Abstract S-methyl-5′-thioadenosine phosphorylase (MTAP) is encoded by the MTAP gene located at 9p21 and is often homozygously co-deleted in cancer together with cyclin dependent kinase 2A (CDKN2A). As a result, MTAP deficiency results in a critical vulnerability of cancer cells towards drugs targeting multiple pathways. MTAP deficiency has also been found to predict poor response to immune checkpoint inhibitors. Ductal adenocarcinoma of the pancreas belongs to a group of tumors with a particularly high rate of MTAP deficiencies. To learn more on potential differences in the tumor-microenvironment and proliferative activity between MTAP deficient and proficient cancers, a tissue microarray containing 378 pancreatic ductal adenocarcinomas were analyzed by an 8-marker multiplex immunohistochemistry approach. With respect to innate immune infiltration, MTAP loss was associated with significantly higher densities of CD68+ macrophages compared to MTAP-retained tumors (p = 0.010), an effect predominantly driven by stromal CD68+PanCK- macrophages (p = 0.005). In contrast, total lymphocyte densities (CD45+CD68-PanCK-) showed only a mild, non-significant increase in MTAP-loss tumors. However, proliferative lymphocytes within the tumor compartment (CD45+CD68-MCM3+Ki67+PanCK+) were significantly enriched in MTAP-loss cases (p = 0.018), while proliferative intraepithelial lymphocytes lacking Ki67 (CD45+CD68-MCM3+PanCK+) showed a trend toward higher density (p = 0.052). Tumor cell proliferative activity was consistently elevated in MTAP-loss tumors, reflected by significantly increased labeling indices in very early (Ki67+MCM3+; p = 0.004), early (MCM3+; p = 0.007), and intermediate (Ki67+MCM3+; p = 0.030) cell-cycle phases. Clinicopathological parameters further modulated these associations. Early-stage tumors (pT1-2) displayed higher lymphocyte densities than pT3-4 tumors (p = 0.025), particularly in MTAP-loss cases, whereas MTAP-retained tumors showed higher densities of proliferating macrophages in pT1-2 than in pT3-4 lesions (p < 0.001). Nodal-negative tumors (pN0) exhibited higher intraepithelial lymphocyte (p = 0.044) and proliferative lymphocyte (p = 0.036) densities than pN+ tumors, most prominently in MTAP-retained cases. Similarly, lower-grade tumors (Grade 1-2) demonstrated significantly higher intraepithelial proliferative lymphocyte densities (p < 0.005) compared to Grade 3 tumors, again largely restricted to MTAP-retained cancers. It is concluded, that MTAP deficiency impacts the immune microenvironment of pancreatic ductal adenocarcinoma by promoting stromal macrophage accumulation and increased proliferation within immune and tumor compartments. Citation Format: Elena Bady, Jan H. Müller, Ronald Simon, Christian Bernreuther, Nina Schraps, Fiete Gehrisch, Natalia Gorbokon, Florian Viehweger, Frank Jacobsen, Guido Sauter, Katharina Möller, Andreas Lübke, Andrea Hinsch, Till S. Clauditz, Eike C. Burandt, Zhihao Huang. MTAP loss reshapes the immune and proliferative landscape of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7404.

BackgroundGlutathione peroxidase 2 (GPX2) has a pivotal role in removing reactive oxygen species (ROS) from cells. Although the number of studies analyzing GPX2 in cancer is still limited, data suggesting a role of altered GPX2 expression in various cancer types are accumulatin.MethodsTo better comprehend the role of GPX2 expression in cancer, GPX2 was analyzed by immunohistochemistry on tissue microarrays containing 18,555 samples from 148 different tumor types.ResultsA total of 95 of 148 tumor categories showed GPX2 expression in at least one case, and 61 tumor categories contained at least one strongly positive case. GPX2 positivity was most seen in colorectal adenocarcinomas (97.9%) and adenomas (100%), non-invasive urothelial carcinomas (88.9-100%), pancreatico-biliary cancers (83.4-94.7%), Brenner tumors of the ovary (89.7%), gastro-esophageal adenocarcinomas (83.1-87.3%), and seminomas (85.6%). Clinically important cancer types with infrequent and often weak GPX2 staining included sarcomas, lymphomas, high-grade serous ovarian carcinomas, prostatic adenocarcinomas, melanomas, mesotheliomas, and renal cell carcinomas. Reduced GPX2 staining was linked to microsatellite instability (MSI, p<0.0001), advanced pT stage (p=0.0044), nodal metastasis (p=0.0093), V1 (p=0.0020) and L1 (p=0.0057) in colorectal adenocarcinoma, invasive growth (p<0.0001), high grade (p=0.0013), nodal metastasis (p=0.0005), V1 (p=0.0004) and L1 (p=0.0001) in urothelial carcinoma, high grade in pancreatic ductal adenocarcinoma (p=0.0233), and MSI in gastric adenocarcinoma (p=0.0325).ConclusionIt is concluded, that GPX2 expression is common in cancer and preferably occurs in tumor entities derived from normal cell types with high GPX2 expression. In these tumors, reduced GPX2 expression is often linked to features of aggressive disease.

Abstract Although renal cell carcinoma (RCC), has become an important target for immune checkpoint therapies, the role of specific immune cell populations and checkpoint expression within its tumor microenvironment remains poorly understood. To learn more on the role of different immune cell populations and their expression of checkpoint proteins, we analyzed a tissue microarray containing 646 RCC samples using a 14-marker multiplex immunohistochemistry approach. Immune cell composition, checkpoint expression, and spatial distribution (stromal vs. intraepithelial) were quantified across 55 spatial immune parameters and evaluated for associations with clinicopathological features. With respect to individual cell types, high densities of FOXP3+ regulatory T-cells (Tregs) were significantly associated with advanced pT stage (p < 0.001), whereas other cell types or overall inflammatory infiltration showed no such association. Increased immune infiltration - specifically CD3+ and CD4+ T-cells - correlated with nodal metastasis (p < 0.05). Both associations were predominantly driven by stromal rather than intraepithelial immune cells. The expression of several checkpoint proteins in multiple cell types was associated with unfavorable tumor phenotype. Higher PD-1 expression on CD4+ (p < 0.001) and CD8+ T-cells (p = 0.03) and higher TIM-3 expression on FOXP3+ T-cells (p = 0.04) and CD11c+ dendritic cells (p = 0.04) were associated with advanced pT stage. High levels of PD-L1 expression on macrophages (p = 0.008), CTLA-4 expression on CD4+ (p = 0.04) and CD8+ (p = 0.005) T-cells, TIM-3 expression on FOXP3+ T-cells (p = 0.04), and PD-1 expression on CD4+ T-cells (p = 0.04) were linked to nodal metastasis. These immune checkpoint associations were likewise predominantly stromal in nature. It is concluded, that Immune cell composition and checkpoint pathway activity correlate with tumor progression and metastatic behavior in kidney cancer. These clinically relevant interactions arise primarily in the stromal compartment, highlighting the stroma as a key immune regulatory niche within the tumor microenvironment of kidney cancer. Citation Format: Zhihao Huang, Jan H. Müller, Ronald Simon, Christian Bernreuther, Nina Schraps, Fiete Gehrisch, Natalia Gorbokon, Florian Viehweger, Frank Jacobsen, Guido Sauter, Katharina Möller, Andreas Lübke, Andrea Hinsch, Till S. Clauditz, Eike C. Burandt, Elena Bady. Clinical impact of intraepithelial versus stromal immune cells in kidney cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7403.

TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) is an inhibitory immune checkpoint molecule which mainly occurs in subsets of inflammatory cells. On the surface membrane of cancer cells TIM-3 may represent a druggable target. To assess the prevalence of TIM-3 expression in cancer, a tissue microarray containing 17, 172 samples from 134 different tumor types and subtypes was analyzed by immunohistochemistry (IHC). TIM-3 expression of tumor cells and macrophages were semiquantitatively recorded. In cancer cells, a membranous TIM-3 positivity was predominantly seen in clear cell (61.9%) and papillary (62.5%) renal cell carcinomas (RCCs) but - less commonly - also in Hodgkin’s (25.0%) and non-Hodgkin’s lymphomas (up to 9.1%), epithelial thymomas (6.9%), ovarian carcinomas (0.7%-1.4%), seminomas (0.4%), and several mesenchymal neoplasms (up to 16.0%). High TIM-3 expression of tumor cells was linked to low grade (p=0.0079), low UICC stage (p=0.0085), low pT (p<0.0001), and pN0 (p=0.0067) in clear cell RCC (ccRCC), and to pM0 (p=0.0177) in papillary RCC (pRCC). TIM-3 expression of macrophages occurred in all tumor entities but its relationship with parameters of aggressive disease varied between entities. A high quantity of TIM-3 positive macrophages was linked to unfavorable tumor parameters such as high grade (p<0.0001), advanced pT stage (p=0.0126), and distant metastasis (p=0.0069) in ccRCC, as well as high grade (p<0.0001), HER2 positivity (p=0.0015), and absence of estrogen (p<0.0001) and progesterone receptor (p=0.0187) expression in invasive breast carcinomas of no special type. A high number of TIM-3 positive macrophages was linked to favorable tumor parameters such as L0 (p=0.0333) and V0 (p=0.0161) in mismatch repair protein proficient colorectal carcinoma, low pT (p=0.0128), L0 (p=0.0333), and V0 (p=0.0161), as well as a lack of rete testis (p=0.0053) and spermatic cord infiltration (p=0.0365) in testicular seminoma. It is concluded that tumor cell TIM-3 expression among epithelial or mesenchymal tumors is largely restricted to RCCs where high expression is associated with parameters of non-aggressive disease. TIM-3 expression in tumor associated macrophages is abundant and its clinical significance varies between tumor entities. Maximilian Lennartz, David Dum, Christian Bernreuther, Patrick Lebok, Guido Sauter, Andreas H Marx, Ronald Simon, Christoph Fraune, Natalia Gorbokon, Eike Burandt, Sarah Minner, Stefan Steurer, Till S Clauditz, Till Krech. TIM-3 expression in human cancer: A tissue microarray study on 17, 172 patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3207.

KDM6A, also known as UTX (ubiquitously transcribed X chromosome tetratricopeptide repeat protein) is a critical epigenetic regulator which is often subject to truncating mutations and subsequent expression loss in cancer. To interrogate the clinical significance of aberrant KDM6A expression, a tissue microarray containing prostate cancers from patients treated by prostatectomy was analyzed by immunohistochemistry. KDM6A immunostaining was weak in 33.5%, moderate in 52.9%, and strong in 7.3% of 13, 747 interpretable tumors while 880 (6.4%) tumors completely lacked KDM6A staining (KDM6A deficiency). The KDM6A status depended on the TMPRSS2:ERG fusion status. Strong KDM6A staining occurred in only 4.5% of ERG IHC negative, but in 11.1% of ERG IHC positive cancers while KDM6A deficiency occurred in 10.5% of ERG negative, but in only 1.4% of ERG positive cancers (p<0.0001 each). High KDM6A expression was linked to advanced pT, high Gleason grade, nodal positivity, and early PSA recurrence (p<0.0001 each). The prognostic role of high KDM6A expression was independent of pre- (Gleason score from biopsy, cT, PSA) and postoperatively (Gleason score from prostatectomy, pT, pN, R status) available parameters in all tumors (p<0.0001) and in ERG negative (p<0.0001) and positive subgroups (p=0.0049). In contrast, the outcome of KDM6A deficient cancers varied substantially between ERG negative (good prognosis) and ERG positive (bad prognosis) subgroups. In conclusion, the striking independent association of high KDM6A expression with poor prognosis suggests an important role of KDM6A expression in cancer cells which may have clinical utility in prostate cancer. The reciprocal role of KDM6A expression losses in ERG positive and negative cancers indicates a variable, cancer subtype dependent clinical effect of KDM6A deficiency. The clinical effects of anti-EZH2 drugs remains may depend on the ERG status in prostate cancer. Neele Heckmann, Natalia Gorbokon, Maximilian Lennartz, Martina Kluth, Eike Burandt, Till S Clauditz, Waldemar Wilczak, Stefan Steurer, Christian Bernreuther, Elena Bady, Lisa Hornsteiner, Ronald Simon, Hans Heinzer, Alexander Haese, Sarah Minner, Guido Sauter, Thorsten Schlomm, Markus Graefen, Florian Viehweger. Prevalence and prognostic role of KDM6A deficiency and overexpression in surgically treated prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4652.

KDM6A (lysine demethylase 6A) is an epigenetic regulator and part of the COMPASS-like complex. Truncating KDM6A mutations commonly occur in cancer and may result in a dependency on EZH2, a potential therapeutic target. Because only one copy of X chromosome genes is normally active in human cells, truncating KDM6A mutations result in a complete expression loss which can be seen by immunohistochemistry. To assess the prevalence of KDM6A expression loss and its role in cancer, a tissue microarray containing 14, 814 samples from 153 different tumor entities was analyzed. In normal tissues, KDM6A staining was ubiquitously seen in nuclei. At least one case with a complete KDM6A expression loss was observed in 58 of 153 tumor categories. KDM6A deficiency predominated in urothelial carcinomas (17.3-42.0%). Tumor entities with a KDM6A deficiency rate of 4-10% included adenocarcinomas of the gallbladder (9.3%), the stomach (up to 8.3%), the pancreas (7.7%), and the prostate (5.9%), endometrioid and serous endometrial carcinoma (up to 7.8%), squamous cell carcinomas (up to 7.5%), papillary renal cell carcinoma (5.1%), hepatocellular carcinoma (4.4%), and malignant melanoma (4.2%), and serous high-grade carcinoma of the ovary (4.1%). Reduced KDM6A expression was associated with advanced pT stage (p=0.0233), high grade (p=0.0002), distant metastasis (p=0.0152), and absence of ER (p<0.0001) and PR (p=0.0045) expression as well as HER2 amplification (p=0.0005) in breast cancer, nodal metastasis in squamous cell carcinomas (p=0.0498), advanced pT stage (p=0.0002), nodal metastasis (p=0.0112), and microsatellite instability (MSI; p=0.0147) in colorectal adenocarcinoma, and with MSI (p=0.0033) in gastric adenocarcinoma. Among gender agnostic cancers, KDM6A deficiency was markedly more common in tumors from males (4.3%) than from females (1.9%; p<0.0001). KDM6A sequencing of 78 bladder cancers revealed truncating mutations in 48.7% and non-truncating mutations in 5.1% of tumors. Truncating but not non-truncating KDM6A mutations were significantly associated with KDM6A expression loss (p<0.0001). All 15 (100%) male patients and 17 of 23 (73.9%) female patients with a truncating KDM6A mutation had a complete KDM6 expression loss. It is concluded that KDM6A deficiency predominates in urothelial neoplasms although it occurs in many different tumor entities, and that both reduced and absent KDM6A correlates with increased aggressiveness. Whether KDM6A deficient cancers are susceptible to EZH2 inhibitors in vivo remains to be seen. Florian Viehweger, Paul Wirth, Natalia Gorbokon, Maximilian Lennartz, Viktor Reiswich, Till Krech, Claudia Hube-Magg, Elena Bady, Lisa Hornsteiner, Guido Sauter, Ronald Simon, Stefan Steurer, Martina Kluth, Andreas H Marx, Andrea Hinsch, Andreas M Luebke, Anne Menz, Clara von Bargen, Frank Jacobsen, Piero Caneve, Ria Schlichter, Sebastian Dwertmann Rico, Simon Kind, Sören Weidemann, Maria Christina Tsourlakis, Waldemar Wilczak, Till S Clauditz, Christian Bernreuther, Sarah Minner. Prevalence of KDM6A deficiency in human cancer: a tissue microarray study on 14, 814 cancers from 153 different tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5294.

Lung cancer is the most frequent tumor related reason for death worldwide. It is hoped that a better understanding of the immune environment can foster the development of new immune checkpoint therapies to improve patient management and survival. To study the composition and prognostic impact of the immune infiltrate in non-small cell lung cancer, a tissue microarray containing 798 lung cancer patients, including 441 lung adenocarcinomas, 219 squamous cell carcinomas, was analyzed by multiplex fluorescence immunohistochemistry. A set of 14 antibodies was used to detect cytotoxic-, T-helper- and regulatory T-cells, B-cells, M2 macrophages, and dendritic cells (CD3, CD4, CD8, FOXP3, CD20, CD11c, CD163) expression of immune checkpoint molecules (TIGIT, PD-1, CTLA-4, TIM-3, PD-L1), and immune cell proliferation (Ki67) as well as panCK as an epithelial cell marker for tumor detection. Comparison with clinical data revealed significant associations between a high fraction of intratumoral CD3+ T-cells (p=0.0182), cytotoxic T-cells (p=0.0192) and dendritic cells (p=0.0080) and favorable prognosis, and between a low fraction of intratumoral CD3+T-cells (p=0.0007) and advanced tumor stage. A high Ki-67 labelling index was linked to poor prognosis (p=0.0002) and nodal positivity in all 798 lung cancer patients (p<0.0001), as well as in the subsets of 441 lung adenocarcinomas (p=0.0002) and 219 lung squamous cell carcinomas (p=0.0015). No prognostic relevance was found for the expression of the immune checkpoint molecules TIGIT, PD-L1, TIM-3, CTLA-4 and PD-1 on the immune cells in our series of cancers. In conclusion, a high T-cell infiltrate is linked to beneficial tumor characteristics in non-small cell lung cancers. Lisa Hornsteiner, Natalia Gorbokon, Jonas B Raedler, Claudia Hube-Magg, Monika Kaszubowski, Nina Schraps, Katharina Möller, Florian Viehweger, Florian Lutz, Till S Clauditz, Christoph Fraune, Eike Burandt, Ronald Simon, Guido Sauter, Martin Reck, Klaus Raabe, Maximilian Lennartz, Stefan Steurer, Elena Bady. Prognostic impact of the immune environment in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6486.

9p21 deletions including CDKN2A belong to the most common chromosomal aberrations in cancer. Most of the topical interest in 9p21 comes S-methyl-5′-thioadenosine phosphorylase (MTAP) which is also included in most 9p21 deletions. Homozygous 9p21 deletions are the major cause for MTAP deficiency which results in a critical vulnerability of cancer cells towards drugs targeting several different pathways. Fluorescence in situ hybridization (FISH) is the gold standard method for deletion detection because single cells are always analyzed independent of any admixture of non-neoplastic cells. To assess the prevalence of heterozygous and homozygous deletions in cancer, a tissue microarray containing 7, 172 samples from 125 different tumor entities was analyzed by FISH. Consecutive sections were immunostained for MTAP and p16. Among 4, 999 evaluable cases, 10.7% had a heterozygous and 7.4% a homozygous deletion. Heterozygous and homozygous did not predominate in the same tumor categories. Homozygous deletions were most frequent in mesotheliomas (up to 47.8%), pancreatic ductal adenocarcinoma (44, 7%), and in urothelial carcinoma (up to 36.4%) while the heterozygous deletions were most common in squamous cell carcinomas (up to 34.3%), leiomyosarcoma (31.4%), adenocarcinomas of the esophagus (30.1%), malignant melanoma (29.4%), and cholangiocarcinoma (21.2%). Accordingly, the proportion of homozygous deletions within the 9p21 deleted cases varied markedly. It was high in mesothelioma (up to 91.7%), urothelial carcinoma (up to 80%), pancreatic ductal adenocarcinoma (76.4%), and mucinous carcinoma of the ovary (66.7%). It was intermediate in squamous cell carcinomas of different organs (10-50%) and adenocarcinoma of the lung (46.3%), and it was particularly low in endometrioid (9.1%) and in high-grade serous (4%) carcinoma of the ovary. Both MTAP and p16 were not detectable in cancers with 9p21 deletion, but otherwise the results differed markedly between these proteins. The expression of MTAP was markedly lower in heterozygously deleted cancers as in wild type cancers (p<0.0001) while p16 expression did not change significantly from normal to heterozygously deleted cases. In summary, these data provide a unique FISH based catalogue of 9p21 deletions in cancer and demonstrate that the proportions of heterozygous and homozygous MTAP deletions vary markedly between tumor entities. The near perfect concordance between homozygous MTAP deletions and absence of MTAP immunostaining further supports the use of IHC for detecting MTAP deficiency. Natalia Gorbokon, Katharina Teljuk, Katharina Möller, Seyma Büyücek, Maximilian Lennartz, Sebastian Dwertmann Rico, Simon Kind, Viktor Reiswich, Florian Viehweger, Elena Bady, Lisa Hornsteiner, Guido Sauter, Ronald Simon, Stefan Steurer, David Dum, Sarah Minner, Frank Jacobsen, Till S Clauditz, Martina Kluth. Prevalence of heterozygous and homozygous 9p21 deletions in human cancer: a tissue microarray study on 4999 tumors from 125 different tumor types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3359.

Objective:The gene lysine demethylase 6A (KDM6A) located on chromosome Xp11 often shows truncating mutations in urothelial carcinoma. Mutations resulting in protein expression loss can be detected by immunohistochemistry (IHC). Methods:A tissue microarray with >2,500 bladder tumors was analyzed by IHC. 78 cancers were sequenced for KDM6A. Results:KDM6A expression loss decreased from 36% of 345 pTaG2 low-grade to 23% of 152 pTaG2 high-grade and 18.5% of 92 pTaG3 tumors (p=0.0004) but not further in pT2-4 cancers (17.2-21.9%). KDM6A staining was unrelated to pT, pN, grade, and overall survival (p>0.1894) in 636 patients with pT2-4 cancers. KDM6A loss was more common in male (22.2%) than in female patients (15.4%; p=0.0067), and in tumors from males with Y-chromosome loss (36.1%) than without Y-loss (16.3%; p<0.0001). A KDM6A loss occurred in all 15 male and in 17 (74%) of 23 female patients with a truncating KDM6A mutation, but only 15 (75%) of 20 male and 17 (81%) of 21 female patients with KDM6A expression loss had a truncating mutation. Conclusions:KDM6A expression loss is frequent in urothelial carcinoma and mostly due to truncating mutations. KDM6A IHC may be a useful tool for the distinction of neoplastic from non-neoplastic urothelial cells in follow-up examinations of patients with KDM6A deficient cancers.

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.

Although there is rising evidence that immune cell subpopulations that are in direct contact with the tumor cells (intraepithelial) can predict response to immune checkpoint therapy and patients' outcome, a comprehensive assessment of intraepithelial immune cells and their spatial interplay is lacking. To assess intraepithelial leukocyte densities, immune checkpoint expression, and spatial interactions in 43 carcinoma entities, 4915 tumor samples in a tissue microarray format were analyzed using a deep learning framework and BLEACH&STAIN multiplex fluorescence immunohistochemistry. This approach enabled single-cell resolution quantification of 21 biomarkers through 7 sequential staining and imaging rounds. Immune and tumor cells were classified into 54 subpopulations. The mean intraepithelial immune cell density of CD8+ cytotoxic T cells, CD4+ T-helper cells, FOXP3+ regulatory T cells, CD20+ B cells, M1/M2 macrophages, and CD11c+ dendritic cells varied markedly between tumor entities and individual tumors. For instance, 88(±90) cells/mm2 were found in tubular breast cancer, 661(±729) cells/mm2 in colorectal cancer, and up to 2325(±2131) cells/mm2 in squamous cell cancers from various origins. Unsupervised cluster analysis revealed a "cluster a" of 634 patients from almost all different tumor entities with an exceptionally high density of intraepithelial immune cells that was characterized by a unique interaction profile along with the highest immune checkpoint expression. Across all analyzed tumor entities, the intraepithelial highly inflamed cluster a was significantly linked to low pathologic tumor stage (P < .001). The data from this study provide a comprehensive characterization of intraepithelial immune cells across 43 different human carcinomas and identified an inflamed pan-cancer phenotype characterized by strong interactions of intraepithelial CD8+ cytotoxic T cells, CD4+ T cells, dendritic cells, and M2 macrophages, along with highest levels of TIM3, PD-1, and CTLA-4 expression that is linked to a favorable tumor phenotype.

The cell adhesions molecule L1 (CD171; L1CAM) was initially characterized as an essential protein for neural development but subsequent studies have pinpointed towards a critical role in cancer. Because of its expression in only few normal tissues and its membranous nature L1CAM is a promising drug target. To better comprehend the role of L1CAM expression in different cancer types, a tissue microarray containing 20,079 samples from 135 different tumor entities and 608 samples of 76 different normal tissue types was analyzed. A membranous L1CAM staining was found in 1175 (9.1%) of the 12888 interpretable tumor samples, including 301 (2.3%) with weak, 569 (4.4%) with moderate, and 305 (2.4%) with strong positivity. A total of 73 of 135 tumor entities showed L1CAM expression in at least one case, and 36 tumor categories included at least one case with strong L1CAM staining. The frequency of L1CAM positivity was particularly high in several subtypes of neural and neuroendocrine neoplasms (11.1-100%), endometrium carcinoma (15.4-54.8%), ovarian cancer (22.0-44.6%), cervical adenocarcinoma (27.3%), malignant melanoma (25.3-31.0%), malignant mesothelioma (20.0-29.2%), adenocarcinomas of the gastrointestinal and the biliopancreatic tractus (9.1-18.9%), and in advanced urothelial tumors (13.0-18.2%). In urinary bladder cancer, there was a significant association between L1CAM expression and invasive tumor growth (pTa vs. pT2-4, p<0.0001). In colorectal adenocarcinoma, L1CAM positivity was linked to left-sided tumor location and microsatellite instability. However, L1CAM staining was unrelated to tumor phenotype in muscle-invasive urothelial carcinoma, clear cell and papillary renal cell carcinoma, serous high-grade carcinoma of the ovary, endometrioid endometrium carcinoma, gastric adenocarcinoma, and in pancreatic adenocarcinoma. It is concluded that many different tumor entities could benefit from L1CAM targeting drugs once these should provide sufficiently safe and efficient. However, L1CAM expression does not appear to confer an aggressive phenotype to affected cancer cells. Seyma Büyücek, Magali Lurati, Katharina Möller, Florian Viehweger, Ria Schlichter, Anne Menz, Andreas M Luebke, Viktor Reiswich, Martina Kluth, Elena Bady, Lisa Hornsteiner, Claudia Hube-Magg, Andrea Hinsch, Florian Lutz, Sören Weidemann, Frank Jacobsen, David Dum, Christian Bernreuther, Patrick Lebok, Guido Sauter, Andreas H Marx, Ronald Simon, Christoph Fraune, Natalia Gorbokon, Eike Burandt, Sarah Minner, Stefan Steurer, Till S Clauditz, Till Krech, Viktoria Chirico, Maximilian Lennartz. L1CAM(CD171) can be highly expressed in many tumor types but lacks strong associations with tumor phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 100.

The stimulator of interferon genes (STING) is expressed on various cell types and tumour entities, where it might enhance antitumoural effects of the immune system and impact tumour angiogenesis. However, the clinical significance of STING expression in tumour cells as compared to tumour-associated inflammatory cells is not fully resolved. To evaluate the clinical significance of STING expression in different cell types of colorectal cancer (CRC), 1,905 patients were analysed by multiplex fluorescence immunohistochemistry in a tissue microarray format in combination with a deep learning algorithm for automated cell detection. High STING expression on tumour was associated with microsatellite instability (MSI, p<0.0001), low tumour stage (pT, p=0.0013), absence of nodal metastasis (pN, p=0.0003) and tumour localisation in the right colon (p<0.0001). Subgroup analysis of tumours with and without MSI did not show associations between STING expression on tumour cells and pT or pN. While the number of CD68- leukocytes and macrophages was related to MSI, low pT ​and pN0, there were significant associations between a high percentage of STING-positive macrophages and CD68- leukocytes to low pT (p<0.0001 each) and the absence of metastases for a high percentage of STING-positive macrophages (p=0.0033). In summary, our data show that high STING expression in tumour cells is strongly linked to MSI while STING expression on macrophages and CD68- leukocytes is tightly linked to the extent of tumour-associated inflammation in CRC.