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.

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.

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.

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.

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.

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.

High density of cytotoxic T-lymphocytes is linked to tumoral PD-L1 expression regardless of the mismatch repair status in colorectal cancer.

BACKGROUND: Immune checkpoint-inhibitors targeting the PD-1/PD-L1 system are FDA approved in microsatellite instable (MSI) or mismatch repair deficient (dMMR) colorectal cancer (CRC). PD-L1 expression is tightly linked to features connected to immune checkpoint inhibitor response, but studies on large subsets of cancers analyzing the correlation between different status of MSI/dMMR, tumor infiltrating lymphocytes and PD-L1 expression are still lacking. METHODS: More than 1800 CRC were analyzed for PD-L1 by immunohistochemistry in a tissue microarray format. Data were compared to MMR, the number of intratumoral CD8+ cytotoxic T-cells, and adverse clinico-pathological parameters. Different cutoff levels for defining PD-L1 positivity in tumor cells (1%, 5%, 10%, and 50%) yielded comparable results. RESULTS: At a cutoff level of 5%, PD-L1 positivity was seen in 5.1% of tumors. PD-L1 was more often positive in dMMR (18.6%) than in MMR proficient (pMMR) cancers (4.1%; p < 0.0001). The number of intratumoral CD8+ lymphocytes was strikingly higher in PD-L1 positive (939.5 ± 118.2) than in PD-L1 negative cancers (310.5 ± 24.8). A higher number of intratumoral CD8+ lymphocytes was found in dMMR CRC (PD-L1 positive: 1999.7 ± 322.0; PD-L1 negative: 398.6 ± 128.0; p < 0.0001) compared to pMMR CRC (PD-L1 positive: 793.2 ± 124.8; PD-L1 negative: 297.2 ± 24.2; p < 0.0001). In dMMR and pMMR CRC, PD-L1 expression in tumor cells was unrelated to tumor stage, lymph node status or lymphatic/venous invasion. PD-L1 positivity in tumor associated immune cells was seen in 47.5% of cases and was significantly linked to high numbers of tumor infiltrating CD8+, low tumor stage, and absence of lymph node metastasis and lymphatic/venous invasion (p < 0.0001 each). CONCLUSION: The data support the previously suggested fact that PD-L1 expression in tumor cells is driven by extensive cytotoxic T-cell infiltration in highly immunogenic dMMR and pMMR CRC. Frequent and intense PD-L1 expression in tumor cells of dMMR CRC may contribute to the high response rates of dMMR CRC to immune checkpoint-inhibitors.

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.

Reduced KLK2 expression is a strong and independent predictor of poor prognosis in ERG-negative prostate cancer.

BACKGROUND: Kallikrein-related peptidase 2 (KLK2)-like KLK3 (prostate-specific antigen [PSA])-belongs to the highly conserved serine proteases of the glandular kallikrein protein family (KLK family). Studies suggested that measurement of KLK2 serum levels advanced the predictive accuracy of PSA testing in prostate cancer. METHODS: To clarify the potential utility of KLK2 as a prognostic tissue biomarker, KLK2 expression was analyzed by immunohistochemistry in more than 12 000 prostate cancers. RESULTS: Normal epithelium cells usually showed weak to moderate KLK2 immunostaining, whereas KLK2 was negative in 23%, weak in 38%, moderate in 35%, and strong in 4% of 9576 analyzable cancers. Lost or reduced KLK2 immunostaining was associated with advanced tumor stage, high Gleason score, lymph node metastasis, increased cell proliferation, positive resection margin, and early PSA recurrence (P < .0001). Comparison with previously analyzed molecular alterations revealed a strong association of KLK2 loss and presence of TMPRSS2:ERG fusion (P < .0001), most of all analyzed common deletions (9 of 11; P ≤ .03), and decreased PSA immunostaining (P < .0001 each). Cancers with combined negative or weak immunostaining of KLK2 and PSA showed worse prognosis than cancers with at least moderate staining of one or both proteins (P < .0001). Multivariate analyses including established preoperative and postoperative prognostic parameters showed a strong independent prognostic impact of KLK2 loss alone or in combination of PSA, especially in erythroblast transformation-specific-negative cancers (P ≤ .006). CONCLUSIONS: Loss of KLK2 expression is a potentially useful prognostic marker in prostate cancer. Analysis of KLK2 alone or in combination with PSA may be useful for estimating cancer aggressiveness at the time of biopsy.

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.

Epithelial splicing regulatory protein 1 and 2 (ESRP1 and ESRP2) upregulation predicts poor prognosis in prostate cancer.

BACKGROUND: Epithelial splicing regulatory protein 1 (ESRP1) and 2 (ESRP2) regulate alternative splicing events of various pre-mRNAs. Some of these targets play a role in cancer-associated processes, including cytoskeleton reorganization and DNA-repair processes. This study was undertaken to estimate the impact of ESRP1 and ESRP2 alterations on prostate cancer patient prognosis. METHODS: A tissue microarray made from 17,747 individual cancer samples with comprehensive, pathological, clinical and molecular data was analyzed by immunohistochemistry for ESRP1 and ESRP2. RESULTS: Nuclear staining for ESRP1 was seen in 38.6% (36.0% low, 2.6% high) of 12,140 interpretable cancers and in 41.9% (36.4% low, 5.3% high) of 12,962 interpretable cancers for ESRP2. Nuclear protein expression was linked to advanced tumor stage, high Gleason score, presence of lymph node metastasis, early biochemical recurrence, and ERG-positive cancers (p < 0.0001 each). Expression of ESRPs was significantly linked to 11 (ESRP1)/9 (ESRP2) of 11 analyzed deletions in all cancers and to 8 (ESRP1)/9 (ESRP2) of 11 deletions in ERG-negative cancers portending a link to genomic instability. Combined ESRPs expression analysis suggested an additive effect and showed the worst prognosis for cancers with high ESRP1 and ESRP2 expression. Multivariate analyses revealed that the prognostic impact of ESRP1, ESRP2 and combined ESRP1/ESRP2 expression was independent of all established pre- and postoperative prognostic features. CONCLUSIONS: Our data show a striking link between nuclear ESRP expression and adverse features in prostate cancer and identifies expression of ESRP1 and/or ESRP2 as independent prognostic markers with a potential for routine application.

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.

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.

High B7-H3 expression is linked to increased risk of prostate cancer progression.

B7-H3 is a member of the B7 superfamily of immune checkpoint molecules. B7-H3 up regulation has been linked to cancer development and progression in many tumors including prostate cancer. To clarify the potential utility of B7-H3 as a prognostic biomarker, B7-H3 expression was analyzed by immunohistochemistry in more than 17 000 prostate cancers. Normal prostatic glands were largely B7-H3 negative, while membranous B7-H3 immunostaining was seen in 47.0% of analyzed cancers. B7-H3 immunostaining was weak in 12.3%, moderate in 21.1% and strong in 13.5% of cases. High B7-H3 expression was associated with pT, Gleason score, lymph node metastasis, high Ki67 labeling index and early prostate-specific antigen recurrence (P < 0.0001 each). High B7-H3 expression was also linked to high androgen receptor expression and TMPRSS2:V-ets avian erythroblastosis virus E26 oncogene homolog (ERG) fusions (P < 0.0001 each). Multivariate analyses showed a strong independent prognostic impact of high B7-H3 expression in all cancers and in the ERG negative subgroup. Comparison with previously analyzed frequent chromosomal deletions revealed a close association with Phosphatase and Tensin Homolog deletions. Analysis of B7-H3, alone or in combination with other markers, might be of clinical utility, especially in the subgroup of ERG negative prostate cancers.

Upregulation of PTTG1 is associated with poor prognosis in prostate cancer.

Pituitary tumor-transforming gene 1 (PTTG1) is a regulator of chromosome stability. PTTG1 overexpression had been associated with tumor aggressiveness in several cancer types. To examine its prognostic utility in prostate cancer, a tissue microarray including 12 427 tumors with clinical and molecular data was analyzed by immunohistochemistry. PTTG1 immunostaining was largely absent in normal prostate epithelial cells. In cancers, staining was considered weak in 5.4%, moderate in 5.6% and strong in 0.8%. Strong staining was linked to advanced pT stage, high classical and quantitative Gleason grade, high Ki67-labeling index (all P < 0.0001) and lymph node metastasis (P = 0.0083). The prognostic impact of PTTG1 expression was independent of established preoperative and postoperative prognostic features. Comparison with molecular features revealed that PTTG1 upregulation was associated with nine of 12 common genomic deletions (P < 0.05), p53 alterations and high androgen receptor levels (P < 0.001 each), but was unrelated to the TMPRSS2:ERG fusion status. In conclusion, these data identify PTTG1 as a strong and independent prognostic feature in prostate cancer. PTTG1 measurement, either alone or in combination with other biomarkers might be instrumental for determining prostate cancer aggressiveness.

Loss of CCAAT-enhancer-binding protein alpha (CEBPA) is linked to poor prognosis in PTEN deleted and TMPRSS2:ERG fusion type prostate cancers.

BACKGROUND: The transcription factor CCAAT-enhancer-binding protein alpha (CEBPA) is a crucial regulator of cell proliferation and differentiation. Expression levels of CEBPA have been suggested to be prognostic in various tumor types. METHODS: Here, we analyzed the immunohistochemical expression of CEBPA in a tissue microarray containing more than 17 000 prostate cancer specimens with annotated clinical and molecular data including for example TMPRSS2:ERG fusion and PTEN deletion status. RESULTS: Normal prostate glands showed moderate to strong CEBPA staining, while CEBPA expression was frequently reduced (40%) or lost (30%) in prostate cancers. Absence of detectable CEBPA expression was markedly more frequent in ERG negative (45%) as compared to ERG positive cancers (20%, P < 0.0001). Reduced CEBPA expression was linked to unfavorable phenotype (P < 0.0001) and poor prognosis (P = 0.0008). Subgroup analyses revealed, that the prognostic value of CEBPA loss was entirely driven by tumors carrying both TMPRSS2:ERG fusions and PTEN deletions. In this subgroup, CEBPA loss was tightly linked to advanced tumor stage (P < 0.0001), high Gleason grade (P < 0.0001), positive nodal stage (0.0003), and early biochemical recurrence (P = 0.0007), while these associations were absent or markedly diminished in tumors with normal PTEN copy numbers and/or absence of ERG fusion. CONCLUSIONS: CEBPA is down regulated in about one third of prostate cancers, but the clinical impact of CEBPA loss is strictly limited to the subset of about 10% prostate cancers carrying both ERG fusion and deletions of the PTEN tumor suppressor. Our findings challenge the concept that prognostic molecular markers may be generally applicable to all prostate cancers.