versus normal tissue findings demonstrated that upregulation and downregulation of CK19 can occur in cancer and that both alterations can be linked to unfavorable phenotypes. CK19 downregulation was linked to high grade (p = 0.0017) and loss of estrogen receptor- and progesterone receptor-expression (p 0.0001 each) in invasive breast carcinoma of no special type. CK19 upregulation was linked to nodal metastases in neuroendocrine tumors and papillary thyroid carcinomas (p 0.05 each) and to poor grade in clear cell renal cell carcinoma (p 0.05). CK19 upregulation was particularly common in squamous cell carcinomas. We concluded that CK19 IHC might separate primary liver cell carcinoma from liver metastases, seminoma from other testicular tumors, and helps in the detection of early neoplastic transformation in squamous epithelium.

Cytokeratin 10 (CK10) is a type I acidic low molecular weight cytokeratin which is mainly expressed in keratinizing squamous epithelium of the skin. Variable levels of CK10 protein have been described in squamous carcinomas of different sites and in some other epithelial neoplasms. To comprehensively determine the prevalence of CK10 expression in normal and neoplastic tissues, a tissue microarray containing 11,021 samples from 131 different tumor types and subtypes was analyzed by immunohistochemistry. CK10 immunostaining was detectable in 41 (31.3 %) of 131 tumor categories, including 18 (13.7 %) tumor types with at least one strongly positive case. The highest rate of positive staining was found in squamous cell carcinomas from various sites of origin (positive in 18.6 %-66.1 %) and in Warthin tumors of salivary glands (47.8 %), followed by various tumor entities known to potentially exhibit areas with squamous cell differentiation such as teratomas (33.3 %), basal cell carcinomas of the skin (14.3 %), adenosquamous carcinomas of the cervix (11.1 %), and several categories of urothelial neoplasms (3.1 %-16.8 %). In a combined analysis of 956 squamous cell carcinomas from 11 different sites of origin, reduced CK10 staining was linked to high grade (p 0.0001) and advanced stage (p = 0.0015) but unrelated to HPV infection. However, CK10 staining was not statistically related to grade (p = 0.1509) and recurrence-free (p = 0.5247) or overall survival (p = 0.5082) in 176 cervical squamous cell carcinomas. In the urinary bladder, CK10 staining occurred more commonly in muscle-invasive (17.7 %) than in non-invasive urothelial carcinomas (4.0 %-6.0 %; p 0.0001). In summary, our data corroborate a role of CK10 as a suitable marker for mature, keratinizing squamous cell differentiation in epithelial tissues. CK10 immunohistochemistry may thus be instrumental for a more objective evaluation of the clinical significance of focal squamous differentiation in cancer.

Purpose Expansion of CD8+ cytotoxic Tlymphocytes is a prerequisite for anti-cancer immune activity and has gained interest in the era of immune checkpoint therapy. Methods To understand the CD8+ T cell dynamics in the tumor microenvironment, we used multiplex fluorescence immunohistochemistry to quantitate CD8+ proliferation (Ki67 co-expression) in tissue microarrays from 1107 colorectal, 642 renal cell, 1066 breast, 375 ovarian, 451 pancreatic and 347 gastric cancer samples. Results The density and the percentage of proliferating (Ki67+) CD8+ T cells were both highly variable between tumor types as well as between patients with the same tumor type. Elevated density and percentage of proliferating CD8+ cytotoxic T cells were significantly associated with favorable tumor parameters such as low tumor stage, negative nodal stage (p ≤ 0.0041 each), prolonged overall survival (p ≤ 0.0028 each) and an inflamed immune phenotype (p = 0.0025) in colorectal cancer and, in contrast, linked to high tumor stage, advanced ISUP/Fuhrman/Thoenes grading (each p ≤ 0.003), shorter overall survival (p ≤ 0.0330 each) and an immune inflamed phenotype (p = 0.0094) in renal cell cancer. In breast, ovarian, pancreatic and gastric cancer the role of (Ki67+)CD8+ Tcells was not linked to clinicopathological data. Conclusion Our data demonstrate a tumor type dependent prognostic impact of proliferating (Ki67+)CD8+ Tcells and an inverse impact in colorectal and renal cell cancer.

Introduction: Mucin 5AC (MUC5AC) belongs to the family of secreted gel-forming mucins. It is physiologically expressed in some normal mucin producing epithelial cells but also in pancreatic, ovarian, and colon cancer cells. The role of MUC5AC expression in cancer is not fully understood. This study was designed to explore the role of MUC5AC for pancreatic cancer progression, its association to microsatellite instability, and its diagnostic utility. Methods: Mucin 5AC expression was studied immunohistochemically in a tissue microarray (TMA) from 532 pancreatic cancers, 61 cancers of the ampulla Vateri, six acinar cell carcinomas and 12 large sections of pancreatitis. Results: Mucin 5AC staining was interpretable in 476 of 599 (79%) arrayed cancers. Staining was completely absent in normal pancreas and pancreatitis, but frequent in pancreatic cancer. Membranous and cytoplasmic MUC5AC expression was most common in pancreatic adenocarcinomas (71% of 423), followed by carcinomas of the ampulla Vateri (43% of 47), and absent in six acinar cell carcinomas. Mucin 5AC expression was unrelated to tumor phenotype (tumor stage, tumor grade, lymph node, and distant metastasis), and microsatellite instability in ductal adenocarcinomas and carcinomas of the ampulla Vateri. Conclusion: Our study indicates that MUC5AC is an excellent biomarker for pancreatic cancer diagnosis, especially to support the sometimes-difficult diagnosis on small biopsies. Mucin 5AC expression is unrelated to pancreatic cancer aggressiveness.

Mucin 5AC (MUC5AC) is a secreted gel-forming mucin expressed by several epithelia. In the colon, MUC5AC is expressed in scattered normal epithelial cells but can be abundant in colorectal cancers. To clarify the relationship of MUC5AC expression with parameters of tumor aggressiveness and mismatch repair deficiency (dMMR) in colorectal cancer, a tissue microarray containing 1812 colorectal cancers was analyzed by immunohistochemistry. MUC5AC expression was found in 261 (15.7%) of 1,667 analyzable colorectal cancers. MUC5AC expression strongly depended on the tumor location and gradually decreased from proximal (27.4% of cecum cancers) to distal (10.6% of rectal cancers; p p p

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: 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.

Desmoglein-3 (Dsg3) is a transmembrane glycoprotein which is preferably found in desmosomes of keratinocytes in squamous epithelium. Both loss and upregulation of Dsg3 have been implicated in cancer progression. To comprehensively evaluate Dsg3 expression in normal and neoplastic tissues, a tissue microarray containing 15,869 samples from 137 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. Dsg3 immunostaining was detectable in 47 (34.3 %) tumor categories including 15 (10.9 %) tumor types with at least one strongly positive case. The highest rate of Dsg3 positivity was found in squamous cell carcinomas from various sites (71.2-97.3 %), basal cell carcinomas of the skin (41.9 %), various tumors from salivary glands (12.9-38.9 %), and in urothelial neoplasms (2.1-20.7 %). Dsg3 positivity in less than 10 % of cases was seen in 23 additional cancer categories. Dsg3 staining was almost always weak and rarely moderate in these tumors. High Dsg3 expression was linked to invasive growth in urothelial carcinoma (p 0.0001), as well as to advanced pT stage (p = 0.0102), nodal metastasis (p = 0.0162), blood vessel infiltration (p = 0.0189) and lymph vessel infiltration (p = 0.0151) in colorectal cancer. Reduced Dsg3 expression was linked to high grade in a cohort of 599 squamous cell carcinomas from 11 different sites of origin (p 0.0001). Associations between Dsg3 immunostaining and clinicopathological features were not found in invasive breast cancer of no special type, ductal adenocarcinomas of the pancreas and in gastric adenocarcinomas. In summary, Dsg3 expression predominates in squamous cell carcinomas and loss of Dsg3 immunostaining goes along with dedifferentiation of these tumors. The identification of focal squamous differentiation in other neoplasms may constitute a diagnostic application of Dsg3 immunohistochemistry.

Combined analysis of cytokeratin 7 (CK7) and cytokeratin 20 (CK20) is often used for assessing the origin of metastatic cancer. To evaluate the diagnostic utility of CK7 and CK20, tissue microarrays containing 15,424 samples from 120 different tumor types and subtypes and 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry. CK7 positivity was seen in 52% (8.7% weak, 5.9% moderate, 37% strong) and CK20 positivity in 23% (5.1% weak, 3.4% moderate, 15% strong) of interpretable tumors. Of 8390 positive tumors, 1181 (14%) showed positivity for CK7 and CK20, 5380 (64%) showed positivity for CK7 alone, and 1829 (22%) showed positivity for CK20 alone. CK20 predominated in gastrointestinal tract, urothelial and Merkel cell carcinomas. CK7 was usually negative in prostate cancer and colorectal cancer. Combined evaluation of CK7/CK20 revealed the best diagnostic utility in CK20 positive tumors, where CK7 negativity is often linked to colorectal origin while CK7 positivity argues for urothelial origin or mucinous ovarian cancer. Associations with unfavorable tumor features were found for cytokeratin 7 loss in breast cancer of no special type, urothelial and renal cell carcinomas, for CK7 overexpression in high-grade serous ovarian and gastric cancer, and for CK20 overexpression in urothelial carcinoma. CK20 loss was linked to MSI in gastric (p = 0.0291) and colorectal adenocarcinoma (p 0.0001). These analyses provide comprehensive data on the frequency of CK7 and CK20 immunostaining - alone or in combination - in human cancers. These data facilitate interpretation of CK7/CK20 immunostaining in cancers.