Prognostic Impact and Spatial Interplay of Immune Cells in Urothelial Cancer.

BACKGROUND: Quantity and the spatial relationship of specific immune cell types can provide prognostic information in bladder cancer. OBJECTIVE: To characterize the spatial interplay and prognostic role of different immune cell subpopulations in bladder cancer. DESIGN, SETTING, AND PARTICIPANTS: A total of 2463 urothelial bladder carcinomas were immunostained with 21 antibodies using BLEACH&STAIN multiplex fluorescence immunohistochemistry in a tissue microarray format and analyzed using a framework of neuronal networks for an image analysis. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Spatial immune parameters were compared with histopathological parameters and overall survival data. RESULTS AND LIMITATIONS: The identification of > 300 different immune cell subpopulations and the characterization of their spatial relationship resulted in numerous spatial interaction patterns. Thirty-nine immune parameters showed prognostic significance in univariate analyses, of which 16 were independent from pT, pN, and histological grade in muscle-invasive bladder cancer. Among all these parameters, the strongest association with prolonged overall survival was identified for intraepithelial CD8+ cytotoxic T cells (time-dependent area under receiver operating characteristic curve [AUC]: 0.70), while stromal CD8+ T cells were less relevant (AUC: 0.65). A favorable prognosis of inflamed cancers with high levels of "exhaustion markers" suggests that TIM3, PD-L1, PD-1, and CTLA-4 on immune cells do not hinder antitumoral immune response in tumors rich of tumor infiltrating immune cells. CONCLUSIONS: The density of intraepithelial CD8+ T cells was the strongest prognostic feature in muscle-invasive bladder cancer. Given that tumor cell killing by CD8+ cytotoxic T lymphocytes through direct cell-to-cell-contacts represents the "terminal end route" of antitumor immunity, the quantity of "tumor cell adjacent CD8+ T cells" may constitute a surrogate for the efficiency of cancer recognition by the immune system that can be measured straightaway in routine pathology as the CD8 labeling index. PATIENT SUMMARY: Quantification of intraepithelial CD8+ T cells, the strongest prognostic feature identified in muscle-invasive bladder cancer, can easily be assessed by brightfield immunohistochemistry and is therefore "ready to use" for routine pathology.

Automated Prognosis Marker Assessment in Breast Cancers Using BLEACH&STAIN Multiplexed Immunohistochemistry.

Prognostic markers in routine clinical management of breast cancer are often assessed using RNA-based multi-gene panels that depend on fluctuating tumor purity. Multiplex fluorescence immunohistochemistry (mfIHC) holds the potential for an improved risk assessment. To enable automated prognosis marker detection (i.e., progesterone receptor [PR], estrogen receptor [ER], androgen receptor [AR], GATA3, TROP2, HER2, PD-L1, Ki67, TOP2A), a framework for automated breast cancer identification was developed and validated involving thirteen different artificial intelligence analysis steps and an algorithm for cell distance analysis using 11+1-marker-BLEACH&STAIN-mfIHC staining in 1404 invasive breast cancers of no special type (NST). The framework for automated breast cancer detection discriminated normal glands from malignant glands with an accuracy of 98.4%. This approach identified that five (PR, ER, AR, GATA3, PD-L1) of nine biomarkers were associated with prolonged overall survival (p ≤ 0.0095 each) and two of these (PR, AR) were found to be independent risk factors in multivariate analysis (p ≤ 0.0151 each). The combined assessment of PR-ER-AR-GATA3-PD-L1 as a five-marker prognosis score showed strong prognostic relevance (p < 0.0001) and was an independent risk factor in multivariate analysis (p = 0.0034). Automated breast cancer detection in combination with an artificial intelligence-based analysis of mfIHC enables a rapid and reliable analysis of multiple prognostic parameters. The strict limitation of the analysis to malignant cells excludes the impact of fluctuating tumor purity on assay precision.

BLEACH&STAIN 15-marker Multiplexed Imaging in 3,098 Human Carcinomas Reveals Six Major PD-L1-driven Immune Phenotypes with Distinct Spatial Orchestration.

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.

Nonredundant Upregulation of CD112R (PVRIG) and PD-1 on Cytotoxic T Lymphocytes Located in T Cell Nests of Colorectal Cancer.

Focal T lymphocyte aggregates commonly occur in colorectal cancer; however, their biological significance is unknown. To study focal aggregates of T lymphocytes, a deep learning-based framework for automated identification of T cell accumulations (T cell nests) was developed using CD8, PD-1, CD112R, and Ki67 multiplex fluorescence immunohistochemistry. To evaluate the clinical significance of these parameters, a cohort of 523 colorectal cancers with clinical follow-up data was analyzed. Spatial analysis of locally enriched CD8+ T cell density and cell-to-cell contacts identified T cell nests in the tumor microenvironment of colorectal cancer. CD112R and PD-1 expressions on CD8+ T cells located in T cell nests were found to be elevated compared with those on CD8+ T cells in all other tumor compartments (P < .001 each). Although the highest mean CD112R expression on CD8+ T cells was observed at the invasive margin, the PD-1 expression on CD8+ T cells was elevated in the center of the tumor (P < .001 each). Across all tissue compartments, proliferating CD8+ T cells showed higher relative CD112R and PD-1 expressions than those shown by non-proliferating CD8+ T cells (P < .001 each). Integration of all available spatial and immune checkpoint expression parameters revealed a superior predictive performance for overall survival (area under the curve, 0.65; 95% CI, 0.60-0.70) compared with the commonly used CD8+ tumor-infiltrating lymphocyte density (area under the curve, 0.57; 95% CI, 0.53-0.61; P < .001). Cytotoxic T cells with elevated CD112R and PD-1 expression levels are orchestrated in T cell nests of colorectal cancer and predict favorable patient outcomes, and the spatial nonredundancy underlies fundamental differences between both inhibitory immune checkpoints that provide a rationale for dual anti-CD112R/PD-1 immune checkpoint therapy.

Automated Ki-67 labeling index assessment in prostate cancer using artificial intelligence and multiplex fluorescence immunohistochemistry.

The Ki-67 labeling index (Ki-67 LI) is a strong prognostic marker in prostate cancer, although its analysis requires cumbersome manual quantification of Ki-67 immunostaining in 200-500 tumor cells. To enable automated Ki-67 LI assessment in routine clinical practice, a framework for automated Ki-67 LI quantification, which comprises three different artificial intelligence analysis steps and an algorithm for cell-distance analysis of multiplex fluorescence immunohistochemistry (mfIHC) staining, was developed and validated in a cohort of 12,475 prostate cancers. The prognostic impact of the Ki-67 LI was tested on a tissue microarray (TMA) containing one 0.6 mm sample per patient. A 'heterogeneity TMA' containing three to six samples from different tumor areas in each patient was used to model Ki-67 analysis of multiple different biopsies, and 30 prostate biopsies were analyzed to compare a 'classical' bright field-based Ki-67 analysis with the mfIHC-based framework. The Ki-67 LI provided strong and independent prognostic information in 11,845 analyzed prostate cancers (p < 0.001 each), and excellent agreement was found between the framework for automated Ki-67 LI assessment and the manual quantification in prostate biopsies from routine clinical practice (intraclass correlation coefficient: 0.94 [95% confidence interval: 0.87-0.97]). The analysis of the heterogeneity TMA revealed that the Ki-67 LI of the sample with the highest Gleason score (area under the curve [AUC]: 0.68) was as prognostic as the mean Ki-67 LI of all six foci (AUC: 0.71 [p = 0.24]). The combined analysis of the Ki-67 LI and Gleason score obtained on identical tissue spots showed that the Ki-67 LI added significant additional prognostic information in case of classical International Society of Urological Pathology grades (AUC: 0.82 [p = 0.002]) and quantitative Gleason score (AUC: 0.83 [p = 0.018]). The Ki-67 LI is a powerful prognostic parameter in prostate cancer that is now applicable in routine clinical practice. In the case of multiple cancer-positive biopsies, the sole automated analysis of the worst biopsy was sufficient. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

PD-L1 expression and CD8 positive lymphocytes in human neoplasms: A tissue microarray study on 11,838 tumor samples.

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.

Loss of TROP2 and epithelial cell adhesion molecule expression is linked to grade progression in pTa but unrelated to disease outcome in pT2-4 urothelial bladder carcinomas.

Introduction: Trophoblast cell surface antigen 2 (TROP2; EpCAM2) is a transmembrane glycoprotein which is closely related to EpCAM (EpCAM; EpCAM1). Both proteins share partial overlapping functions in epithelial development and EpCAM expression but have not been comparatively analyzed together in bladder carcinomas. TROP2 constitutes the target for the antibody-drug conjugate Sacituzumab govitecan (SG; TrodelvyTM) which has been approved for treatment of metastatic urothelial carcinoma by the United States Food and Drug administration (FDA) irrespective of its TROP2 expression status. Methods: To evaluate the potential clinical significance of subtle differences in TROP2 and EpCAM expression in urothelial bladder cancer, both proteins were analyzed by multiplex fluorescence immunohistochemistry in combination with a deep-learning based algorithm for automated cell detection on more than 2,700 urothelial bladder carcinomas in a tissue microarray (TMA) format. Results: The staining pattern of TROP2 and EpCAM were highly similar. For both proteins, the staining intensity gradually decreased from pTa G2 low grade (TROP2: 68.8±36.1; EpCAM: 21.5±11.7) to pTa G2 high grade (64.6±38.0; 19.3±12.2) and pTa G3 (52.1±38.7; 16.0±13.0, p<0.001 each). In pT2-4 carcinomas, the average TROP2 and EpCAM staining intensity was intermediate (61.8±40.9; 18.3±12.3). For both proteins, this was significantly lower than in pTa G2 low grade (p<0.001 each) but also higher than in pTa G3 tumors (p=0.022 for TROP2, p=0.071 for EpCAM). Within pT2-4 carcinomas, the TROP2 and EpCAM staining level was unrelated to pT, grade, UICC-category, and overall or tumor-specific patient survival. The ratio TROP2/EpCAM was unrelated to malignant phenotype and patient prognosis. Conclusion: Our data show that TROP2 and EpCAM expression is common and highly interrelated in urothelial neoplasms. Despite of a progressive loss of TROP2/EpCAM during tumor cell dedifferentiation in pTa tumors, the lack of associations with clinicopathological parameters in pT2-4 cancer argues against a major cancer driving role of both proteins for the progression of urothelial neoplasms.

T-Cell Density at the Invasive Margin and Immune Phenotypes Predict Outcome in Vulvar Squamous Cell Cancer.

Background: Although quantification of tumor infiltrating lymphocytes (TILs) has become of increasing interest in immuno-oncology, only little is known about TILs infiltration in the tumor microenvironment and its predictive value in vulvar cancer. Methods: Immunohistochemistry and automated digital image analysis was applied to measure the densities of CD3+ (DAKO, #IR503) and CD8+ (DAKO, #IR623) TILs at the invasive margin and in the center of 530 vulvar squamous cell cancers. Results: An elevated density of CD3+ T-cell at the invasive margin was significantly associated with low tumor stage (p = 0.0012) and prolonged survival (overall survival [OS] p = 0.0027, progression free survival [PFS] p = 0.024) and was independent from tumor stage, nodal stage, grade, and HPV-status in multivariate analysis (p < 0.05). The prognostic impact of CD3+ cells in the center of the tumor was weaker compared to the invasive margin (OS p = 0.046, PFS 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 (p = 0.0071) and PFS (p = 0.0027). Conclusion: Our data demonstrate a high prognostic value of CD3+ T-cells at the invasive margin and immune phenotypes in vulvar squamous cell cancer.

Semi-automated validation and quantification of CTLA-4 in 90 different tumor entities using multiple antibodies and artificial intelligence.

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+ cells between different tumor entities. To quantify CTLA-4+ cells, 4582 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. 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). 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). 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.

Prevalence of proliferating CD8+ cells in normal lymphatic tissues, inflammation and cancer.

CD8+ cytotoxic T-lymphocytes are essential components of the anti-tumor immunity. To better understand the expansion of CD8+ T-cells we used multiplex fluorescence immunohistochemistry to study Ki67+CD8+ cells in normal lymphoid tissues, selected inflammatory diseases and cancers in 41 large sections/ microenvironment tissue microarrays (TMAs) as well as 765 samples in a conventional TMA format. The evaluation of more than 20 different compartments of normal lymphoid tissues revealed that the percentage of proliferating (ki67+) CD8+ cells did commonly not exceed 3%. In inflammations, the percentage of Ki67+CD8+ cells was more variable and higher compared to normal tissues. In cancers, the percentage of Ki67+CD8+ cells was higher in the tumor center than at the invasive margin. In the tumor center of 765 colorectal cancers, the density of Ki67+CD8+ cells and the percentage of proliferating CD8+ cytotoxic T-cells was significantly associated with microsatellite instability (p<0.0001), pT (p<0.0002) and pN category (p<0.0098). In summary, these data show that the percentage of Ki67+CD8+ cells is usually at a baseline proliferation rate below 3% in healthy secondary lymphoid organs. This rate is often markedly higher in inflammatory and neoplastic diseases compared to normal tissues. The striking link with unfavorable tumor features in colorectal cancer suggest a potential clinical utility of assessing the percentage of Ki67+CD8+ cells to predict patients outcome.

Prognostic role of proliferating CD8+ cytotoxic Tcells in human cancers.

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.

High level of EZH2 expression is linked to high density of CD8-positive T-lymphocytes and an aggressive phenotype in renal cell carcinoma.

PURPOSE: Enhancer of zeste homolog 2 (EZH2), the catalytic part of the Polycomb repressive complex 2 (PRC2), has a prognostic role in renal cell carcinoma (RCC) and was recently shown to modulate the immune response by reducing tumor cell immunogenicity. METHODS: To investigate whether the prognostic role of EZH2 might be driven by a modified immune environment, more than 1800 RCCs were analyzed in a tissue microarray for EZH2 expression and CD8 positive lymphocytes were quantitated by automated digital imaging. RESULTS: EZH2 positivity was found in 75.2% of 1603 interpretable tumors. In clear cell RCC, high EZH2 expression was significantly linked to high ISUP, Furmann, and Thoenes grade (p < 0.0001 each), advanced stage (p < 0.0001), nodal (p = 0.0190) and distant metastasis (p < 0.0001) as well as shortened overall (p < 0.0027) and recurrence free survival (p < 0.0001). The density of CD8+ cells varied from 0 to 5048 cells/mm2 (Median 120 cells/mm2). A high CD8+ count was significantly associated with high ISUP, Fuhrmann, and Thoenes grade (p < 0.0001 each), advanced tumor stage (p = 0.0041), distant metastasis (p = 0.0026) as well as reduced overall survival (p = 0.0373) and recurrence free survival (p = 0.0450). The density of CD8+ cells continuously increased with raising EZH2 levels (p < 0.0001). CONCLUSION: Our data support a striking prognostic role of both EZH2 expression and the density of CD8+ cells in RCC. The tight relationship of EZH2 expression and CD8+ cell counts in RCC is consistent with models suggesting that EZH2 overexpression can be caused by high lymphocyte content in certain tumor types. Such a mechanism could explain the unique finding of high lymphocyte counts driving poor prognosis in RCC patients.

Prevalence of CD8+ cytotoxic lymphocytes in human neoplasms.

PURPOSE: Immune checkpoint inhibitors have recently been approved by the US FDA as first and/or second line therapy in a subset of cancer types. Recent evidence suggests that the quantity of tumor infiltrating lymphocytes (TILs) influences the likelihood of response to immune checkpoint inhibitors. Here, we set out to assess the density of CD8+ lymphocytes in a wide range of different cancer types and subtypes. METHODS: The density of CD8+ lymphocytes was compared across different cancer types using tissue microarrays (TMAs) composed of up to 50 tumor samples each from 84 different cancer types and subtypes. In total 2652 cancers and 608 normal tissues were successfully analyzed by CD8 immunohistochemistry followed by automated image analysis of digitized slides. RESULTS: We found that the median CD8+ lymphocyte counts ranged from 6 cells/mm2 in pleomorphic adenoma up to 1573 cells/mm2 in Hodgkin's lymphoma. The CD8 counts were generally lower in normal tissues compared to cancer tissues. Blood vessels of the spleen were the only non-lymphatic tissue staining positive for CD8. Tumor types approved for checkpoint inhibitor therapy, including malignant melanoma (81), muscle invasive urothelial carcinoma (119), small cell lung cancer (120), clear cell renal cell cancer (153), squamous cell carcinoma (189) and adenocarcinoma of the lung (328) as well as Hodgkin's lymphoma (1573) were all ranking among the upper half of our list. Comparably high CD8 densities (median cells/mm2) were also found in several rare and aggressive cancer types including Merkel cell carcinoma (70), angiosarcoma (95), anaplastic thyroid cancer (156) and embryonal carcinoma of the testis (186). In 73 of the 84 analyzed cancer types, the highly variable CD8 counts occasionally exceeded the average CD8 count of tumors for which checkpoint inhibitors have been approved. CONCLUSION: These data support the concept that among most tumor types at least some individual cancers may benefit from treatment with immune checkpoint inhibitors.

MMR Deficiency is Homogeneous in Pancreatic Carcinoma and Associated with High Density of Cd8-Positive Lymphocytes.

BACKGROUND: Microsatellite instability (MSI) has emerged as a predictive biomarker for immune checkpoint inhibitor therapy. Cancer heterogeneity represents a potential obstacle for the analysis of predicitive biomarkers. MSI has been reported in pancreatic cancer, but data on the possible extent of intratumoral heterogeneity are lacking. METHODS: To study MSI heterogeneity in pancreatic cancer, a tissue microarray (TMA) comprising 597 tumors was screened by immunohistochemistry with antibodies for the mismatch repair (MMR) proteins MLH1, PMS2, MSH2, and MSH6. RESULTS: In six suspicious cases, large section immunohistochemistry and microsatellite analysis (Bethesda panel) resulted in the identification of 4 (0.8%) validated MSI cases out of 480 interpretable pancreatic ductal adenocarcinomas. MSI was absent in 55 adenocarcinomas of the ampulla of Vater and 7 acinar cell carcinomas. MMR deficiency always involved MSH6 loss, in three cases with additional loss of MSH2 expression. Three cancers were MSI-high and one case with isolated MSH6 loss was MSS in PCR analysis. The analysis of 44 cancer-containing tumor blocks revealed that the loss of MMR protein expression was always homogeneous in affected tumors. Automated digital image analysis of CD8 immunostaining demonstrated markedly higher CD8 + tumor infiltrating lymphocytes in tumors with (mean = 685, median = 626) than without (mean = 227; median = 124) MMR deficiency (p < 0.0001), suggesting a role of MSI for immune response. CONCLUSIONS: Our data suggest that MSI occurs early in a small subset of ductal adenocarcinomas of the pancreas and that immunohistochemical MMR analysis on limited biopsy or cytology material may be sufficient to estimate MMR status of the entire cancer mass.

Loss of cytoplasmic survivin expression is an independent predictor of poor prognosis in radically operated prostate cancer patients.

Survivin is an inhibitor of apoptosis. Aberrant survivin expression occurs in malignant tumors and has often been linked to unfavorable patient outcome. Here we analyzed 12 432 prostate cancers by immunohistochemistry. Survivin immunostaining was regularly expressed at high levels in normal prostate epithelium but expression was often reduced in prostate cancers. Among 9492 evaluable prostate cancers, 9% expressed survivin strongly, 19% moderately, 28% weakly, and 44% lacked it. Loss of cytoplasmic survivin was seen in advanced tumor stage, higher Gleason score, preoperative PSA levels, and Ki-67 labeling index, and associated with earlier PSA recurrence (P < .0001). Survivin loss was significantly more common in cancers carrying TMPRSS2:ERG fusions (61% survivin negative) than in ERG wild-type cancers (32% survivin negative; P < .0001). Multivariate analysis revealed that reduced cytoplasmic survivin expression predicted poor prognosis independent from Gleason score, pT, pN, and serum PSA level. This was valid for ERG-positive and ERG-negative cancers. Survivin expression loss even retained its prognostic impact in 1020 PTEN deleted cancers, a group that is already characterized by dismal patient prognosis. In conclusion, reduced survivin expression is associated with more aggressive tumors and inferior prognosis in prostate cancer.

High RSF1 protein expression is an independent prognostic feature in prostate cancer.

Background: Remodelling and spacing factor 1 (RSF1) is involved in the regulation of chromatin remodelling and represents a potential therapeutic target. High RSF1 expression has been linked to adverse tumour features in many cancer types, but its role in prostate cancer is uncertain.Methods: In this study, RSF1 expression was analysed by immunohistochemistry on a tissue microarray with 17,747 prostate cancers.Results: Nuclear RSF1 staining of 16,456 interpetable cancers was considered strong, moderate, weak and negative in 25.2%, 48.7%, 5.3% and 20.8% of cancers respectively. Positive RSF1 expression was associated with advanced tumour stage, high Gleason grade, lymph node metastasis (p < .0001 each), early biochemical recurrence (p < .0003) and more frequent in the ERG positive than in the ERG negative subset (88% versus 71%; p < .0001). Subset analysis revealed, that associations between RSF1 expression and unfavourable tumour phenotype and PSA recurrence were present in both subgroups but stronger in the ERG negative than in the ERG positive subset. The univariate Cox proportional hazard ratio for PSA recurrence-free survival for strong versus negative RSF1 expression was a weak 1.60 compared with 5.91 for the biopsy Gleason grade ≥4 + 4 versus ≤3 + 3. The positive association of RSF1 protein detection with deletion of 3p13, 10q23 (PTEN), 12p13, 16q23, and 17p13 (p < .0001 each) suggest a role of high RSF1 expression in the development of genomic instability.Conclusion: In summary, the results of our study identify RSF1 as an independent prognostic marker in prostate cancer with a particularly strong role in ERG negative cases.

Upregulation of SPDEF is associated with poor prognosis in prostate cancer.

SAM pointed domain-containing Ets transcription factor (SPDEF), a member of the ETS transcription factor family, has been associated with prostate cancer development; however, its role in tumour development and progression is controversial. In the present study, SPDEF expression was analysed on a tissue microarray with >12,000 prostate cancer samples. SPDEF expression levels were higher in most prostate cancer samples than in normal prostate epithelium, suggesting SPDEF was upregulated in cancer. Nuclear SPDEF expression was identified in 80% of prostate cancer samples, and considered weak in 26.4%, moderate in 40.1% and strong in 13.5% of cases. SPDEF positivity was significantly associated with tumour stage, Gleason grade, lymph node metastasis and PSA recurrence (all P<0.0001). SPDEF overexpression was more common in ERG positive (94%) than in ERG negative cancer (69%; P<0.0001). Elevated SPDEF expression predicted poor prognosis independent from established prognostic parameters, including Gleason grade, pT, pN, serum PSA level and nodal status (P<0.01). In summary, SPDEF overexpression was associated with aggressive behaviour, particularly in ERG negative prostate cancer, and may have potential for clinical application.

Expression of the immune checkpoint receptor TIGIT in seminoma.

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

Immune Exclusion Is Frequent in Small-Cell Carcinoma of the Bladder.

Small-cell cancer of the urinary bladder is a rare but highly aggressive disease. It is currently unclear whether immune checkpoint therapies that have been approved for urothelial carcinomas will also be efficient in small-cell carcinomas. In this study, we analyzed potential predictors of response including PD-L1 expression and the quantity and location of tumor-infiltrating lymphocytes (TILs) in 12 small-cell and 69 "classical" urothelial cancers by immunohistochemistry. The analysis revealed that small-cell carcinomas were characterized by the virtual absence of PD-L1 expression and an "immune-excluded" phenotype with only a few TILs in the center of the tumor (CT). In small-cell carcinomas, the average immune cell density in the CT (CD3: 159 ± 206, CD8: 87 ± 169 cells/mm2) was more than 3 times lower than that in the urothelial carcinomas (CD3: 625 ± 800, p < 0.001; CD8: 362 ± 626 cells/mm2, p = 0.004) while there was no significant difference in the immune cell density at the invasive margin (IM) (small-cell carcinomas CD3: 899 ± 733, CD8: 404 ± 433 cells/mm2; urothelial carcinomas CD3: 1167 ± 1206, p = 0.31; CD8: 582 ± 864 cells/mm2, p = 0.27). Positive PD-L1 staining was found in 39% of urothelial cancers, but in only 8% of small-cell bladder cancer cases (p = 0.04). Concordant with these data, a sharp decrease of PD-L1 positivity from >80% to 0% positive cells and of TILS in the CT from 466-1063 CD3-positive cells/mm2 to 50-109 CD3-positive cells/mm2 was observed in two cancers with clear-cut progression from "classical" urothelial to small-cell carcinoma. In conclusion, these data demonstrate that small-cell bladder cancer commonly exhibits an immune-excluded phenotype.