KLK7 expression in human tumors: a tissue microarray study on 13,447 tumors.

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 < 0.0001) in colorectal adenocarcinoma, nodal metastasis in hepatocellular carcinoma (p = 0.0382), advanced pathological tumor stage in papillary thyroid cancer (p = 0.0132), and low grade of malignancy in a cohort of 719 squamous cell carcinomas from 11 different sites of origin (p < 0.0001). 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.

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.

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.

Cancer-Associated Fibroblasts Induce Proliferation and Therapeutic Resistance to Everolimus in Neuroendocrine Tumors through STAT3 Activation.

INTRODUCTION: Cancer-associated fibroblasts (CAF) have been identified as relevant contributors to cancer progression and drug resistance in many tumors. Although neuroendocrine tumors (NET) are often associated with a strong stromal reaction, no study has addressed whether CAF are involved in progression and therapeutic resistance in NET. The aim of this study was to characterize the role of CAF in NET. METHODS: We established primary CAF cultures derived from NET liver metastases to study the effect on NET cell lines NT-3 and BON. Immunohistochemistry was performed on tissue sections of primary and metastatic NET tissue. RESULTS: Immunohistochemistry identified CAF dispersed in between tumor cells and within fibrotic bands separating tumor cell clusters in NET. Stimulating NET cells with CAF decreased expression of SSTR2 and chromogranin A and induced expression of CXCR4. CAF induced a 2.3-fold increase in proliferation and completely reversed the response to everolimus in NT-3 cells. We identified STAT3 as the main signaling pathway induced by CAF. STAT3 targeting by small interfering RNA knockdown and inhibitors prevented CAF-induced proliferation and restored everolimus responsiveness. STAT3 activation in NET tissue was associated with decreased chromogranin A expression, increased Ki-67 index, and decreased 5-year overall and progression-free survival. CAF directly influence proliferation and therapeutic response in NET cells. CONCLUSION: Identifying STAT3 as the contributing pathway of this so far neglected tumor-stroma interaction has the potential to become a new therapeutic target to halt tumor growth and to restore therapeutic responsiveness in NET.

GATA3 Expression in Human Tumors: A Tissue Microarray Study on 16,557 Tumors.

INTRODUCTION: GATA3 is a transcription factor involved in epithelial cell differentiation. GATA3 immunostaining is used as a diagnostic marker for breast and urothelial cancer but can also occur in other neoplasms. METHODS: To evaluate GATA3 in normal and tumor tissues, a tissue microarray containing 16,557 samples from 131 different tumor types and subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. RESULTS: GATA3 positivity was found in 69 different tumor types including 23 types (18%) with at least one strongly positive tumor. Highest positivity rates occurred in noninvasive papillary urothelial carcinoma (92-99%), lobular carcinoma (98%), carcinoma of no special type of the breast (92%), basal cell carcinoma of the skin (97%), invasive urothelial carcinoma (73%), T-cell lymphoma (23%), adenocarcinoma of the salivary gland (16%), squamous cell carcinoma of the skin (16%), and colorectal neuroendocrine carcinoma (12%). In breast cancer, low GATA3 staining was linked to high pT stage (p = 0.03), high BRE grade (p < 0.0001), HER2 overexpression (p = 0.0085), estrogen and progesterone receptor negativity (p < 0.0001 each), and reduced survival (p = 0.03). CONCLUSION: Our data demonstrate that GATA3 positivity can occur in various tumor entities. Low levels of GATA3 reflect cancer progression and poor patient prognosis in breast cancer.

Pattern of MUC6 expression across 119 different tumor types: A tissue microarray study on 15 412 tumors.

Mucin 6 (MUC6) is a secreted gel-forming mucin covering the surfaces of gastrointestinal and other tissues. Published work demonstrates that MUC6 can also be expressed in several cancer types and can aid in the distinction of different tumor entities. To systematically analyze MUC6 expression in normal and cancerous tissues, a tissue microarray containing 15 412 samples from 119 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. At least a weak MUC6 positivity was seen in 50 of 119 (42%) tumor entities. Thirty-three tumor entities included tumors with strong positivity. MUC6 immunostaining was most frequent in mucinous carcinomas of the breast (44%), adenocarcinomas of the stomach (30%-40%) and esophagus (35%), and neuroendocrine carcinomas of the colon. Strong MUC6 staining was linked to advanced pT stage (p = 0.0464), defective mismatch repair status and right-sided tumor location (p < 0.0001 each) in colorectal cancer, as well as to high tumor grade (p = 0.0291), nodal metastasis (p = 0.0485), erb-b2 receptor tyrosine kinase 2 positivity (p < 0.0001) and negative estrogen receptor (p = 0.0332)/progesterone receptor (p = 0.0257) status in breast carcinomas of no special type. The broad range of tumor types with MUC6 expression limits the utility of MUC6 immunohistochemistry for the distinction of different tumor types.

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.

Cytokeratin 7 and cytokeratin 20 expression in cancer: A tissue microarray study on 15,424 cancers.

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.

Trophoblast Cell Surface Antigen 2 Expression in Human Tumors: A Tissue Microarray Study on 18,563 Tumors.

INTRODUCTION: Trophoblast cell surface antigen 2 (TROP2) is the target of sacituzumab govitecan, an antibody-drug conjugate approved for treatment of triple negative breast cancer and urothelial carcinoma. METHODS: A tissue microarray containing 18,563 samples from 150 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by TROP2 immunohistochemistry. RESULTS: TROP2 positivity was found in 109 tumor categories, including squamous cell carcinomas of various origins, urothelial, breast, prostate, pancreatic, and ovarian cancers (>95% positive). High TROP2 expression was linked to advanced stage (p = 0.0069) and nodal metastasis (p < 0.0001) in colorectal cancer as well as to nodal metastasis in gastric adenocarcinoma (p = 0.0246) and papillary thyroid cancer (p = 0.0013). Low TROP2 expression was linked to advanced stage in urothelial carcinoma (p < 0.0001), high pT (p = 0.0024), and high grade (p < 0.0001) in breast cancer, as well as with high Fuhrmann grade (p < 0.0001) and pT stage (p = 0.0009) in papillary renal cell carcinomas. CONCLUSION: TROP2 is expressed in many epithelial neoplasms. TROP2 deregulation can be associated with cancer progression in a tumor-type dependent manner. Since anti-TROP2 cancer drugs have demonstrated efficiency, they may be applicable to a broad range of tumor entities in the future.

Cytokeratin 10 (CK10) expression in cancer: A tissue microarray study on 11,021 tumors.

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.

Diagnostic and prognostic impact of cytokeratin 18 expression in human tumors: a tissue microarray study on 11,952 tumors.

BACKGROUND: Cytokeratin 18 (CK18) is an intermediate filament protein of the cytokeratin acidic type I group and is primarily expressed in single-layered or "simple" epithelial tissues and carcinomas of different origin. METHODS: To systematically determine CK18 expression in normal and cancerous tissues, 11,952 tumor samples from 115 different tumor types and subtypes (including carcinomas, mesenchymal and biphasic tumors) as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. RESULTS: CK18 was expressed in normal epithelial cells of most organs but absent in normal squamous epithelium. At least an occasional weak CK18 positivity was seen in 90 of 115 (78.3%) tumor types. Wide-spread CK18 positivity was seen in 37 (31.9%) of tumor entities, including adenocarcinomas of the lung, prostate, colon and pancreas as well as ovarian cancer. Tumor categories with variable CK18 immunostaining included cancer types arising from CK18 positive precursor cells but show CK18 downregulation in a fraction of cases, tumor types arising from CK18 negative precursor cells occasionally exhibiting CK18 neo-expression, tumors derived from normal tissues with variable CK18 expression, and tumors with a mixed differentiation. CK18 downregulation was for example seen in renal cell cancers and breast cancers, whereas CK18 neo-expression was found in squamous cell carcinomas of various origins. Down-regulation of CK18 in invasive breast carcinomas of no special type and clear cell renal cell carcinomas (ccRCC) was related to adverse tumor features in both tumors (p ≤ 0.0001) and poor patient prognosis in ccRCC (p = 0.0088). Up-regulation of CK18 in squamous cell carcinomas was linked to high grade and lymph node metastasis (p < 0.05). In summary, CK18 is consistently expressed in various epithelial cancers, especially adenocarcinomas. CONCLUSIONS: Down-regulation or loss of CK18 expression in cancers arising from CK18 positive tissues as well as CK18 neo-expression in cancers originating from CK18 negative tissues is linked to cancer progression and may reflect tumor dedifferentiation.

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.

A non-diploid DNA status is linked to poor prognosis in renal cell cancer.

PURPOSE: DNA ploidy measurement has earlier been suggested as a potentially powerful prognostic tool in many cancer types, but the role in renal tumors is still unclear. METHODS: To clarify its prognostic impact, we analyzed the DNA content of 1320 kidney tumors, including clear cell, papillary and chromophobe renal cell carcinoma (RCC) as well as renal oncocytoma and compared these data with clinico-pathological parameters and patient prognosis. RESULTS: A non-diploid DNA content was seen in 37% of 1276 analyzable renal tumors with a striking predominance in chromophobe carcinoma (74.3% of 70 cases). In clear cell carcinoma, a non-diploid DNA content was significantly linked to high-grade (ISUP, Fuhrman, Thoenes; p < 0.0001 each), advanced tumor stage (p = 0.0011), distant metastasis (p < 0.0001), shortened overall survival (p = 0.0010), and earlier recurrence (p < 0.0001). In papillary carcinoma, an aberrant DNA content was significantly linked to high Fuhrman grade (p = 0.0063), distant metastasis (p = 0.0138), shortened overall survival (p = 0.0010), and earlier recurrence (p = 0.0003). CONCLUSION: In summary, the results of our study identify a non-diploid DNA content as a predictor of an unfavorable prognosis in clear cell and papillary carcinoma.

Immunohistochemically detectable thyroglobulin expression in extrathyroidal cancer is 100% specific for thyroidal tumor origin.

Thyroglobulin is a secreted 660 kDa glycoprotein produced by thyroid follicular cells used in diagnostic pathology to secure or exclude a thyroidal origin of metastases of unknown primary tumors. This study was performed to estimate specificity of thyroglobulin immunohistochemistry. 9974 tumor samples from 109 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format. Thyroglobulin was strongly expressed in all normal thyroid samples but not in any other normal tissues. Thyroglobulin immunostaining was detected in 99.1% of 106 thyroid adenomas, 98.1% of 364 papillary, 95.2% of 147 follicular, and 7.5% of 40 anaplastic thyroid cancers. Twelve of 15 thyroid samples that were thyroglobulin negative on TMAs showed at least a weak focal thyroglobulin positivity in corresponding large sections, suggesting higher sensitivity of large section analysis. Thyroglobulin positivity in one diffuse large B-cell lymphoma of the thyroid, one chondrosarcoma metastasis to the thyroid, and 42.4% of 92 medullary thyroid cancers was considered to be caused by diffusion of thyroidal colloid from destroyed or even intact adjacent follicles. Thyroglobulin positivity was, however, not seen in 6403 extrathyroidal tumors from 104 different tumor types and subtypes. Our data demonstrate a complete specificity of positive thyroglobulin immunostaining for thyroid origin in tumor tissues obtained from extrathyroidal locations. However, for all tumors located within the thyroid, false positivity can occur as a result of tissue contamination by thyroglobulin rich thyroid colloid from adjacent normal tissue.

Elevated MUC5AC expression is associated with mismatch repair deficiency and proximal tumor location but not with cancer progression in colon cancer.

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 < 0.0001). MUC5AC expression was also strongly linked to dMMR. dMMR was found in 21.3% of 169 cancers with MUC5AC positivity but in only 4.6% of 1051 cancers without detectable MUC5AC expression (p < 0.0001). A multivariate analysis showed that dMMR status and tumor localization predicted MUC5AC expression independently (p < 0.0001 each). MUC5AC expression was unrelated to pT and pN status. This also applied to the subgroups of 1136 proficient MMR (pMMR) and of 84 dMMR cancers. The results of our study show a strong association of MUC5AC expression with proximal and dMMR colorectal cancers. However, MUC5AC expression is unrelated to colon cancer aggressiveness.

Loss of the adhesion molecule CEACAM1 is associated with early biochemical recurrence in TMPRSS2:ERG fusion-positive prostate cancers.

Altered expression of the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) has been linked to adverse tumor features in various cancer types. To better understand the role of CEACAM1 in prostate cancer, we analyzed a tissue microarray containing tumor spots from 17,747 prostate cancer patients by means of immunohistochemistry. Normal prostate glands showed intense membranous CEACAM1 positivity. Immunostaining was interpretable in 13,625 cancers and was considered high in 28%, low in 43% and absent in 29% of tumors. Low and lost CEACAM1 expression was strongly linked to adverse tumor features including high classical and quantitative Gleason grade, lymph node metastasis, advanced tumor stage, positive surgical margin, a high number of genomic deletions and early biochemical recurrence (p < 0.0001 each). Subset analysis of molecularly defined cancer subsets revealed that these associations were strongest in V-ets avian erythroblastosis virus E26 oncogene homolog (ERG) fusion-positive cancers and that CEACAM1 loss was prognostic even in tumors harboring genomic deletions of the phosphatase and tensin homolog tumor suppressor (p < 0.0001). Multivariate analysis suggested that CEACAM1 analysis can provide independent prognostic information beyond established prognosis parameters at the stage of the initial biopsy when therapy decisions must be taken. In conclusion, loss of CEACAM1 expression predicts poor prognosis in prostate cancer and might provide clinically useful prognostic information particularly in cancers harboring the TMPRSS2:ERG fusion.

Upregulation of the transcription factor TFAP2D is associated with aggressive tumor phenotype in prostate cancer lacking the TMPRSS2:ERG fusion.

BACKGROUND: TFAP2D is a transcription factor important for modulating gene expression in embryogenesis. Its expression and prognostic role in prostate cancer has not been evaluated. METHODS: Therefore, a tissue microarray containing 17,747 prostate cancer specimens with associated pathological, clinical, and molecular data was analyzed by immunohistochemistry to assess the role of TFAP2D. RESULTS: TFAP2D expression was typically increased in prostate cancer as compared to adjacent non-neoplastic glands. TFAP2D staining was considered negative in 24.3% and positive in 75.7% of 13,545 interpretable cancers. TFAP2D staining was significantly linked to advanced tumor stage, high classical and quantitative Gleason grade, lymph node metastasis, and a positive surgical margin (p ≤ 0.0045). TFAP2D positivity was more common in ERG fusion positive (88.7%) than in ERG negative cancers (66.8%; p < 0.0001). Subset analyses in 3776 cancers with and 4722 cancers without TMPRSS2:ERG fusion revealed that associations with tumor phenotype and patient outcome were largely driven by the subset of ERG negative tumors. Multivariate analysis did not identify TFAP2D protein expression levels as a robust independent prognostic parameter. Positive TFAP2D immunostaining was significantly associated with 10 of 11 previously analyzed chromosomal deletions in ERG negative cancers (p ≤ 0.0244 each) indicating that elevated TFAP2D expression parallels genomic instability in prostate cancer. CONCLUSION: These data demonstrate that TFAP2D protein overexpression is linked to prostate cancer progression and genomic instability in ERG negative prostate cancers.

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.

High homogeneity of MMR deficiency in ovarian cancer.

OBJECTIVE: Mismatch repair (MMR) deficiency and Bethesda panel microsatellite instability (MSI) are increasingly analyzed to identify tumors that might benefit from immune checkpoint inhibitors, but tumor heterogeneity is a potential obstacle for such analyses. In ovarian cancer, data on intratumoral heterogeneity of MMR deficiency/MSI are lacking. METHODS: N = 582 ovarian cancers were screened for MMR deficiency by immunohistochemistry (IHC) on a tissue microarray. 10 cases suspect for MMR deficiency were identified among 478 interpretable cancers and repeated IHC on large sections combined with polymerase chain reaction (PCR)-based MSI analysis validated MMR deficiency/MSI in 9 of these tumors. RESULTS: MMR deficiency/MSI was predominantly seen in endmetrioid cancers (8 of 35, 23%) and also in 1 of 358 serous carcinomas (0.3%), but was absent in 34 mucinous carcinomas, 23 clear cell carcinomas, 17 malignant mixed Mullerian tumors (carcinosarcomas), and 11 mixed carcinomas. MMR deficiency involed protein loss of PMS2/MLH1 in 6 cases and of MSH2 and/or MSH6 in 3 cases. 7 MMR deficient cancers were MSI-high (all endometrioid), one was MSI-low (endometrioid) and one cancer with unequivocal MMR protein loss exhibited microsatellite stability (serous). MLH1 promotor methylation was observed in 4 of 5 endometrioid cancers with MLH1 protein loss. Immunostaining of all available cancer-containing tissue blocks (n = 114) of tumors with confirmed MMR deficiency/MSI revealed uniform MMR status throughout the entire tumor mass. CONCLUSIONS: Our data show that MSI is present in a substantial proportion of endometrioid ovarian cancers but can also occur in other tumor subtypes. MMR deficiency/MSI typically involves the entire tumor mass, suggesting that MMR inactivation occurs early in tumorigenesis in a subset of ovarian cancers.

Claudin-1 upregulation is associated with favorable tumor features and a reduced risk for biochemical recurrence in ERG-positive prostate cancer.

BACKGROUND: Claudin-1 is a membrane-tight junction protein and important for the sealing of the paracellular cleft in epithelial and endothelial cells. Differential expression of Claudin-1 is linked to disease outcome in various cancers. MATERIAL AND METHODS: To evaluate the potential relevance of Claudin-1 expression in prostate cancer, a tissue microarray containing samples of 17,747 tumors with annotated clinico-pathological and molecular data was immunohistochemically analyzed for Claudin-1 expression. RESULTS: In normal prostate, glandular cells were always Claudin-1-negative while there was a strong staining of gland-surrounding basal cells. In contrast to normal prostatic glands, a positive Claudin-1 immunostaining, was found, however, in 38.7% of 12,441 interpretable cancers and was considered weak in 12.7%, moderate in 13.2%, and strong in 12.8% of cases. Positive Claudin-1 immunostaining was associated with favorable tumor features like low pT (p = 0.0032), low Gleason grade (p< 0.0001), and a reduced risk of PSA recurrence (p = 0.0005). A positive Claudin-1 staining was markedly more frequent in ERG-positive (63%) than in ERG-negative cancers (23%; p < 0.0001). Subset analyses revealed that all associations of Claudin-1 expression and favorable phenotype and prognosis were driven by ERG-positive cancers. Multivariate analyses revealed, however, that even in ERG-positive cancers, the prognostic impact of high Claudin-1 expression was not independent of established clinico-pathological parameters. Comparison with 12 previously analyzed chromosomal deletions identified conspicuous associations with PTEN and 12p13 deletions potentially indicating functional interactions. CONCLUSION: These data identify a peculiar role for Claudin-1 in prostate cancer. The protein is overexpressed in a fraction of prostate cancers and increased Claudin-1 expression levels predict a favorable prognosis in ERG-positive cancer.