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Purpose: The identification of a robust IHC marker to predict the response to antiangiogenic bevacizumab in ovarian cancer is of high clinical interest. VEGF-A, the molecular target of bevacizumab, is expressed as multiple isoforms with pro- or antiangiogenic properties, of which VEGF-A165b is the most dominant antiangiogenic isoform. The balance of VEGF-A isoforms is closely related to the angiogenic capacity of a tumor and may define its vulnerability to antiangiogenic therapy. We investigated whether the expression of VEGF-A165b could be related to the effect of bevacizumab in advanced ovarian cancer patients. Experimental Design: Formalin-fixed paraffin-embedded tissues from 413 patients of the ICON7 multicenter phase III trial, treated with standard platinum-based chemotherapy with or without bevacizumab, were probed for VEGF-A165b expression by IHC. Results: In patients with low VEGF-A165b expression, the addition of bevacizumab to standard platinum-based chemotherapy significantly improved progression-free (HR: 0.727; 95% CI, 0.538–0.984; P = 0.039) and overall survival (HR: 0.662; 95% CI, 0.458–0.958; P = 0.029). Multivariate analysis showed that the addition of bevacizumab in low VEGF-A165b–expressing patients conferred significant improvements in progression-free survival (HR: 0.610; 95% CI, 0.446–0.834; P = 0.002) and overall survival (HR: 0.527; 95% CI, 0.359–0.775; P = 0.001), independently from established risk factors. Conclusions: We demonstrate for the first time that bevacizumab may differentially improve the prognosis of advanced ovarian cancer patients with low expression of VEGF-A165b, an antiangiogenic VEGF-A splice variant. We envision that this novel biomarker could be implemented into routine diagnostics and may have direct clinical implications for guiding bevacizumab-related treatment decisions in advanced ovarian cancer patients.

Purpose:The identification of a robust IHC marker to predict the response to antiangiogenic bevacizumab in ovarian cancer is of high clinical interest. VEGF-A, the molecular target of bevacizumab, is expressed as multiple isoforms with pro- or antiangiogenic properties, of which VEGF-A165b is the most dominant antiangiogenic isoform. The balance of VEGF-A isoforms is closely related to the angiogenic capacity of a tumor and may define its vulnerability to antiangiogenic therapy. We investigated whether the expression of VEGF-A165b could be related to the effect of bevacizumab in advanced ovarian cancer patients.Experimental Design:Formalin-fixed paraffin-embedded tissues from 413 patients of the ICON7 multicenter phase III trial, treated with standard platinum-based chemotherapy with or without bevacizumab, were probed for VEGF-A165b expression by IHC.Results:In patients with low VEGF-A165b expression, the addition of bevacizumab to standard platinum-based chemotherapy significantly improved progression-free (HR: 0.727; 95% CI, 0.538–0.984; P = 0.039) and overall survival (HR: 0.662; 95% CI, 0.458–0.958; P = 0.029). Multivariate analysis showed that the addition of bevacizumab in low VEGF-A165b–expressing patients conferred significant improvements in progression-free survival (HR: 0.610; 95% CI, 0.446–0.834; P = 0.002) and overall survival (HR: 0.527; 95% CI, 0.359–0.775; P = 0.001), independently from established risk factors.Conclusions:We demonstrate for the first time that bevacizumab may differentially improve the prognosis of advanced ovarian cancer patients with low expression of VEGF-A165b, an antiangiogenic VEGF-A splice variant. We envision that this novel biomarker could be implemented into routine diagnostics and may have direct clinical implications for guiding bevacizumab-related treatment decisions in advanced ovarian cancer patients.

Supplementary Figure from Bevacizumab May Differentially Improve Prognosis of Advanced Ovarian Cancer Patients with Low Expression of VEGF-A165b, an Antiangiogenic VEGF-A Splice Variant

Background Ten–eleven translocation methylcytosine dioxygenase 1 (TET1) is involved in DNA demethylation and transcriptional regulation, plays a key role in the maintenance of stem cell pluripotency, and is dysregulated in malignant cells. The identification of cancer stem cells (CSCs) driving tumor growth and metastasis is the primary objective of biomarker discovery in aggressive prostate cancer (PCa). In this context, we analyzed TET1 expression in PCa. Methods A large-scale immunohistochemical analysis of TET1 was performed in normal prostate (NOR) and PCa using conventional slides (50 PCa specimens) and tissue microarrays (669 NOR and 1371 PCa tissue cores from 371 PCa specimens). Western blotting, RT-qPCR, and 450 K methylation array analyses were performed on PCa cell lines. Genome-wide correlation, gene regulatory network, and functional genomics studies were performed using publicly available data sources and bioinformatics tools. Results In NOR, TET1 was exclusively expressed in normal cytokeratin 903 (CK903)–positive basal cells. In PCa, TET1 was frequently detected in alpha-methylacyl-CoA racemase (AMACR)–positive tumor cell clusters and was detectable at all tumor stages and Gleason scores. Pearson’s correlation analyses of PCa revealed 626 TET1-coactivated genes (r > 0.5) primarily encoding chromatin remodeling and mitotic factors. Moreover, signaling pathways regulating antiviral processes (62 zinc finger, ZNF, antiviral proteins) and the pluripotency of stem cells were activated. A significant proportion of detected genes exhibited TET1-correlated promoter hypomethylation. There were 161 genes encoding transcription factors (TFs), of which 133 were ZNF-TFs with promoter binding sites in TET1 and in the vast majority of TET1-coactivated genes. Conclusions TET1-expressing cells are an integral part of PCa and may represent CSCs with oncogenic potential.

Background Renal cell carcinoma (RCC) is a heterogeneous disease comprising histologically defined subtypes. For therapy selection, precise subtype identification and individualized prognosis are mandatory, but currently limited. Our aim was to refine subtyping and outcome prediction across main subtypes, assuming that a tumor is composed of molecular features present in distinct pathological subtypes. Methods Individual RCC samples were modeled as linear combination of the main subtypes (clear cell (ccRCC), papillary (pRCC), chromophobe (chRCC)) using computational gene expression deconvolution. The new molecular subtyping was compared with histological classification of RCC using the Cancer Genome Atlas (TCGA) cohort (n = 864; ccRCC: 512; pRCC: 287; chRCC: 65) as well as 92 independent histopathologically well-characterized RCC. Predicted continuous subtypes were correlated to cancer-specific survival (CSS) in the TCGA cohort and validated in 242 independent RCC. Association with treatment-related progression-free survival (PFS) was studied in the JAVELIN Renal 101 (n = 726) and IMmotion151 trials (n = 823). CSS and PFS were analyzed using the Kaplan–Meier and Cox regression analysis. Results One hundred seventy-four signature genes enabled reference-free molecular classification of individual RCC. We unambiguously assign tumors to either ccRCC, pRCC, or chRCC and uncover molecularly heterogeneous tumors (e.g., with ccRCC and pRCC features), which are at risk of worse outcome. Assigned proportions of molecular subtype-features significantly correlated with CSS (ccRCC (P = 4.1E − 10), pRCC (P = 6.5E − 10), chRCC (P = 8.6E − 06)) in TCGA. Translation into a numerical RCC-R(isk) score enabled prognosis in TCGA (P = 9.5E − 11). Survival modeling based on the RCC-R score compared to pathological categories was significantly improved (P = 3.6E − 11). The RCC-R score was validated in univariate (P = 3.2E − 05; HR = 3.02, 95% CI: 1.8–5.08) and multivariate analyses including clinicopathological factors (P = 0.018; HR = 2.14, 95% CI: 1.14–4.04). Heterogeneous PD-L1-positive RCC determined by molecular subtyping showed increased PFS with checkpoint inhibition versus sunitinib in the JAVELIN Renal 101 (P = 3.3E − 04; HR = 0.52, 95% CI: 0.36 − 0.75) and IMmotion151 trials (P = 0.047; HR = 0.69, 95% CI: 0.48 − 1). The prediction of PFS significantly benefits from classification into heterogeneous and unambiguous subtypes in both cohorts (P = 0.013 and P = 0.032). Conclusion Switching from categorical to continuous subtype classification across most frequent RCC subtypes enables outcome prediction and fosters personalized treatment strategies.

Bladder cancer is one of the more common malignancies in humans and the most expensive tumor for treating in the Unites States (US) and Europe due to the need for lifelong surveillance. Non-invasive tests approved by the FDA have not been widely adopted in routine diagnosis so far. Therefore, we aimed to characterize the two putative tumor suppressor genes ECRG4 and ITIH5 as novel urinary DNA methylation biomarkers that are suitable for non-invasive detection of bladder cancer. While assessing the analytical performance, a spiking experiment was performed by determining the limit of RT112 tumor cell detection (range: 100&ndash, 10,000 cells) in the urine of healthy donors in dependency of the processing protocols of the RWTH cBMB. Clinically, urine sediments of 474 patients were analyzed by using quantitative methylation-specific PCR (qMSP) and Methylation Sensitive Restriction Enzyme (MSRE) qPCR techniques. Overall, ECRG4-ITIH5 showed a sensitivity of 64% to 70% with a specificity ranging between 80% and 92%, i.e., discriminating healthy, benign lesions, and/or inflammatory diseases from bladder tumors. When comparing single biomarkers, ECRG4 achieved a sensitivity of 73%, which was increased by combination with the known biomarker candidate NID2 up to 76% at a specificity of 97%. Hence, ITIH5 and, in particular, ECRG4 might be promising candidates for further optimizing current bladder cancer biomarker panels and platforms.

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA (TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa.

The annual symposium of the German Research Association for Bladder Carcinoma (DFBK) was organized on February 7th and 8th, 2020, in Dusseldorf. On the first day, eight international guest speakers invited by the DFBK and the Department of Urology of the Heinrich Heine University Dusseldorf presented the current state of research on bladder cancer (BC). Topics were genomic changes and molecular classification in non-muscle-invasive and muscle-invasive BC, prospects and limits of proteome technology in urine diagnostics, function of chromatin regulators in bladder carcinogenesis, cellular reactions to aneuploidy, organoid technology and biobanking, as well as novel aspects of immunotherapy for BC. The second day was dedicated to new results and ideas of the DFBK members on BC pathomechanisms, diagnostics and therapeutic approaches, and most importantly, discussions on the further development of collaborative projects. Additional information is available at http://www.forschungsverbund-blasenkarzinom.de.

In this study, we aimed to identify a DNA methylation pattern suitable for prognosis assessment of muscle-invasive bladder cancer and to investigate metastasis-associated processes regulated by DNA methylation. Genome-wide methylation analysis was performed on 23 muscle-invasive bladder tumors by microarray analysis. Validation was performed by the qAMP technique in two different patient cohorts (n = 32 and n = 100). mRNA expression was analyzed in 12 samples. Protein expression was determined using tissue microarrays of 291 patients. Bladder cancer cell lines T24 and 253JB-V were used for functional analyses. Microarray analyses revealed KISS1R, SEPT9 and CSAD as putative biomarkers with hypermethylation in node-positive tumors. The combination of the three genes predicted the metastatic risk with sensitivity of 73% and specificity of 71% in cohort 1, and sensitivity of 82% and specificity of 54% in cohort 2. mRNA expression differences were detected for KISS1R (p = 0.04). Protein expression of KISS1R was significantly reduced (p

The German Bladder Cancer Association (DFBK) invited its members to the 3rd annual meeting 2012 in Hannover 4 years after the official founding. The meeting was directed to discuss the progress of ongoing and newly initiated projects and collaborations. In this article we will introduce current research activities and collaborations of the DFBK and would like to invite interested researchers to join this national interdisciplinary research association. The aim of the DFBK is to initiate interdisciplinary collaboration and to support scientific discussions among its members. For further information please visit our website at www.forschungsverbund-blasenkarzinom.de.

measured using RT-qPCR. Results from cohort A were used to develop a model with (combinations of) the four genes based on the comparative CT method, and the model was validated in the independent cohort B. RESULTS: The HOXC6-DLX1 score model resulted in the highest area-under-the-curve (AUC) of 0.75 (95% confidence interval: 0.70-0.80) for the diagnosis of high-grade PCa upon prostate biopsy, with a specificity of 36% and 91% sensitivity. The HOXC6-DLX1 score outperformed PCA3 in both cohorts (cohort A: AUC 0.75 vs. 0.65; cohort B: 0.73 vs. 0.62). Furthermore, the HOXC6-DLX1 score was significantly correlated with the Gleason score upon biopsy. Subgroup analysis confirmed the added value of this urine test for the prediction of high-grade PCa in patients with low serum PSA values (

Currently used clinicopathological parameters are insufficient for a reliable prediction of metastatic risk and disease-free survival (DFS) of patients with clear-cell renal cell carcinoma (ccRCC). To identify prognostic genes, the expression profiles of primary ccRCC obtained from patients with different DFS--eight synchronously, nine metachronously and seven not metastasized tumors--were determined by genome-wide expression analyses. Synchronously and metachronously metastasized primary ccRCC differed in the expression of 167 genes. Thirty-six of these genes were also differentially expressed in synchronously vs. metachronously developed pulmonary metastases analyzed in a previous study. Because of their DFS-associated deregulation that is concordant in metastases and primary ccRCC, these genes are potentially functionally involved in metastatic tumor growth and are also prognostically useful. A prognostic impact was confirmed for the genes CD31, EDNRB and TSPAN7 at the mRNA level (n=86), and for TSPAN7 at the protein level (n=106). Patients with a higher gene expression of EDNRB or TSPAN7, or with TSPAN7-positive vessels in both cores investigated on tissue microarrays had a significantly longer DFS and tumor-specific survival (TSS). Patients with a higher CD31 gene expression showed a significantly longer TSS. EDNRB was an independent prognostic marker for the DFS. CD31, EDNRB and TSPAN7 had an independent impact on the TSS. In summary, comparative analysis of primary tumors and metastases is appropriate to identify independent prognostic markers in ccRCC. Gene expression of CD31 and EDNRB, and endothelial TSPAN7 protein level are potentially useful to improve outcome prediction because of their independent prognostic impact.

Prostate cancer (PCa) is the most common noncutaneous cancer diagnosis and the second leading cause of cancer-related deaths among men in the United States. Effective treatment modalities for advanced metastatic PCa are limited. Immunotherapeutic strategies based on T cells and antibodies represent interesting approaches to prevent progression from localized to advanced PCa and to improve survival outcomes for patients with advanced disease. CD8+ cytotoxic T lymphocytes (CTLs) efficiently recognize and destroy tumor cells. CD4+ T cells augment the antigen-presenting capacity of dendritic cells and promote the expansion of tumor-reactive CTLs. Antibodies mediate their antitumor effects via antibody-dependent cellular cytotoxicity, activation of the complement system, improving the uptake of coated tumor cells by phagocytes, and the functional interference of biological pathways essential for tumor growth. Consequently, several tumor-associated antigens (TAAs) have been identified that represent promising targets for T cell- or antibody-based immunotherapy. These TAAs comprise proteins preferentially expressed in normal and malignant prostate tissues and molecules which are not predominantly restricted to the prostate, but are overexpressed in various tumor entities including PCa. Clinical trials provide evidence that specific immunotherapeutic strategies using such TAAs represent safe and feasible concepts for the induction of immunological and clinical responses in PCa patients. However, further improvement of the current approaches is required which may be achieved by combining T cell- and/or antibody-based strategies with radio-, hormone-, chemo- or antiangiogenic therapy.

Context MicroRNAs (miRNA) are noncoding RNAs that post-transcriptionally regulate gene expression. Their altered expression and function have been observed in most urologic cancers. MiRNAs represent potential disease biomarkers and novel therapeutic targets. Objective To review and evaluate the evidence implicating miRNAs in the pathogenesis of prostate cancer (PCa), bladder cancer (BCa), and renal cancer. Evidence acquisition A systematic review was performed using PubMed and Embase to search for reports using strings for microRNA, non-coding RNA, cancer, prostate, bladder, and renal cancer. Identified manuscripts were retrieved and references searched. Selected studies were required to concentrate on the role of miRNA in these urologic cancers. Evidence synthesis We reviewed articles that focus on this topic. More than 40 miRNAs have been implicated in urologic cancer and many target common carcinogenic pathways. In particular, apoptosis avoidance, cell proliferation, epithelial-to-mesenchymal transition, angiogenic signalling, and the generation of androgen independence are targeted or facilitated by more than one miRNA. Little work has been done to evaluate the translational applications for this knowledge to date. Novel therapeutic strategies have been developed and are under investigation to selectively modulate miRNAs; such work would potentially enable personalised tumour therapy. Conclusions MiRNAs appear to be important modulators of urologic cancer. Their expression is frequently altered in these tumours, and many are functionally implicated in their pathogenesis. They require evaluation to determine the translational role and therapeutic potential for this knowledge.

Ein vorrangiges Ziel des nationalen Netzwerkes „Deutscher Forschungsverbund Blasenkarzinom e.V.“ ist es, eine Plattform zu bieten, auf der wissenschaftliche Aktivitaten im Bereich der Blasenkarzinomforschung gebundelt und kommuniziert werden. Einen fundierten Uberblick uber aktuelle Arbeiten auf diesem Forschungsgebiet bot die Jahrestagung der Deutschen Gesellschaft fur Urologie vom 22. bis 25. September 2010 in Dusseldorf. In diesem Artikel werden einige der interessanten Beitrage zum Thema Blasenkarzinomforschung inhaltlich zusammengefasst und kommentiert. Ein Schwerpunkt wurde insbesondere auf das Urothelkarzinom im Bereich der Uropathologie und die praklinische Forschung gelegt.

Iron nanoparticles encapsulated by carbon are protected from reactions with their environment avoiding oxidation in ambient conditions and thus, preserving their magnetic properties. Such particles are good candidates for magnetic fluid hyperthermia. When graphite shells are present, acidic treatments allow the formation of carboxylic groups on the nanoparticle surface. Those carboxylic groups can be used for further complexation with the drug cisplatin. We show the possibility of loading cisplatin on such nanoparticles and that the loading is dependent on the degree of surface functionalization. The drug release is dependent on time and temperature, making it ideal for applications involving hyperthermia. We show the possibility of applying hyperthermia in vitro using these nanoparticles. When loaded with cisplatin a stronger cytotoxic effect is observed. Such particles could be potentially used as multimodal anti-cancer agents for therapies based on the synergistic effect of chemotherapy and hyperthermia.

Background: The urokinase plasminogen activator (uPA) system is one of the best-investigated protease systems, both under physiological and pathological conditions, including various types of cancer. However, effects of co-expression of members of the uPA system in soft-tissue sarcoma (STS) patients at the protein level in both tumour tissue and serum have not been investigated yet. Methods: We examined 82 STS patients for protein levels of uPA, PAI-1and uPAR in tumour tissue and serum by ELISA. Results: A significant correlation between high antigen levels of uPA, PAI-1 or uPAR in tumour tissue, and of uPAR in serum, with poor outcome of STS patients was found for the first time. Most strikingly, we observed an additive effect of combined uPA, PAI-1 or uPAR levels in tumour tissue extracts with uPAR levels in serum on patients' prognosis. High uPA/uPAR, PAI-1/uPAR and uPAR/uPAR antigen levels in tumour tissue/serum were associated with a 5.9-fold, 5.8-fold and 6.2-fold increased risk of tumour-related death (P=0.003, 0.001 and 0.002, respectively) compared with those patients who displayed low levels of the respective marker combination. Conclusion: As expression of members of the uPA system in tumour tissue and serum is additively correlated with prognosis of STS patients, our results suggest that combinations of these biomarkers can identify STS patients with a higher risk of tumour-related death.

Carbon-encapsulated magnetic nanoparticles are a new class of materials where the core magnetic nanoparticle is protected from reactions with its environment by graphite shells. Having a structure similar to carbon nanotubes, these nanoparticles could be potentially functionalized using methods which are already applied to those structures. We present the effects of acidic treatments based on HCl, HNO3, and H2SO4 on these nanoparticles highlighting the impact on their magnetic and surface properties. We show that acidic treatments based on HNO3 can be successfully applied for the generation of carboxylic groups on the surface of the nanoparticles. Using methylamine as a model, we demonstrate that these functional groups can be used for further functionalization with amino-containing biomolecules via diimide-activated amidation.

Due to their particular magnetic properties, nanoparticles of metallic iron are promising candidates for magnetic fluid hyperthermia when compared to the commonly used iron oxides. However, the difficulty of handling these structures in ambient conditions without oxidation hinders its practical application. In this work, iron filled carbon nanotubes non-covalently functionalized by human serum albumin are studied as potential agents for hyperthermia. Here the iron is encapsulated inside of the carbon shells and protected from reactions with its environment. Besides protecting the iron and biological environment against each other, the carbon shells can also work as an interface for conjugation with other biological molecules of interest. In order to assess if such structures could induce any toxic effect in human cell cultures, we have probed its biocompatibility on a dosage and time dependent manner by measuring metabolic activity, cell proliferation, cell cycle distribution and apoptosis. Our results have shown that those nanotubes strongly associate with cells within a short incubation period and do not pose any significant toxic effect. The magnetic properties of iron filled carbon nanotubes in biological environment, i.e., associated to cells, have been studied and a possible rotation as a function of the applied magnetic field is discussed. Our initial findings encourage the further study of these structures as potential hyperthermia agents.

Selected transcript markers as well as their combinations were analyzed on minimal prostate tissue specimens with regard to their diagnostic potential. Artificial prostate biopsies from RPE explants were used for evaluation and optimization of the techniques used followed by application to diagnostic prostate needle core biopsies. Minimal prostate specimens were cryopreserved and processed with standardized methods. The RNA amount of a half of each biopsy was sufficient for the analysis of 11 marker genes and one reference gene (TBP) using quantitative PCR assays.The relative transcript amounts obtained were included in several analyses including calculations for each single marker gene like median overexpression rate as well as marker combinations. Two optimized mathematical models based on relative expression levels of EZH2, hepsin, PCA3, prostein, and TRPM8 were evaluated with regard to their diagnostic potential. Compared to single marker analyses these models show higher sensitivity and specificity for prostate cancer detection.Thus biomolecular prostate cancer identification may represent a suitable diagnostic tool to supplement conventional techniques on prostate biopsies. Furthermore, an extension of this approach to PCa prognosis and the transfer to urine samples appear very promising.

Objectives The absence of effective therapies for advanced prostate cancer has entailed an intensive search for novel treatments. This review presents an overview of specific immunotherapeutic strategies for prostate cancer. Methods Current literature was reviewed regarding the identification of tumor antigens and the design of T-cell– and antibody-based immunotherapy for prostate cancer. The PubMed database was searched using the key words antibodies, clinical trials, dendritic cells, immunotherapy, prostate cancer, and T cells. Results T cells and antibodies are powerful components of the specific antitumor immune response. CD8 + cytotoxic T lymphocytes (CTLs) efficiently destroy tumor cells. CD4 + T cells improve the antigen-presenting capacity of dendritic cells (DCs) and support the stimulation of tumor-reactive CTLs. Monoclonal antibodies exhibit their antitumor effects via antibody-dependent cellular cytotoxicity and complement activation. Consequently, much attention has been given to the identification of tumor antigens that represent attractive targets for specific immunotherapy. Several prostate cancer-related antigens were described and used in clinical trials. Such studies were based on the administration of peptides, proteins, or DNA. Furthermore, men with prostate cancer were vaccinated with peptide-, protein-, or RNA-loaded DCs, which display an extraordinary capacity to induce tumor-reactive T cells. Monoclonal antibodies directed against surface antigens were also used. Clinical trials revealed that immunotherapeutic strategies represent safe and feasible concepts for the induction of immunologic and clinical responses in men with prostate cancer. Conclusions Specific immunotherapy represents a promising treatment modality for prostate cancer. Further improvement of the current approaches is required and may be achieved by combining T-cell– and antibody-based vaccination strategies with radio-, hormone-, chemo-, or antiangiogenic therapy.

As individual risk assessment mainly depends on the correct prediction of the tumor's biological behavior, primary diagnosis plays a key role in the clinical management of prostate cancer patients. Prostate core needle biopsy, as a primary diagnostic tool, should not only confirm clinical suspicion but also supply the urologist with information which is necessary for risk-adapted therapy. The experience and competence of both the urologist and the pathologist are crucial for the quality of prostate core needle biopsy diagnosis. Optimized handling and submission of prostate core needle biopsy specimens by the urologist to the pathologist are of outstanding importance for improving the number of cancer cases detected. Increasing availability of molecular markers leads to the necessity of developing new tissue sampling procedures which allow prostate core needle biopsy specimens to be simultaneously studied histologically and by molecular approaches.

Cancer stem cells can play an important role in tumorigenesis and tumor progression. However, it is still difficult to detect and isolate cancer stem cells. An alternative approach is to analyse stem cell-associated gene expression. We investigated the coexpression of three stem cell-associated genes, Hiwi, hTERT and survivin, by quantitative real-time-PCR in 104 primary soft-tissue sarcomas (STS). Multivariate Cox's proportional hazards regression analyses allowed correlating gene expression with overall survival for STS patients. Coexpression of all three stem cell-associated genes resulted in a significantly increased risk of tumor-related death. Importantly, tumors of patients with the poorest prognosis were of all four tumor stages, suggesting that their risk is based upon coexpression of stem cell-associated genes rather than on tumor stage.

Dendritic cells (DCs) essentially contribute to the induction and regulation of innate and adaptive immunity. Based on these important properties, DCs may profoundly influence tumor progression in patients. However, little is known about the role of distinct human DC subsets in primary tumors and their impact on clinical outcome. In the present study, we investigated the characteristics of human 6-sulfo LacNAc (slan) DCs in clear cell renal cell carcinoma (ccRCC). slanDCs have been shown to display various tumor-directed properties and to accumulate in tumor-draining lymph nodes from patients. When evaluating 263 ccRCC and 227 tumor-free tissue samples, we found increased frequencies of slanDCs in ccRCC tissues compared to tumor-free tissues. slanDCs were also detectable in the majority of 24 metastatic lymph nodes and 67 distant metastases from ccRCC patients. Remarkably, a higher density of slanDCs was significantly associated with a reduced progression-free, tumor-specific or overall survival of ccRCC patients. Tumor-infiltrating slanDCs displayed an immature phenotype expressing interleukin-10. ccRCC cells efficiently impaired slanDC-induced T-cell proliferation and programming as well as natural killer (NK) cell activation. In conclusion, these findings indicate that higher slanDC numbers in ccRCC tissues are associated with poor prognosis. The induction of a tolerogenic phenotype in slanDCs leading to an insufficient activation of innate and adaptive antitumor immunity may represent a novel immune escape mechanism of ccRCC. These observations may have implications for the design of therapeutic strategies that harness tumor-directed functional properties of DCs against ccRCC.

The development of T-cell-based immunotherapies of cancer largely depends on the availability of tumour-associated antigens capable of eliciting tumour-directed cytotoxic T-cell responses. In prostate cancer, the number of antigens defined as suitable targets of cytotoxic T lymphocytes (CTLs) is still limited. Recently, prostein was identified as a transmembrane protein that is highly restricted to prostate tissues. In our study, prostein transcripts were found to be abundant in both malignant and nonmalignant prostate tissue samples. To identify immunogenic CD8+ T-cell epitopes, human leucocyte antigen-A(*)0201-binding peptides were selected from the amino-acid sequence of prostein and were used for the in vitro stimulation of CD8+ T lymphocytes. Specific CTLs were raised against the prostein-derived peptide CLAAGITYV that were capable of lysing prostate cancer cells, indicating that this peptide is naturally generated by tumour cells. Our data suggest that prostein is a suitable candidate to be included in a T-cell-based immunotherapy of prostate cancer.

BACKGROUND New concepts for the immunotherapy of prostate carcinoma (PCa) largely depend on the identification of suitable target antigens that are present in a high percentage of prostate tumors. Their expression in normal tissues should be restricted to the prostate and they should be immunogenic in vivo. The number of antigens displaying these properties is still limited. Here, we identify for the first time an immunogenic peptide derived from the prostate-specific protein transient receptor potential-p8 (trp-p8) that is recognized by cytotoxic T lymphocytes (CTLs) from PCa patients. METHODS To determine the abundance of trp-p8 in prostate tumors, the expression level of trp-p8 mRNA was quantitatively analyzed in a panel of prostate cancer tissues. Trp-p8-derived human leukocyte antigen (HLA)-A*0201-restricted peptides were selected and tested for the in vitro activation of CTLs when loaded on autologous dendritic cells (DCs). RESULTS Trp-p8 mRNA was found to be expressed in all prostate tumors and in the corresponding normal prostate tissue. Of five selected trp-p8-derived peptides, only peptide GLMKYIGEV was shown to activate specific CTLs, which effectively lysed PCa cells confirming the endogenous generation and presentation of this peptide by tumor cells. CONCLUSIONS Our results suggest this antigen as a suitable target for the T-cell-based immunotherapy of PCa. Prostate 56: 270–279, 2003. © 2003 Wiley-Liss, Inc.

Identification of TAAs recognized by CD8(+) CTLs paved the way for new concepts in cancer therapy. In view of the heterogeneity of tumors and their diverse escape mechanisms, CTL-based cancer therapy largely depends on an appropriate number of TAAs. In prostate cancer, the number of antigens defined as suitable targets of CTLs remains rather limited. PSCA is widely distributed in prostate cancer. In this report, we define immunogenic peptides of PSCA which are recognized by circulating CD8(+) T cells from prostate cancer patients and able to activate CTLs in vitro. Screening the amino acid sequence of PSCA for peptides containing a binding motif for HLA-A*0201 resulted in 8 candidate peptides. Specificity and affinity of peptide binding were verified in a competition assay. Frequencies of CD8(+) T lymphocytes reactive against selected epitopes were determined in the blood of prostate cancer patients using the ELISPOT assay. Increased frequencies were revealed for CD8(+) T cells recognizing the peptides ALQPGTALL and AILALLPAL. CTLs from prostate cancer patients were raised against these 2 peptides in vitro when presented by autologous DCs. They specifically recognized peptide-pulsed T2 target cells and prostate cancer cells that were HLA-A*0201- and PSCA-positive, indicating that these peptides were naturally generated by tumor cells. These data suggest that PSCA is a promising target for the immunotherapy of prostate cancer.

Human Pin1, a peptidyl-prolyl cis/trans isomerase with high specificity to -Ser/Thr(PO(3)H(2))-Pro- motifs, is required for cell cycle progression. In an effort to design reversible Pin1 inhibitors by using a substrate structure based approach, a panel of peptides were applied to systematically analyze the minimal structural requirements for Pin1 substrate recognition. Pin1 catalysis (k(cat)/K(m)5 mM(-1) s(-1)) for Ala-Pro, Ser-Pro, and Ser(PO(3)H(2))-Pro was detected using direct UV-visible spectrophotometric detection of prolyl isomerization, while weak competitive inhibition of Pin1 by these dipeptides was observed (K(i)1 mM). Substrates with chain lengths extending from either the P2 to P1' or the P1 to P2' subsite gave k(cat)/K(m) values of 100 mM(-1) s(-1) for Ala-Ser(PO(3)H(2))-Pro and 38 mM(-1) s(-1) for Ser(PO(3)H(2))-Pro-Arg. For both Pin1 and its yeast homologue Ess1, the optimal subsite recognition elements comprise five amino acid residues with the essential Ser(PO(3)H(2)) in the middle position. The resulting substrate Ac-Ala-Ala-Ser(PO(3)H(2))-Pro-Arg-NH-4-nitroanilide possesses a very low cis/trans interconversion barrier in the presence of either Pin1 or Ess1, with k(cat)/K(m) = 9300 mM(-1) s(-1) and 12000 mM(-1) s(-1), respectively. The D-Ser(PO(3)H(2)) residue preceding proline could serve as a substrate-deactivating determinant without compromising ground state affinity. Similarly, substitution of the amide bond preceding proline with a thioxo amide bond produces a potent inhibitor. Pin1 is reversibly inhibited by such substrate analogue inhibitors with IC(50) values in the low micromolar range. The D-amino acid containing inhibitor also exhibits remarkable stability against phosphatase activity in cell lysate.