Stimulator of Interferon Genes Protein (STING) Expression in Cancer Cells: A Tissue Microarray Study Evaluating More than 18,000 Tumors from 139 Different Tumor Entities.

Stimulator of interferon genes protein (STING) activates the immune response in inflammatory cells. STING expression in cancer cells is less well characterized, but STING agonists are currently being evaluated as anticancer drugs. A tissue microarray containing 18,001 samples from 139 different tumor types was analyzed for STING by immunohistochemistry. STING-positive tumor cells were found in 130 (93.5%) of 139 tumor entities. The highest STING positivity rates occurred in squamous cell carcinomas (up to 96%); malignant mesothelioma (88.5%-95.7%); adenocarcinoma of the pancreas (94.9%), lung (90.3%), cervix (90.0%), colorectum (75.2%), and gallbladder (68.8%); and serous high-grade ovarian cancer (86.0%). High STING expression was linked to adverse phenotypes in breast cancer, clear cell renal cell carcinoma, colorectal adenocarcinoma, hepatocellular carcinoma, and papillary carcinoma of the thyroid (p < 0.05). In pTa urothelial carcinomas, STING expression was associated with low-grade carcinoma (p = 0.0002). Across all tumors, STING expression paralleled PD-L1 positivity of tumor and inflammatory cells (p < 0.0001 each) but was unrelated to the density of CD8+ lymphocytes. STING expression is variable across tumor types and may be related to aggressive tumor phenotype and PD-L1 positivity. The lack of relationship with tumor-infiltrating CD8+ lymphocytes argues against a significant IFN production by STING positive tumor cells.

TRPS1 is a Highly Sensitive Marker for Breast Cancer: A Tissue Microarray Study Evaluating More Than 19,000 Tumors From 152 Different Tumor Entities.

Trichorhinophalangeal syndrome 1 (TRPS1) is a nuclear protein highly expressed in breast epithelial cells. TRPS1 immunohistochemistry (IHC) has been suggested as a breast cancer marker. To determine the diagnostic and prognostic utility of TRPS1 IHC, tissue microarrays containing 19,201 samples from 152 different tumor types and subtypes were analyzed. GATA3 IHC was performed in a previous study. TRPS1 staining was seen in 86 of 152 tumor categories with 36 containing at least one strongly positive case. TRPS1 staining predominated in various types of breast carcinomas (51%-100%), soft tissue tumors (up to 100%), salivary gland tumors (up to 46%), squamous cell carcinomas (up to 35%), and gynecological cancers (up to 40%). TRPS1 positivity occurred in 1.8% of 1083 urothelial neoplasms. In invasive breast carcinoma of no special type, low TRPS1 expression was linked to high grade ( P = 0.0547), high pT ( P < 0.0001), nodal metastasis ( P = 0.0571), loss of estrogen receptor and progesterone receptor expression ( P < 0.0001 each), and triple-negative status ( P < 0.0001) but was unrelated to patient survival ( P = 0.8016). In squamous cell carcinomas from 11 different sites, low TRPS1 expression was unrelated to tumor phenotype. Positivity for both TRPS1 and GATA3 occurred in 47.4% to 100% of breast cancers, up to 30% of salivary gland tumors, and 29 (0.3%) of 9835 tumors from 134 other cancer entities. TRPS1 IHC has high utility for the identification of cancers of breast (or salivary gland) origin, especially in combination with GATA3. The virtual absence of TRPS1 positivity in urothelial neoplasms is useful for the distinction of GATA3-positive urothelial carcinoma from breast cancer.

Cadherin-17 (CDH17) expression in human cancer: A tissue microarray study on 18,131 tumors.

Cadherin-17 (CDH17) is a membranous cell adhesion protein predominantly expressed in intestinal epithelial cells. CDH17 is therefore considered a possible diagnostic and therapeutic target. This study was to comprehensively determine the expression of CDH17 in cancer and to further assess the diagnostic utility of CDH17 immunohistochemistry (IHC). A tissue microarray containing 14,948 interpretable samples from 150 different tumor types and subtypes as well as 76 different normal tissue types was analyzed by IHC. In normal tissues, a membranous CDH17 staining was predominantly seen in the epithelium of the intestine and pancreatic excretory ducts. In tumors, 53 of 150 analyzed categories showed CDH17 positivity including 26 categories with at least one strongly positive case. CDH17 positivity was most common in epithelial and neuroendocrine colorectal neoplasms (50.0%-100%), other gastrointestinal adenocarcinomas (42.7%-61.6%), mucinous ovarian cancer (61.1%), pancreatic acinar cell carcinoma (28.6%), cervical adenocarcinoma (52.6%), bilio-pancreatic adenocarcinomas (40.5-69.8%), and other neuroendocrine neoplasms (5.6%-100%). OnIy 9.9% of 182 pulmonary adenocarcinomas were CDH17 positive. In colorectal adenocarcinomas, reduced CDH17 staining was linked to high pT (p = 0.0147), nodal metastasis (p = 0.0041), V1 (p = 0.0025), L1 (p = 0.0054), location in the right colon (p = 0.0033), and microsatellite instability (p < 0.0001). The CDH17 expression level was unrelated to tumor phenotype in gastric and pancreatic cancer. In summary, our comprehensive overview on CDH17 expression in human tumors identified various tumor entities that might often benefit from anti-CDH17 therapies and suggest utility of CDH17 IHC for the distinction of metastatic gastrointestinal or bilio-pancreatic adenocarcinomas (often positive) from primary pulmonary adenocarcinomas (mostly negative).

Steroidogenic Acute Regulatory Protein Is a Useful Marker for Sex-Cord-Stroma Tumors and Normal and Neoplastic Adrenocortical Tissue.

CONTEXT.­: Steroidogenic acute regulatory (StAR) protein is a mitochondrial transport protein with a critical regulatory role for steroid hormone production. The tissue distribution of StAR expression is limited to few human normal tissues. OBJECTIVE.­: To assess the diagnostic and prognostic value of StAR immunohistochemistry analysis. DESIGN.­: A tissue microarray containing 19 202 samples from 152 different tumor types and subtypes and 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. RESULT.­: StAR immunostaining occurred in 198 (1.2%) of the 17 135 analyzable tumors. StAR expression was observed in 27 of 152 tumor categories, 9 of which included at least 1 strongly positive case. The highest rate of StAR positivity occurred in Leydig cell tumors of the testis and the ovary (100%), steroid cell tumors of the ovary (100%), adrenocortical carcinomas (93%) and adenomas (87%), Sertoli-Leydig cell tumors (67%) and granulosa cell tumors of the ovary (56%), as well as seminomas (7%). Nineteen other tumor entities showed-a usually weak-StAR positivity in less than 6% of cases. A comparison with preexisting Melan-A (a melanocyte antigen) data revealed that StAR was more often positive in adrenocortical neoplasms and in Leydig cell tumors while StAR (but not Melan-A) was negative in Sertoli cell tumors. CONCLUSIONS.­: Our data provide a comprehensive overview on the patterns of StAR immunostaining in human tumors and suggest a diagnostic utility of StAR immunohistochemistry for supporting a diagnosis of Leydig cell tumors or of normal or neoplastic adrenocortical tissue.

Prostein expression in human tumors: a tissue microarray study on 19,202 tumors from 152 different Tumor entities.

BACKGROUND: Prostein (P501S), also termed solute carrier family 45 member 3 (SLC45A3) is an androgen regulated protein which is preferentially expressed in prostate epithelial cells. Because of its frequent expression in prostate cancer, prostein was suggested a diagnostic prostate cancer marker. METHODS: In order to comprehensively assess the diagnostic utility of prostein immunohistochemistry, a tissue microarray containing 19,202 samples from 152 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types was analyzed by immunohistochemistry. RESULTS: Prostein immunostaining was typically cytoplasmic, granular and perinuclear. Prostein positivity was seen in 96.7% of 419 prostate cancers including 78.3% with strong staining. In 16,709 extra-prostatic tumors, prostein positivity was observed in 7.2% of all cases but only 0.3% had a strong staining. Overall, 50 different extra-prostatic tumor categories were prostein positive, 12 of which included at least one strongly positive case. Extra-prostatic tumors with highest rates of prostein positivity included different subtypes of salivary gland tumors (7.6-44.4%), neuroendocrine neoplasms (15.8-44.4%), adenocarcinomas of the gastrointestinal tract (7.3-14.8%), biliopancreatic adenocarcinomas (3.6-38.7%), hepatocellular carcinomas (8.1%), and adenocarcinomas of other organs (up to 21%). CONCLUSIONS: Our data provide a comprehensive overview on prostein expression in human cancers. Prostein is a highly sensitive prostate cancer marker occurring in > 96% of prostate cancers. Because prostein can also be expressed in various other tumor entities, classifying of a tumor mass as a prostate cancer should not be based on prostein positivity alone.

GAD2 Is a Highly Specific Marker for Neuroendocrine Neoplasms of the Pancreas.

Glutamate decarboxylase 2 (GAD2) is the most important inhibitory neurotransmitter and plays a role in insulin-producing ß cells of pancreatic islets. The limitation of GAD2 expression to a few normal cell types makes GAD2 a potential immunohistochemical diagnostic marker. To evaluate the diagnostic utility of GAD2 immunohistochemistry, a tissue microarray containing 19,202 samples from 152 different tumor entities and 608 samples of 76 different normal tissue types was analyzed. In normal tissues, GAD2 staining was restricted to brain and pancreatic islet cells. GAD2 staining was seen in 20 (13.2%) of 152 tumor categories, including 5 (3.3%) tumor categories containing at least 1 strongly positive case. GAD2 immunostaining was most commonly seen in neuroendocrine carcinomas (58.3%) and neuroendocrine tumors (63.2%) of the pancreas, followed by granular cell tumors (37.0%) and neuroendocrine tumors of the lung (11.1%). GAD2 was only occasionally (<10% of cases) seen in 16 other tumor entities including paraganglioma, medullary thyroid carcinoma, and small cell neuroendocrine carcinoma of the urinary bladder. Data on GAD2 and progesterone receptor (PR) expression (from a previous study) were available for 95 pancreatic and 380 extrapancreatic neuroendocrine neoplasms. For determining a pancreatic origin of a neuroendocrine neoplasm, the sensitivity of GAD2 was 64.2% and specificity 96.3%, while the sensitivity of PR was 56.8% and specificity 92.6%. The combination of PR and GAD2 increased both sensitivity and specificity. GAD2 immunohistochemistry is a highly useful diagnostic tool for the identification of pancreatic origin in case of neuroendocrine neoplasms with unknown site of origin.

Diagnostic Role and Prognostic Impact of PSAP Immunohistochemistry: A Tissue Microarray Study on 31,358 Cancer Tissues.

Prostate-specific acid phosphatase (PSAP) is a marker for prostate cancer. To assess the specificity and prognostic impact of PSAP, 14,137 samples from 127 different tumor (sub)types, 17,747 prostate cancers, and 76 different normal tissue types were analyzed via immunohistochemistry in a tissue microarray format. In normal tissues, PSAP staining was limited to the prostate epithelial cells. In prostate cancers, PSAP was seen in 100% of Gleason 3 + 3, 95.5% of Gleason 4 + 4, 93.8% of recurrent cancer under androgen deprivation therapy, 91.0% of Gleason 5 + 5, and 31.2% of small cell neuroendocrine cancer. In non-prostatic tumors, PSAP immunostaining was only found in 3.2% of pancreatic neuroendocrine tumors and in 0.8% of diffuse-type gastric adenocarcinomas. In prostate cancer, reduced PSAP staining was strongly linked to an advanced pT stage, a high classical and quantitative Gleason score, lymph node metastasis, high pre-operative PSA levels, early PSA recurrence (p < 0.0001 each), high androgen receptor expression, and TMPRSS2:ERG fusions. A low level of PSAP expression was linked to PSA recurrence independent of pre- and postoperative prognostic markers in ERG-negative cancers. Positive PSAP immunostaining is highly specific for prostate cancer. Reduced PSAP expression is associated with aggressive prostate cancers. These findings make PSAP a candidate marker for prognostic multiparameter panels in ERG-negative prostate cancers.

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.

4-Aminopyridine is a promising treatment option for patients with gain-of-function KCNA2-encephalopathy.

Developmental and epileptic encephalopathies are devastating disorders characterized by epilepsy, intellectual disability, and other neuropsychiatric symptoms, for which available treatments are largely ineffective. Following a precision medicine approach, we show for KCNA2-encephalopathy that the K+ channel blocker 4-aminopyridine can antagonize gain-of-function defects caused by variants in the KV1.2 subunit in vitro, by reducing current amplitudes and negative shifts of steady-state activation and increasing the firing rate of transfected neurons. In n-of-1 trials carried out in nine different centers, 9 of 11 patients carrying such variants benefitted from treatment with 4-aminopyridine. All six patients experiencing daily absence, myoclonic, or atonic seizures became seizure-free (except some remaining provoked seizures). Two of six patients experiencing generalized tonic-clonic seizures showed marked improvement, three showed no effect, and one worsening. Nine patients showed improved gait, ataxia, alertness, cognition, or speech. 4-Aminopyridine was well tolerated up to 2.6 mg/kg per day. We suggest 4-aminopyridine as a promising tailored treatment in KCNA2-(gain-of-function)­encephalopathy and provide an online tool assisting physicians to select patients with gain-of-function mutations suited to this treatment.

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.

Increased Cytoplasmic CD138 Expression Is Associated with Aggressive Characteristics in Prostate Cancer and Is an Independent Predictor for Biochemical Recurrence.

Syndecan-1 (CD138) is a transmembrane proteoglycan expressed in various normal and malignant tissues. It is of interest due to a possible prognostic effect in tumors and its role as a target for the antibody-drug conjugate indatuximab ravtansine. Here, we analyzed 17,747 prostate cancers by immunohistochemistry. Membranous and cytoplasmic CD138 staining was separately recorded. In normal prostate glands, CD138 staining was limited to basal cells. In cancers, membranous CD138 positivity was seen in 19.6% and cytoplasmic CD138 staining in 11.2% of 12,851 interpretable cases. A comparison with clinico-pathological features showed that cytoplasmic CD138 staining was more linked to unfavorable tumor features than membranous staining. Cytoplasmic CD138 immunostaining was associated with high tumor stage (p < 0.0001), high Gleason grade (p < 0.0001), nodal metastases (p < 0.0001), positive surgical margin (p < 0.0001), and biochemical recurrence (p < 0.0001). This also holds true for both V-ets avian erythroblastosis virus E26 oncogene homolog (ERG) fusion positive and ERG fusion negative tumors although the cytoplasmic CD138 expression was markedly more frequent in ERG positive than in ERG negative tumors (p < 0.0001). Comparison with 11 previously analyzed chromosomal deletions identified a conspicuous association between cytoplasmic CD138 expression and 8p deletions (p < 0.0001) suggesting a possible functional interaction of CD138 with one or several 8p genes. Multivariate analysis revealed the cytoplasmic CD138 expression as an independent prognostic parameter in all cancers and in the ERG positive subgroup. In summary, our study indicates the cytoplasmic CD138 expression as a strong and independent predictor of poor prognosis in prostate cancer. Immunohistochemical measurement of CD138 protein may thus-perhaps in combination with other parameters-become clinically useful in the future.

Secreted Frizzled-Related Protein 4 (SFRP4) Is an Independent Prognostic Marker in Prostate Cancers Lacking TMPRSS2: ERG Fusions.

Secreted frizzled-related protein 4 (SFRP4) controls WNT signaling and is thought to play a role for tumor aggressiveness. Here, we analyzed a tissue microarray containing 11,152 prostate cancers with pathological, clinical and molecular data by immunohistochemistry. SFRP4 expression was higher in cancer than in non-neoplastic acinar cells. SFRP4 staining was seen in 64.9% of tumors and classified as weak in 33.2%, moderate in 23.9% and strong in 7.8% of cancers. SFRP4 overexpression was linked to advanced tumor stage, high classical/quantitative Gleason grade (p < 0.0001 each), lymph node metastasis (p = 0.0002), and a positive surgical margin (p = 0.0017). SFRP4 positivity was markedly more frequent in ERG positive (77.4%) than in ERG negative cancers (57.4% p < 0.0001). Subset analyses in 2725 cancers with and 3592 cancers without TMPRSS2:ERG fusion revealed that associations with tumor phenotype and patient outcome were largely driven by the subset of ERG negative tumors. In a multivariate analysis including various postoperative and prognostic clinico-pathological features, SFRP4 protein expression emerged as an independent prognostic parameter in ERG negative cancers. SFRP4 immunostaining was significantly linked with 10 of 11 previously analyzed chromosomal deletions (p < 0.05 each). In conclusion, high SFRP4 immunostaining is associated with poor prognosis and genomic instability in ERG negative prostate cancers.

High CHK2 protein expression is a strong and independent prognostic feature in ERG negative prostate cancer.

Checkpoint kinase 2 (CHK2) is a serine-threonine kinase with a role in DNA repair, cell cycle arrest or apoptosis in response to DNA damage. Both reduced and increased CHK2 expression has been described in different tumour types with impact on patient prognosis. To evaluate prevalence and significance of altered CHK2 expression in prostate cancer, a tissue microarray containing 17,747 tumours was analysed by immunohistochemistry. Nuclear CHK2 immunostaining was absent or weak in benign prostate epithelium but often more prominent in cancers. CHK2 immunostaining was considered weak in 38.8%, moderate in 33.6% and strong in 11.2% of prostate cancers. High CHK2 expression was strongly associated with TMPRSS2:ERG fusions (p<0.0001). Subgroup analysis of ERG positive and negative cancers revealed that high CHK2 staining was significantly linked to advanced tumour stage, high Gleason score, positive nodal status, positive surgical margin, high preoperative PSA (p<0.0001 each) and early prostate-specific antigen (PSA) recurrence (p=0.0001) in the subset of ERG negative cancers, while most of these associations were absent in ERG positive cancers. In ERG negative cancers, high CHK2 expression was an independent predictor of patient prognosis, even if parameters were included that were only available postoperatively. High CHK2 expression was also linked to presence of chromosomal deletions, high level of androgen receptor expression, positive p53 immunostaining, and high Ki-67 labelling index. These provide further in vivo evidence for previously described functional interactions. In summary, high CHK2 expression is linked to adverse tumour features and independently predicts early biochemical recurrence in ERG negative prostate cancer. CHK2 measurement, either alone or in combination, might be of clinical utility in this prostate cancer subgroup.

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.

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.

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.

Integrating Tertiary Gleason 5 Patterns into Quantitative Gleason Grading in Prostate Biopsies and Prostatectomy Specimens.

BACKGROUND: Presence of small (tertiary) Gleason 5 pattern is linked to a higher risk of biochemical recurrence in prostate cancer. It is unclear, however, how to integrate small Gleason 5 elements into clinically relevant Gleason grade groups. OBJECTIVE: To analyze the prognostic impact of Gleason 5 patterns in prostate cancer and to develop a method for integrating tertiary Gleason 5 patterns into a quantitative Gleason grading system. DESIGN, SETTING, AND PARTICIPANTS: Prostatectomy specimens from 13 261 consecutive patients and of 3295 matched preoperative biopsies were available. Percentages of Gleason 3, 4, and 5 had been recorded for each cancer. Outcome measurements and statistical analysis: RESULTS AND LIMITATIONS: Our data demonstrate that minimal Gleason 5 areas have strong prognostic impact in Gleason 7 carcinomas, while further expansion of the Gleason 5 pattern population has less impact. We thus defined an integrated quantitative Gleason score (IQ-Gleason) by adding a lump score of 10 to the percentage of unfavorable Gleason pattern (Gleason 4/5) if any Gleason 5 was present and by adding another 7.5 points in case of a Gleason 5 fraction >20%. There was a continuous increase of the risk of prostate-specific antigen recurrence with increasing IQ-Gleason. This was also true for subgroups with identical Cancer of the Prostate Risk Assessment Postsurgical scores (p<0.0001) or Gleason grade groups (p<0.0001). CONCLUSIONS: The IQ-Gleason represents a simple and efficient approach for combining both quantitative Gleason grading and tertiary Gleason grades in one highly prognostic numerical variable. PATIENT SUMMARY: Prostatectomy specimens (13 261) were analyzed to estimate the relevance of small Gleason 5 elements in prostate cancers. Even the smallest Gleason 5 areas markedly increased the risk of prostate-specific antigen recurrence after surgery. Larger fractions of Gleason 5 patterns had less further impact on prognosis. Based on this, a numerical Gleason score (integrated quantitative Gleason score) was defined by the percentages of Gleason 4 and 5 patterns, enabling a refined estimate of patient prognosis.

Clinical Utility of Quantitative Gleason Grading in Prostate Biopsies and Prostatectomy Specimens.

BACKGROUND: Gleason grading is the strongest prognostic parameter in prostate cancer. Gleason grading is categorized as Gleason ≤ 6, 3 + 4, 4 + 3, 8, and 9-10, but there is variability within these subgroups. For example, Gleason 4 components may range from 5-45% in a Gleason 3 + 4 = 7 cancer. OBJECTIVE: To assess the clinical relevance of the fractions of Gleason patterns. DESIGN, SETTING, AND PARTICIPANTS: Prostatectomy specimens from 12823 consecutive patients and of 2971 matched preoperative biopsies for which clinical data with an annual follow-up between 2005 and 2014 were available from the Martini-Klinik database. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: To evaluate the utility of quantitative grading, the fraction of Gleason 3, 4, and 5 patterns seen in biopsies and prostatectomies were recorded. Gleason grade fractions were compared with prostatectomy findings and prostate-specific antigen recurrence. RESULTS AND LIMITATIONS: Our data suggest a striking utility of quantitative Gleason grading. In prostatectomy specimens, there was a continuous increase of the risk of prostate-specific antigen recurrence with increasing percentage of Gleason 4 fractions with remarkably small differences in outcome at clinically important thresholds (0% vs 5%; 40% vs 60% Gleason 4), distinguishing traditionally established prognostic groups. Also, in biopsies, the quantitative Gleason scoring identified various intermediate risk groups with respect to Gleason findings in corresponding prostatectomies. Quantitative grading may also reduce the clinical impact of interobserver variability because borderline findings such as tumors with 5%, 40%, or 60% Gleason 4 fractions and very small Gleason 5 fractions (with pivotal impact on the Gleason score) are disclaimed. CONCLUSIONS: Quantitative Gleason pattern data should routinely be provided in addition to Gleason score categories, both in biopsies and in prostatectomy specimens. PATIENT SUMMARY: Gleason score is the most important prognostic parameter in prostate cancer, but prone to interobserver variation. The results of our study show that morphological aspects that define the Gleason grade in prostate cancer represent a continuum. Quantitation of Gleason patterns provides clinically relevant information beyond the traditional Gleason grading categories ≤ 3 + 3, 3 + 4, 4 + 3, 8, 9 -1 0. Quantitative Gleason scoring can help to minimize variations between different pathologists and substantially aid in optimized therapy decision-making.